Azura Amid (Eds.) - Recombinant Enzymes - From Basic Science To Commercialization-Springer International Publishing (2015) PDF

Recombinant Enzymes—From Basic Science
to Commercialization
Azura Amid
Editor
Recombinant Enzymes—
From Basic Science
to Commercialization
1 3
Editor
Azura Amid
Department of Biotechnology Engineering
Kulliyyah of Engineering
International Islamic University Malaysia
Kuala Lumpur
Malaysia
ISBN 978-3-319-12396-7 ISBN 978-3-319-12397-4 (eBook)

DOI 10.1007/978-3-319-12397-4
Springer Cham Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014955170
© Springer International Publishing Switzerland 2015

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To my parents, husband, Nurulss
and Amiruddin
You are my strength. May Allah bless us
Ameen
v
Preface
Recombinant enzyme is a substance that has many applications in industry. Studies

on the productivity, efficiency, durability and its applications have been conducted
by many researchers worldwide. However, suitable and focus research on a larger
scale production is rarely discussed in detail because it is kept as secret by giant
companies producing recombinant enzyme. Often the good findings on the latest
discoveries in the laboratory were put on shelf and not suitable to be commercial-
ized. Therefore, this book aims to share basic science that should be emphasized
by the researchers so that their bench scale results will translates into a commercial
product in the future.
In the first chapter, the reader will be introduced to the recombinant enzyme and
why it is so important in our daily lives. This chapter is also discussing the impor-
tance of research and commercialization strategies. The second chapter highlights
the recombinant DNA strategies in producing recombinant enzyme clone.
The fourth and fifth chapters discuss in more detailed studies that need to be
carried out on the recombinant enzyme especially on the characterization and puri-
fication stages. When the basic rules have been strengthened, research methods in
the up-stream processing of fermentation condition including media formulation
that is able to produce high productivity is discusses in the fifth chapter, followed
by strategies to scale up the production volume to higher scale is discuss in Chap. 7.
Later, Chap. 8 discuss the downstream processing of recombinant enzyme. In
this chapter there are four stages of processes that need to carry out to ensure that
the resulting recombinant enzyme has high quality and purity. To ensure a product
is suitable to be produced in large scale manufacturing plant, engineering design is
required. Chapter nine highlights procedure on how to produce engineering design
along with appropriate software. The important of economic and environmental
evaluation analysis carried out by the software are also discussed.
Finally, Chaps. 10, 11 and 12 bring readers to the case study of recombinant bro-
melain. This enzyme has been cloned by recombinant DNA methods and has gone
through phases of many research stages those discussed in chapters two to nine.
Finally, this book would not be a reality if there are no supports from all parties
involved, namely editors, authors and publisher. We hope this book will encourage
researchers to keep continue their basic research related to recombinant enzyme
vii
viii Preface
and finally able to commercialize it. This book is dedicated to all recombinant
enzyme researchers especially those who are in Malaysia. Editor dreams that one
day Malaysia will have our own recombinant enzyme manufacturing plant where
many students and biochemical engineer will be able to practice their knowledge.
A. Amid
Contents
1 Introduction to Recombinant Enzyme Pre-commercialization�� 1

Azura Amid
2 Recombinant Enzyme: Cloning and Expression�� 11

Azura Amid and Norhidayah Hassan
3 Common Laboratory Procedure�� 19

Azura Amid, Mohd Jamil Aizat Jamaluddin
and Muhd Ezza Faize Othman
4 Characterization of Recombinant Enzymes�� 41

Farah Fadwa Ben Belgasem and Hamzah Mohd. Salleh
5 Purification of Recombinant Protein for Industrial Use�� 61

Faridah Yusof
6 Recombinant-Enzyme Fermentation�� 81

Azura Amid, Nurul Azira Ismail and Mohd Jamil Aizat Jamaluddin
7 Scaling-Up Recombinant Enzyme Fermentation�� 99

Azlin Suhaida Azmi, Sarina Sulaiman, Nor Fadhillah Mohamed Amin
and Fathilah Ali
8 Downstream Processing of Recombinant Enzymes

for Commercialization�� 115
Fadzilah Adibah Abdul Majid
9 Economic and Environmental Evaluation of Recombinant

Enzyme Production�� 129
Mohd Jamil Aizat Jamaluddin, Azlin Suhaida Azmi, Sarina Sulaiman,
Dzun Noraini Jimat, Muhd. Ezza Faiez Othman and Azura Amid
ix
x Contents
10 Case Study: Recombinant Bromelain Selection�� 143

Azura Amid, Nurul Azira Ismail and Zatul Iffah Mohd Arshad
11 Case Study: Recombinant Bromelain Cloning,

Characterization and Upstream Processes�� 159
12 Case Study: Recombinant Bromelain Downstream Processing�� 175

Azura Amid, Zatul Iffaf Mohd Arshad and Muhd Ezza Faiez Othman
Index�� 187
Chapter 1
Introduction to Recombinant Enzyme
Pre-commercialization
Azura Amid
Abstract This chapter discusses a number of matters involving the commercializa-

tion of recombinant enzymes. The chapter begins with an introduction of recombi-
nant enzymes and an explanation of why they should be commercialized, the factors
that encourage academics to commercialize product, the methods that can be used in
the commercialization process and the obstacles in the commercialization process.
Keywords File patent · Investor · Joint-venture · Key performance index · Pre-

commercialization grant · Recombinant proteases · Spin-off-compnay · Xylanase
1.1 Recombinant Enzyme
1.1.1 What?
A recombinant enzyme is an enzyme that is produced through recombinant DNA

technology. First, the gene for the specific enzyme is isolated from a natural
source, such as bacteria, animals, plants or humans. The specific gene is then
ligated into the expression vector and transformed into a suitable host. The host
selection is often based on the final application. Researchers are advised to choose
a host that is easiest to manipulate in advance, such as E. coli, before choosing
a host that is difficult to use, such as mammalian cells. Moreover, the fermenta-
tion cost for E. coli is much less than for mammalian cells. Furthermore, there
are modified strains of E. coli that have posttranslational modification pathways,
such as those reported by Chen and co–workers [1]. The recombinant enzyme is
produced either as an intercellular or extracellular protein and must undergo a pro-
cess of purification and formulation before it can be used in further applications.
A. Amid ()
Biomolecular and Bioprocess Engineering Research Unit, Department of Biotechnology
Engineering, Faculty of Engineering, International Islamic University Malaysia, P.O. Box 10,
50728 Kuala Lumpur, Malaysia
e-mail: azuraamid@iium.edu.my
© Springer International Publishing Switzerland 2015 1
A. Amid (ed.), Recombinant Enzymes–From Basic Science to Commercialization,
DOI 10.1007/978-3-319-12397-4_1
2 A. Amid
The purification process has been facilitated by the availability of protein-tagging

technology. The tagged heterologous enzyme is captured by the anti-tag during the
purification process
1.1.2 The Differences Between Natural and Recombinant

Enzyme
Natural and recombinant enzymes are indistinguishable by the naked eye. How-
ever, recombinant enzymes have been genetically modified to be more effective at
the desired temperatures, pH, or other conditions. Recombinant enzymes may also
be resistant to various inhibitors of enzyme activity (e.g., harsh chemicals), which
improves their use and efficiency in industrial or home applications.
1.1.3 Applications
Recombinant enzymes have similar applications as the natural enzyme. All of the
functions of a natural enzyme can also be performed by recombinant enzymes.
There are many recombinant enzymes used in the medical field. These enzymes
include recombinant insulin for diabetics, recombinant growth hormone for treat-
ing growth hormone deficiency in children and recombinant Factor VIII for treat-
ing hemophilia. Recombinant enzymes are also used in the food industry. For
example, recombinant chymosin is used to manufacture cheese and recombinant
bovine somatotropin is used to increase the production of high quality milk in
cows.
Recombinant enzymes are also used in the detergent industry where recombinant
proteases are used to remove stubborn dirt. Recombinant proteases are also widely
used in the cosmetic industry because proteases can enhance the penetration of ac-
tive ingredients into the skin. Furthermore, proteases are included in livestock feed
to produce more protein. Proteases also function as a natural meat tenderizer. In ad-
dition to proteases, recombinant lipase is also a common enzyme used in industrial
applications. For example, lipase is used in the manufacture of Roquefort cheese
and lipid-rich wastewater treatment [2]. Hasan and co-workers mentioned the use of
several microbial lipases in the following industrial applications: detergents, food,
flavor additives, biocatalytic resolution of pharmaceuticals, esters and amino acid
derivatives, fine chemical production, agrochemicals, biosensors, bioremediation,
cosmetics and perfume production [3]. Another important recombinant enzyme is
pectinase. Pectinase is used in fruit and vegetable processing, citrus processing,
wine making, coffee and tea fermentation, paper making, and textile processing as
well as in animal feed and laboratory equipment [4].
1 Introduction to Recombinant Enzyme Pre-commercialization 3
1.2 Why Choose Recombinant Enzyme
1.2.1 Shorter Production Time Using Fermentation

and Purification Technology
Many proteases can be found in fruits, such as kiwi, papaya, fig and pineapple.
The continued production of these enzymes from fruits will be very time consum-
ing. Moreover, fruit is preferred as a food and is not an ideal commercial enzyme
source. Therefore, recombinant enzymes are an alternative and bacteria can be used
as a host to grow and produce the enzyme. Bacteria require 12–24 h to produce en-
zymes, which is less than the 2–3 months required to harvest fruit. The purification
process for enzymes using recombinant DNA technology is less tedious because the
enzyme may have be a fusion tagged protein that easily binds to a special matrix
during purification.
1.2.2 Production of Rare and Difficult Enzymes For Industry

Application
Recombinant DNA technology and protein engineering offer a superior method of

modifying enzymes, especially for industrial applications that involve high tem-
perature and high pH. Not all natural enzymes are compatible with this industrial
processes requirement. Thus, by using recombinant DNA technology and protein
engineering one can tailor the modified enzyme according to industrial require-
ments. One example of this type of enzyme is thermostable xylanase, which is used
in the pulp and paper industry. To produce pulp the wood is treated at a high tem-
perature and basic pH. Most xylanases can only partly fulfill these requirements.
Thus, a thermostable recombinant enzyme is a better choice [5].
1.2.3 H
alal Issues for Proteins Isolated From Human
or Animal Tissue
Several industrial enzymes are produced from non-halal sources. For example, in-
sulin for the treatment of diabetes and lipase for lipid modification have a porcine
base [6]. All products used by the Muslim community must be from a halal and
“toyibah” source. Therefore, it is advantageous to use recombinant enzymes pro-
duced by microorganisms that are accepted as safe.
4 A. Amid
1.3 Reason for Commercialization
1.3.1 Researchers Satisfaction
Commercialization is generally an unpopular activity for academics because it

does not affect their key performance index. The factors included in the key perfor-
mance index are the number of publications, teaching quality, amount of profit from
consultation activities, research grants and quality of service delivered to society.
Therefore, many academics who are trying to commercialize their findings are mo-
tivated by the satisfaction of moving their research product to market.
The commercialization process is not easy for a busy academic that has teach-
ing, research and management obligations. An academician has to learn to become
an entrepreneur and businessman, which are new skills that are outside of their
expertise area.
The main motivation for many academics involved in the commercialization pro-
cess is the sense of satisfaction derived from sharing their research with the community.
1.3.2 Researcher Key Performance Index
Commercialization activities do not directly contribute to the key performance in-

dex (KPI) of an academic. However, some activities related to commercialization
indirectly affects the overall KPI score. For example, the patent should be filed in
advance to ensure the research output is not replicated by other groups. A patent
contributes to the KPI.
The government often gives pre-commercialization grants to high impact proj-
ects prior to the commercialization phase. Examples of grants given by the Malay-
sian government are the Techno-fund under the Ministry of Science, Technology
and Innovation (MOSTI) and the Prototype Research Grant Scheme (PRGS) under
the Ministry of Education (MOE). Successfully getting this type of grant will in-
crease the KPI.
1.3.3 Revenue Generation
Governments allocate money to universities to conduct fundamental research. If

the fundamental research is successful and the results can be commercialized then
the taxes paid by companies producing the product return to the government and
provide financial support to new fundamental projects.
1.3.4 Market Demands
One key factor that academics must consider at the start of a research project is the
needs and problems of society. An important objective of research is to solve a cur-
rent problem by finding a remedy for important issues. Examples of major problems
that need solutions include the following: treatments for infectious diseases, cures
for metabolic disorders, solutions to the problem of distance communication and
improvement of national defense technologies.
The correct problem must be given priority when developing new research so
that fundamental research results can lead to commercialization.
The markets are often large when the product solves the daily problems of life.
For example, a solution to diabetes has a wide market in Malaysia because more
than 11 million Malaysians suffered from diabetes in 2011 alone. This number does
not include patients from Southeast Asia, Asia Pacific, and Europe. Therefore, an
accurate problem statement is very important in fundamental research destined for
eventual commercialization.
1.3.5 Innovation Refinement
The effectiveness of a product cannot be validated on the small scale of a laboratory

because solutions that work in the lab do not necessarily or appropriately transition
to an industrial scale. For example, enzyme production in a laboratory in small
quantities can be formulated into powder form using lyophilization. However, an
industrial scale freeze dryer is absolutely not appropriate because it would increase
the cost of production and this method requires electricity and it takes time. Thus,
on an industrial scale the production of enzymes in powder form should be per-
formed using a spray dryer. However, the spray dryer method may deactivate the
enzyme when high temperatures are applied to convert the enzyme from a liquid to
a powder form. These types of modifications must be made to the product and are
considered innovation refinements.
1.3.6 Recognition
It is undeniable that when an academician successfully commercializes a product

they will be recognized by colleagues and community leaders. They will also be
an example to colleagues and may be occasionally invited to give lectures on their
experience in commercialization and transitioning products to the market.
6 A. Amid
1.3.7 Financial Rewards
When a product is successfully commercialized there are certainly financial re-

wards for the innovator. However, the rewards depend on whether the product is
commercialized in the form of licensing or as a joint venture spin-off company.
1.4 Method of Commercialization
1.4.1 Licensing
There are two important things you need to know about licensing. First is the licens-
ee. The licensee is the manufacturer who rents the idea. Second, are the products
that you want to rent. A researcher needs to identify the product that will be licensed
and how long the product will be licensed. With licensing, your licensee invests all
of their money and takes all of the financial risk. The manufacturers advertise the
product, market it, manufacture it and put their money into the project. If for some
reason they do not perform, you get your invention back and then you are free to
license it to someone else. By licensing, the researcher does not need to raise money
or run a company. A common royalty rate for consumer products is often 3–10 %
of the wholesale price. To ensure that nobody loss money, the researcher needs a
licensing attorney to write the licensing contract.
The advantages of choosing licensing to commercialize research products in-
clude not needing capital to operate a business and access to a mass market because
the manufacturer already has a strong network. Additionally, more time is needed to
run a business than license it to the manufacturer.
Licensing also has disadvantages, such as the fact that the license agreement is
normally for a considerable period of time and that there may be an annual mini-
mum royalty required. Additionally, a new technology may become available that
makes the licensed opportunity obsolete.
1.4.2 Joint-Venture Or Spin-Off-Company
A spin-off company is a company that does business using the university or research
institution intellectual property, and the university may or may not have equity in
the company. The running royalty and upfront fees will be paid to the university
and researcher based on the IP rights and negotiations. This company is called a
joint-venture spin-off company if the university has equity in the company. In this
case, the dividends generated from the successful operation of the company will
be distributed to the participating organizations or individuals in proportion to the
amount of equity they hold in the company.
1.5 Strategies for Commercialization
1.5.1 Identifying Suitable Product
Prior to commercializing a biotechnology product, the researcher must choose

the right products. What are the main factors that should be given priority in the
selection of the right product to be commercialized? To ensure successful commer-
cialization for any biotechnology product, researchers must address an important
issue that enables the products to fulfill the user’s need. The users of a product may
consist of a number of parties. For example, the users of a pharmaceutical product
are patients, physicians and vendors [7]. Researchers must also identify the market
size of their invention, similar products that are available in the market and how
the invention is better than products already in the market. A researcher may use a
market report of for desired product area by referring to the market report data pro-
vided by the Data Group (www.datagroup.asia), Trade Key (www.tradekey.com)
or Docstocs (http://www.docstoc.com/). After reviewing all of the information, the
researcher can decide whether his/her product is suitable for commercialization.
1.5.2 File Patent For Product Protection?
Is it true that researchers must file a patent to protect their ideas from being stolen
by other parties? Which idea is the most appropriate to protect with a patent? Not
all ideas require a patent because not all ideas will have good market opportunities
or will be suitable for commercialization. Filing a patent is not cheap and neither
is maintaining the patent. Therefore, obtaining a patent makes sense only if there
is a reason to believe more money will be made than spent. Instead of obtaining a
patent, the researcher may also choose to have a trade secret, as an alternative, and
this option is free. Many large companies use trade secrets to protect their idea. For
example, Coca-Cola uses a secret recipe. Veer and Jell [8] found that individual
inventors, small firms and universities have different motives for their patents. Uni-
versities are willing to license their patents, while small companies use patents as
signals to investors. Small companies use patents to secure access to capital and
grow the business.
1.5.3 Promoting and Marketing
The most challenging part of commercialization for researchers is marketing. The

personality of an entrepreneur is totally different from an academician. While an
academician is interested in answering questions to make a perfect product, an en-
trepreneur needs to ensure that they constantly offer new products quickly and must
seek new ways to get products to the market [9]. A research exhibition or innovation
8 A. Amid
session may help promote researcher innovation output to investors. In Malaysia,

there are many chances to promote products to investors, such as during the Malay-
sia International Technology Expo and BioMalaysia, which are organized almost
every year. Researchers are also encouraged to participate in international exhibi-
tions, such as the Geneva Inventions, which is also organized every year. However,
it is important not to share everything about your idea because an idea does not
belong to anybody unless it is protected.
1.5.4 Links with Industries
There are many grants available in Malaysia that try to match researchers with
industry players, such as the Techno Fund (under the Ministry of Science, Tech-
nology and Innovation and the Ministry of Agriculture) and MyLABS (under the
Ministry of Education). This idea is brilliant, but this opportunity has been mis-
used by some parties to manipulate the grants. A clear plan must be established
between the collaborator and the researcher so that the final objective will be ac-
complished during the given time and both parties will receive the rewards of
commercialization.
1.5.5 Identifying Possible Investor
In an effort to make Malaysia a high-income nation, the Agensi Inovasi Malaysia

(AIM) was established. AIM provides an environment for researchers to commer-
cialize their products. The services provided by AIM include providing opportuni-
ties for the researcher to find suitable investors. The AIM commercialization office
PlaTCOM Ventures provides a platform for researchers to have their idea commer-
cialized, and it has a list of angel investors willing to invest in a potential product.
The Technology Strategy Board in the United Kingdom and Development Innova-
tion Ventures in the United State of America have similar functions.
1.5.6 Identifying and Applying For Commercialization Grant
In addition to AIM, the Malaysian Technology Development Corporation

(MTDC) also promotes the adoption of technologies by local companies via com-
mercialization activities for local inventions. There are four types of grants avail-
able, and these include the Commercialization of Research & Development Fund
(CRDF), Technology Acquisition Fund (TAF), Business Growth Fund (BGF) and
Business Start-up Fund (BSF). The most appropriate grants for researchers are
CRDF and BSF.
1.6 Obstacle During the Pre-commercialization Stage
1.6.1 Each Product Is Unique
One guarantee is there is no single product that will have the same standard operat-
ing procedure (SOP) that can be applied to commercialize another product. Each
product is unique, and researchers need unique approaches. In the case of recombi-
nant enzymes, some enzymes might not be expressed by E. coli and need eukaryote
hosts.
1.6.2 Bench Scale Procedures Are Not Suitable

When Scaling-Up
One example of a bench scale procedure that is not suitable for larger scale produc-
tion is host cell disruption to produce recombinant enzymes. Homogenization or
continuous sonication procedures are more suitable when handling larger volume
of host cells. However, batch sonication procedures are tedious and take more time.
The important factors require attention when handling larger cell volumes are the
processing time and manufacturing cost. Researchers must try to minimize these
two factors to the lowest level as possible.
1.6.3 Fine-Tuning Each Unit Processes
When new scale-up equipment is used, optimum processes for each unit must be
identified. Therefore, engineering concepts for optimizing and scale-up processing
will be applied. The best solution is to ensure that you have an engineer in your
team to address this problem. For recombinant enzyme production, a biochemical
engineer is the most appropriate candidate to bring into your research team.
1.6.4 Unethical Business Competitors
There is no doubt there are times when researchers feel frustrated when the results
of studies that have consisted of years are reproduced by unethical competitors. It is
frustrating when the product has been marketed with the same design and name. If
this happens, do not panic; look at the product strengths that helped the competitor
decide to copy your product. Then, increase the quality of your product because a
smart buyer will prefer a higher quality product. There is also an option to use all
of the legal protections available to protect your IP. The design of the logo, bottle
and packaging are valuable assets that must be protected. An annual market survey
10 A. Amid
is useful for identifying changes that might show your product is copied. It is also
important to make sure the product service is high quality and your customer feels
they are appreciated
References
1. Chen N, Hong FL, Wang HH, Yuan QH, Ma WY, Gao XN, et al (2012) Modified recombinant
proteins can be exported via the Sec pathway in Escherichia coli. PLoS One 7:e42519
2. Bŕigida AIS, Amaral PFF, Coelho MAZ, Gonc, alves LRB (2013) Lipase from yarrowia lipo-
lytica: production, characterization and application as an industrial biocatalyst. J Mol Catal B
Enzym 101:148–158
3. Hasan F, Shah AA, Hameed A (2006) Industrial applications of microbial lipases. Enzyme
Microb Tech 39:235–51
4. Alimardani-Theuil P, Gainvors-Claisse A, Duchiron F (2011) Yeasts: an attractive source
of pectinases-from gene expression to potential applications: a review. Process Biochem
46:1525–37
5. Haki GD, Rakshit SK (2003) Developments in industrially important thermostable enzymes: a
review. Bioresour Technol 89:17–34
6. Mendes AA, Oliveira PC, de Castro HF (2012) Properties and biotechnological applications of
porcine pancreatic lipase. J Mol Catal B Enzym 78:119–34
7. Shimasaki C (2014) Biotechnology entrepreneurship: starting, managing, and leading biotech
companies. Academic Press, Elsevier Inc. Oxford, U.K.
8. Veer T, Jell F (2012) Contributing to markets for technology? A comparison of patent filing
motives of individual inventors, small companies and universities. Technovation 32:513–22
9. O’Cass A, Sok P (2013) Exploring innovation driven value creation in b2b service firms: the
roles of the manager, employees, and customers in value creation. J Bus Res 66:1074–84
Chapter 2
Recombinant Enzyme: Cloning and Expression
Azura Amid and Norhidayah Hassan
Abstract This chapter will discuss the general strategies of cloning recombinant
enzymes from identifying a suitable DNA template to choosing the correct host
expression system. However, there is no standard operating procedure for cloning
a recombinant enzyme. The best strategies should be based on the researcher’s cre-
ativity and experiences.
Keywords Commercialization grant · cDNA · Cloning · Genomic · Host ·

NA-CBZ-L-lysine p-nitrophenyl ester (LNPE) · Polymerase chain reaction · Vector
2.1 Cloning
There are general and basic steps in DNA cloning. First, the desired DNA of inter-
est is ‘cut’ from the source organism using restriction enzymes. Then, the DNA
is ‘pasted’ into a vector and the ends of the DNA are ligated with the vector. The
vector is then introduced into a host cell, which is often a bacterium or yeast and
the cells are grown by a process called fermentation. The host cells will copy the
vector DNA along with their own DNA and create multiple copies of the inserted
DNA. Lastly, the vector DNA is isolated (or separated) from the host cell DNA and
purified. The DNA that has been ‘cut’ and ‘pasted’ from an organism into a vector
is called recombinant DNA, and DNA cloning is also known as recombinant DNA
technology.
There are many cloning methods that can be chosen according to the objective
of the research. cDNA cloning and genomic cloning are used for most research
studies. Table 2.1 lists the advantages and disadvantages of each cloning method.
A. Amid ()
N. Hassan
Kolej University Insaniah, Kuala Ketil, Kedah, Malaysia
e-mail: dayang_2712@yahoo.com
DOI 10.1007/978-3-319-12397-4_2
12 A. Amid and N. Hassan
Table 2.1 The comparison between cDNA cloning and genomic cloning
Cloning strategy Advantages Disadvantages
cDNA cloning • The entire coding region can be • Any regulatory information
obtained in one vector and trans- related to the introns may be
formed into bacteria for expression lost
• Can obtain the exact coding region • A source of mRNA from a tis-
because the intron sequences are sue that expresses the desired
unnecessary produce must be found and
• Proteins of interest can be produced mRNA is difficult to work with
in large quantities, greatly simplify- because it easily degrades
ing the task of protein purification
Genomic cloning • To study a gene in its natural host, • The gene sequences are
the whole gene is needed and it is extremely long and it will have
easily obtained to be pieced together in several
• The intron sequence may contain clones
enhancers that enhance the tran- • If the aim is to obtain a protein
scription of genes in a gene cluster product, the gene cloned
• Can use any DNA source from any contains introns and therefore
tissue. This is because aside from cannot be expressed in bacteria
cells of the immune system, all tis-
sues contain the same DNA
For commercialization purposes, the enzymes produced must be in an active

form so that they can be expressed by the new host. Therefore, cDNA cloning is
more suitable for this objective.
2.1.1 cDNA Cloning
cDNA (short for complementary DNA or copy DNA) is a DNA copy of an RNA,
and the starting material is usually mRNA. The first cDNA was cloned almost two
decades ago (Watson and Demmer, n.d). The construction and screening of cDNA
libraries has become one of the fundamental procedures in molecular biology. cDNA
cloning allows researchers to obtain a sequence of DNA that directs the production
of a specific protein and any procedure that optimizes cloning will be beneficial.
The development of high efficiency cloning systems provided by commercial com-
panies (e.g., Invitrogen, Promega and Clontech) has allowed the construction of a
cDNA library to become a straightforward procedure. The principle behind cDNA
synthesis is that an mRNA population isolated from a specific developmental stage
should contain mRNAs specific for any protein expressed during that stage. Thus,
if the mRNA can be isolated, then the gene can be studied. mRNA cannot be cloned
directly, but a DNA copy of the mRNA can be cloned. This conversion is accom-
plished by the action of reverse transcriptase and DNA polymerase. The reverse
transcriptase makes a single-stranded DNA copy of the mRNA. DNA polymerases
cannot initiate synthesis de novo and depends on the presence of a primer. Many
2 Recombinant Enzyme: Cloning and Expression 13
Fig. 2.1 Simplified cDNA

cloning strategy Tissue /cells
RNA extraction
Isolated RNA
cDNA synthesis
Synthesized cDNA
PCR
amplification
PCR product
DNA fragment
recombinant vector insertion
mRNAs have a poly-A tail at the 3′ end so oligo-dT is frequently used to prime
DNA synthesis. The production of a second strand of DNA involves exposure of the
DNA/RNA hybrid to a combination of RNAase-H and DNA polymerase. RNAase-
H has the ability to cause single-stranded nicks in the RNA, and DNA polymerase
can then use these single-stranded nicks to initiate second strand DNA synthesis.
Finally, the double stranded product is introduced into an appropriate plasmid or
lambda vector. The flow diagram of these mechanisms is shown in Fig. 2.1.
Another strategy for cDNA synthesis employs ribonuclease H, which recognizes
the RNA component of a DNA-RNA hybrid and cleaves the RNA at a number
of non-specific sites to produce short oligoribonucleotides attached to the cDNA.
These fragments serve as primers for the polymerase to synthesize the second strand
cDNA. DNA Polymerase I is used because the 5′-3′ exonuclease activity is needed
to remove RNA from the front of the enzyme. The newly synthesized strands of
cDNA are joined in a vector by ligation using T4 ligase (+ ATP).
A cDNA library is a set of clones representing many mRNAs in a given cell type
at a given time. Such libraries can contain tens of thousands of different clones. In
general, this means that it is representative of all of the mRNAs present in a par-
ticular tissue. The technique to synthesize the cDNA is the same as described previ-
ously. However, probing or screening of the library is required to determine which
plaques contain the gene of interest. A probe is a piece of DNA or RNA that is used
to detect specific nucleic acid sequences by hybridization (binding of two nucleic
acid chains by base pairing). Once a particular DNA fragment is identified, it can be
isolated and amplified to determine its sequence. If the partial sequence of a gene
is known and its entire sequence must be determined, then the probe should contain
the known sequence of the gene of interest. A detailed review on cDNA cloning was
provided by Harbers [1].
In addition to constructing the library to make clones, there are other more direct
approaches. PCR cloning can be used for cloning both the genome and cDNA, and
this approach avoids constructing a library.
There are two strategies commonly used in PCR cloning. The first is to treat
the PCR products and blunt-end the PCR fragments prior to cloning. It is not
easy to clone PCR products by simple blunt-end ligation into blunt-ended plas-
mid vectors. This difficulty arises because some thermostable DNA polymerases,
including Taq DNA Polymerase, add a single nucleotide base extension to the
3′-end of blunt DNA in a template-independent fashion. The most common base
added is adenine, and this produces an ‘A overhang’. However, this strategy of-
ten results in low cloning efficiencies. The second strategy is to add restriction
enzyme recognition sites to the ends of PCR primers. The PCR product is then
digested and cloned into the desired vector. The amplicons generated with Taq
DNA Polymerase typically have A overhangs, and this leads to the method re-
ferred to as T-vector cloning. In essence, the plasmid cloning vector is engineered
to contain 3′-T overhangs that match the 3′-A overhang of the amplicon. The A-
tailed amplicon is directly ligated to the T-tailed plasmid vector; therefore, there
is no need for further enzymatic treatment of the amplicon other than the action
of DNA Ligase.
The inclusion of control reactions is essential for monitoring the success of
PCR reactions. Wherever possible, a positive control should be included to en-
sure the PCR conditions used can successfully amplify the target sequence. PCR
is extremely sensitive and requires only a few copies of target template, so a
negative control containing no template DNA should always be included to en-
sure that the solutions used for PCR have not become contaminated with the
template DNA.
Polymerase Chain Reaction (PCR) has revolutionized cDNA cloning, and in
some circumstances, it is not necessary to construct a library for the isolation of
a particular cDNA clone. If some sequence information is available, then it is pos-
sible to clone the cDNA by PCR. It is worth an attempt because this approach is
much faster and simpler than building and screening a cDNA library. This method
is suitable if the target is one particular cDNA clone containing one DNA copy of
one mRNA and the sequence is accessible. If this method fails to provide the clone,
then a conventional library can be constructed.
There are several factors to consider when devising a cloning strategy and choos-
ing between constructing a cDNA library or cloning by reverse transcription coupled
PCR (RT-PCR). The factors are the abundance of the particular mRNA in the source
organism, the length of mRNA and the screening factor (Watson and Demmer, n.d).
Additionally, the vector used for cloning is also important because it will determine
the nature of the recombinant enzyme produced. To produce a recombinant enzyme,
an expression vector is chosen. There are many expression vectors available in the
market, and each has advantages (Fig. 2.2).
Fig. 2.2 Strategies of PCR cloning; blunt-end and TA cloning (www.invitrogen.com)
Vector Host
Bacteria system e.g. E. coli
Tag protein gene Yeast
Promoterr Insect cell
Mammalia cell
Expression vector
Selection
marker
Plant cell
Fig. 2.3 Outline of the recombinant protein cloning
2.2 Vector for Cloning
The major activity of cloning will involve the selection of the suitable vector and
expression host, as discussed thoroughly by Overton [2]. Figure 2.3 is a modifica-
tion of the figure in the Overton study [2]. The focus of this subsection is to discuss
vectors that are suitable for expression in E.coli hosts because there are too many
vectors available depending on the host type. A researcher will initiate the cloning
experiment using E.coli as a host, so it is worth using a vector that can be expressed
in E. coli. Durani and co-workers discussed the expression vectors in detail and
specifically noted ligation-free, traceless and tag-switching plasmids [3]. Addition-
ally, Rosano and Ceccarelli [4] discussed suitable strategies to choose the correct
vector. There is no specific vector that is given priority in the selection of expres-
sion vectors. However, there are several key factors that should be considered to
ensure active recombinant enzyme production. First, the selected vector must have
high plasmid stability throughout fermentation. This is facilitated by the availabil-
ity of antibiotic resistance genes. Examples of antibiotics that are frequently used
are ampicillin, kanamycin, chloramphenicol and tetracycline. Second, the origin of
replication should ensure the production of a high copy number of plasmid and this
is necessary for large scale protein purification [3]. Next, there should be a clon-
ing region or multiple cloning site in the vector where the insert will be ligated to
the vector. There are five main methods of ligating into vectors, and they include
the following: (1) ligation-based cloning; (2) ligation-independent cloning; (3) re-
combinogenic cloning (Gateway); (4) SLiCE cloning; and (5) TOPO cloning. As
mentioned earlier in this chapter, the insertion method is also unique to the gene
of interest, and there is no standard operating procedure for ligating the gene into
the vector. This step always depends on the researcher’s creativity as discussed by
Hartley [5].
Other factors that need to be addressed are the transcription promoters and fusion
tags. According to Durani [3], there are 3 main factors to consider when choosing
a good promoter, and these factors include the following: (1) the promoter must be
strong and able to express the recombinant protein in the cell; (2) the promoter must
be able to express the product without adverse effects on the growth of the host cell;
an (3) the promoter should be easily inducible either by a chemical or other method
such as thermal induction. Recently [5, 6], auto-induction media was introduced.
This system requires little user intervention during fermentation from the inocu-
lation stage until cell harvesting. Blommel [6] claimed that this strategy is based
on E. coli metabolic control, which automatically performs a shift from growth to
recombinant protein expression and omits biomass monitoring for the correct time
of inducer addition. These advantages are ideal for the large scale production of
recombinant enzymes.
2.3 Host for the Recombinant Enzyme
Rosano and Ceccarelli [4] mentioned that before starting with other expression
hosts, it is important to first try an E. coli strain because it offers dozen of candi-
date strains that might be suitable for your enzyme expression. Bacterial expression
systems for heterologous protein production are attractive because of their ability
to grow rapidly and at a high density, low cost and high productivity. E. coli fa-
cilitates protein expression because it is simple and inexpensive and a well-known
genetic system. Bacteria can also be utilized with a large number of biotechnology
tools. However, there are few major drawbacks encountered during the expression
of heterologous proteins in E. coli. These problems include the formation of inclu-
sion bodies, incorrect protein folding and degradation of the heterologous protein,
especially when the heterologous enzyme is originally isolated from prokaryotes.
Other expression systems, such as yeast, insect and mammalian systems, might be
suitable if a researcher experiences any of these problems.
Table 2.2 Advantages and disadvantages of bacteria, yeast and mammalian host cell systems in
cloning recombinant proteins
System Type/strain Advantages Disadvantages References
Bacteria E. coli • Fast growing • Limited post- [11, 12]
Bacillus subtilis • Inexpensive to culture translational
Streptomyces spp. • Easily scalable modification
• Robust cellular • Limited protein
structure size and complexity
• Usually require
protein extraction
and refolding
• Traces of endo-
toxin in gram-
negative species
Yeast 7 strains • Post-translational • Glycosylation pat- [11, 13]
Saccharomyces modification tern dissimilar to
cerevisiae, • Secretion humans
Pichia pastoris, • Fast growing • Complex scaling
Hansenula poly- • Inexpensive to culture up fermentation
morpha (Pichia • Robust cellular
angusta), structure
Arxula • Synthesis of complex
adeninivorans, and multi subunit
Kluyveromyces proteins
lactis,
Yarrowia lipolytica,
Schizosaccharomy-
ces pombe
Mamma- Chinese ovary • Innate secretion • Slow growing [11]
lian hamster (CHO) mechanism • Requires expensive
Murine cell lines • Synthesis of complex, and complex media
Monkey cell lines multisubunit proteins • Sensitive to
Human cell lines • Greatest post-trans- osmotic shock and
lational modification shear stress
capabilities • Can harbor human
pathogens
As of January 2009, there were 151 protein-based recombinant pharmaceuti-

cals licensed by the US Food and Drug Administration and European Medicines
Agency. Of the protein products, 30 % were from E. coli, 19 % were from yeast,
39 % were from mammalian cell lines and 11 % were from hybridoma cells [7].
Table 2.2 describes the advantages and disadvantages of each system. The final
cloning strategy is still based on researcher creativity and experiences. Overton [2]
suggested that although bacterial systems have many disadvantages, advances in
bacterial post-translational modifications [8, 9] and protein release systems (such as
periplasmic release and secretion systems) [10] will enable further improvements to
be made by simplifying protein production processes and enabling even more rapid
syntheses of target proteins.
References
1. Harbers M (2008) The current status of cDNA cloning. Genomics 91:232–242

2. Overton TW (2014) Recombinant protein production in bacterial hosts. Drug Discov Today
19:590–601
3. Durani V, Sullivan BJ, Magliery TJ (2012) Simplifying protein expression with ligation-free,
traceless and tag-switching plasmids. Protein Expres Purif 85:9–17
4. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: ad-
vances and challenges. Front Microbiol 5:172
5. Hartley JL (2006) Cloning technologies for protein expression and purification. Curr Opin
Biotechnol 17:359–366
6. Blommel PG, Becker KJ, Duvnjak P, Fox BG (2007) Enhanced bacterial protein expression
during auto-induction obtained by alteration of lac repressor dosage and medium composi-
tion. Biotechnol Progr 23:585–598
7. Ferrer-Miralles N, Domingo-Espin J, Corchero JL, Vazquez E, Villaverde A (2008) Micro-
bial factories for recombinant pharmaceuticals. Microb Cell Fact 8:17
8. Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia
coli. Nat Biotechnol 22(11):1399–1408
9. Vera A, Gonzalez-Montalban N, Aris A, Villaverde A (2007) The conformational quality of
insoluble recombinant proteins is enhanced at low growth temperatures. Biotechnol Bioeng
96:1101–1106
10. French C, KeshavarzMoore E, Ward JM (1996) Development of a simple method for the
recovery of recombinant proteins from the Escherichia coli periplasm. Enzyme Micro Tech
19:332–338
11. Mohamed VP (2014) Development of effective CHO-K1 host system targeting at nutrient-
regulated Insulin-like Growth Factor 1 (IGF-1) pathway. International Islamic University
Malaysia, Kuala Lumpur
12. Nicholl DST (2009) An Introduction to genetic engineering. International Student Edition.
Cambridge University Press, Singapore
13. Celik E, Calik P (2012) Production of recombinant proteins by yeast cells. Biotechnol Adv
30:1108–1118
Chapter 3
Common Laboratory Procedure
Azura Amid, Mohd Jamil Aizat Jamaluddin and Muhd Ezza Faize Othman
Abstract This chapter presents all common laboratory procedures or protocols that
researchers normally perform during studies of recombinant enzyme production.
The principle and theory behind each experiment is presented to help the reader
understand each step that is conducted in an experiment so the reader will be able to
select and modify the procedures according to the type of enzyme they are working
with. Examples of procedures and the results obtained during recombinant brome-
lain production are used to develop readers’ understanding.
Keywords Casein · Colorimetric protein assay · Protein assay · Turbidity
3.1 Introduction
There are many experiments involved with recombinant enzyme research. How-
ever, when dealing with the production of recombinant enzymes, there are a few
experiments that are typically repeatedly performed throughout the pre-commer-
cialization stage. Among the popular analyses that are commonly conducted are
bacterial growth analysis, total protein content, sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE) and enzyme activity assays. The listed as-
says are important because they will help researchers to calculate the productivity
of recombinant enzyme fermentation.
A. Amid ()
M. J. A. Jamaluddin · M. E. F. Othman
Department of Biotechnology Engineering, Faculty of Engineering, International Islamic
University Malaysia, P.O. Box 10, 50728 Kuala Lumpur, Malaysia
e-mail: jameeljamaluddin@hotmail.com
M. E. F. Othman
e-mail: white_tasbih@yahoo.com
DOI 10.1007/978-3-319-12397-4_3
20 A. Amid et al.
Fig. 3.1 Schematic diagrams on bacterial growth observation by turbidity. (From chemwiki.ucda-
vis.edu)
3.2 Estimating Bacterial Growth by Turbidity Measurement
Turbidity measurement is used for this type of research because it provides fast re-
sults. As bacteria multiply in a liquid medium, the medium becomes turbid. There-
fore, the more turbid the medium becomes, the greater the number of bacteria that
are present. The instrument used in this protocol is a spectrophotometer.
3.2.1 Principle
According to Tortora et al. [1], a beam of light is transmitted through a collimator

(a lens), a monochromator (to select the suitable wavelength) and finally a bacte-
rial suspension to a light-sensitive detector (Fig. 3.1). Once the number of bacteria
increases, less light will reach the detector, and the instrument will indicate the
absorbance or optical density (OD).
3.2.2 Objective of Experiment
This experiment aims to quantitate the amount of bacteria in the culture and plot a
graph of OD600 versus time of fermentation.
3.2.3 Materials and Methods
Consumable item
Disposable pipette tips (200 µL and 1 mL)
Cuvette (15 mL)
Equipment
No Equipment Usage
1 Pipettes and dispenser (200 µL) To add any solution into cuvette
2 Spectrophotometer (A600 nm) To read absorbance at 600 nm
3 Common Laboratory Procedure 21
Chemicals and Reagents

No Chemicals Manufacturer
1 Bacteria culture NA
2 Luria broth Sigma-Aldrich, St. Louis, USA
Methodology
1. Add 1 ml of sterile Luria broth into a cuvette and read the OD600. Set this as a
blank for other samples.
2. Take 1 ml of sample for every hour and add into a cuvette and read the OD600.
3. Plot a graph of OD600 against time.
3.2.4 Results and Discussion
Table 3.1 shows the raw data collected during our observation of the absorbance
reading of a bacterial culture. The raw data gathered in Table 3.1 is then processed
and translated into a graph. The graph (Fig. 3.2) shows that the bacterial growth is
at the lag phase from the inoculation time until 4 h, and then, the culture entered the
log phase until 11 h of fermentation. The bacterial growth reached a maximum level
(OD600 = 6.128) before the culture entered its death phase.
3.3 Total Protein Assay
3.3.1 Principle
The BRADFORD protein assay was first reported by Bradford [2] in 1976. It is a col-
orimetric protein assay that is based on the binding between the hydrophobic pockets
of the protein sample at the non-polar region of the dye through van der Waals forces.
Table 3.1 Raw data presenting absorbance of bacteria culture at OD600 during 12 h of fermentation
Hour OD600 Average Std. Deviation
R1 R2 R3
1 0.037 0.04 0.031 0.036 0.003
2 0.048 0.044 0.042 0.044 0.002
3 0.097 0.091 0.093 0.093 0.002
4 0.0286 0.32 0.331 0.226 0.140
5 1.272 1.099 1.29 1.220 0.086
6 1.71 2.476 2.058 2.081 0.313
7 2.606 3.08 2.818 2.834 0.193
8 3.614 3.806 3.808 3.742 0.090
9 4.594 5.221 4.933 4.916 0.256
10 5.364 5.31 6.32 5.664 0.463
11 6.812 6.741 5.102 6.218 0.789
12 5.151 6.649 4.908 5.569 0.769
22 A. Amid et al.
Fig. 3.2 Processed data of bacteria culture absorbance versus the fermentation time
The binding stabilizes the blue form of the Coomassie dye. Therefore, the amount of
the complex present in solution is a measure of the protein concentration and is esti-
mated by the absorbance reading. The absorbance spectrum for the dye is at 595 nm.
This experiment aims to measure the amount of total protein produced by bacterial
fermentation.
3.3.3 Materials and Method
Consumable items
Cuvette
Microcentrifuge tube (2 mL)
Equipment
No Equipment Usage
1 Pipettes and dispenser (20 & 200 µL) To add any solution into tube
Chemical and reagents

No Chemical Manufacturer
1 Bradford reagent Bio Basic, Canada
2 Bovine Serum Albumin (BSA) Merck, Germany
Fig. 3.3 Standard curve develop and use to calculated total protein content
Methodology
1. Prepare 1 mg/mL BSA as a stock.
2. Dilute this BSA to five different concentrations from 0–100 µg/mL in a 2 mL
final volume.
3. Add 800 µL of diluted BSA into a microcentrifuge tube and then add 200 µL of
Bradford reagent into the same tube.
4. Gently mix the solution by inverting few times and then leave at room tempera-
ture for 15 min.
5. Measure the absorbance at 595 nm using a spectrophotometer.
6. Use sample buffer as a blank.
7. Record the absorbance reading, and plot a graph of the concentration of BSA
versus absorbance.
8. Repeat step 3, but this time replace BSA with the protein sample.
9. Calculate the amount of total protein using the calibration curve drawn based on
the BSA standard (Fig. 3.3).
3.3.4 Results (Table 3.2)
To calculate the total protein, use the below equation, which is deduced from the
equation on the graph:
y + 0.1704
x=
0.1342
If the absorbance for a protein sample is 0.456, the total protein is
0.456 + 0.1704
x=
0.1342
x = 4.66 µg
24 A. Amid et al.
Table 3.2 Raw data obtained for total protein content using BSA as standard
Protein content (µg) A595
1 2 Average
0 0 0 0
10 0.098 0.105 0.102
20 0.203 0.174 0.189
40 0.351 0.338 0.345
60 0.491 0.490 0.491
80 0.638 0.669 0.654
100 0.783 0.784 0.784
3.4 Enzyme Activity Assays
In principle, an enzyme activity assay is used to determine the quality of the process
in each unit operation during pre-commercialization of a recombinant enzyme. For
example, the fermentation process is not considered worthwhile if it produces high
biomass, but the expressed protein is not in an active form or the spray-drying pro-
cess finally decreases the enzyme activity more than 30 %. Enzyme activity assays
essentially involve allowing the reaction of an enzyme on a selected substrate in a
suitable reaction buffer at a certain temperature and for a certain incubation period.
The enzyme will catalyze a specific substrate [3], and thus different enzymes will
require different substrates for the enzyme activity assay to be performed. Table 3.3
below shows examples of common enzyme assays based on the enzyme tested. The
enzyme activity assay procedure in this chapter is based on bromelain, a protease.
Therefore, the substrate chosen will be specific for a protease, such as Na-CBZ-
L-lysine p-nitrophenyl ester (LNPE) (synthetic substrate) and casein (natural sub-
strate) (Table 3.3).
Table 3.3 Common substrates and brief methods for enzyme activity measurement using the
spectrophotometric approach
Enzyme Substrate Temperature Incubation pH Wavelength
(°C) time (min) (nm)
Bromelain Na-CBZ-L-Lysine p-Nitro- 25 5 4.6 340
(protease) phenyl Ester (LNPE)
Protease Casein 37 10 7.5 660
Lipase pNP-palmitate 40 5 8.0 410
Cellulase Glucose (HK) 37 120 5 340
Xylanase RBB-Xylan 40 10 6 590
α-Amylase Soluble starch 20 3 6.9 540
Maltase Maltose 25 30 6.0 340
Pepsin Hemoglobin 37 10 2 280
Na-CBZ-L-Lysine p-Nitrophenyl Ester + Bromelain> p-Nitrophenol + Na-CBZ-L-Lysine
Fig. 3.4 Chemical reaction of LNPE with bromelain
3.4.1 Enzyme Activity Assay Using LNPE as Substrate
3.4.1.1 Principle
This procedure follows Sigma’s continuous spectrophotometric rate determination

protocol (Sigma-Aldrich, Germany). In this assay, Na-CBZ-L-Lysine p-Nitrophenyl
Ester (LNPE) acts as a substrate in the bromelain (protease) reaction. When brome-
lain digests LNPE, the p-nitrophenol and Nα-CBZ-L-Lysine are released from the
reaction (Fig. 3.4). p-Nitrophenol has a color that is quantifiable and measured as
an absorbance value using a spectrophotometer at 340 nm absorbance. The greater
the amount of p-Nitrophenol that is released from LNPE, the more color generated,
and the stronger the bromelain activity is. The absorbance values per minute gener-
ated by the blank will be subtracted from the absorbance values per minute of the
bromelain or sample activity. One unit activity of bromelain represents one unit of
enzyme releasing 1.0 µmole of p-nitrophenol from Nα-CBZ-L-lysine p-nitrophenol
ester per minute at pH 4.6 at 25 °C.
3.4.1.2 Objective of Experiment
The objective of this experiment is to measure bromelain enzyme activity.
3.4.1.3 Materials and methods
Consumable item
Falcon tube (15 mL)
Equipment
No Equipment Usage
1 Pipettes and dispenser (200 µL) To add any solution into tube
2 Dispenser (2.6 and 2.7 mL) To add any solution into e tube
4 pH meter To read pH
26 A. Amid et al.
Chemicals and reagents

1 Sodium acetate, trihydrate Sigma-Aldrich, St. Louis, USA
2 Potassium chloride Sigma-Aldrich, St. Louis, USA
3 L-Cysteine, hydrochloride, monohydrate Sigma-Aldrich, St. Louis, USA
4 LNPE Sigma-Aldrich, St. Louis, USA
5 Acetonitrile Sigma-Aldrich, St. Louis, USA
6 1 M HCl Sigma-Aldrich, St. Louis, USA
7 Commercial bromelain (3–7 units/mg) Sigma-Aldrich, St. Louis, USA
Methodology
Reagent preparation
1. 100 mL of Reagent A (30 mM Sodium Acetate Buffer with 100 mM Potassium
Chloride and 1.0 mM L-Cysteine), pH 4.6, at 25 °C.
a. Add 40.8 mg of sodium acetate trihydrate into 50 mL of deionized dH2O.
b. Add 17.6 mg of L-cysteine hydrochloride monohydrate into the solution.
c. Add 745.5 mg of potassium chloride into the solution.
d. Adjust pH 4.6 with 1 M HCl at 25 °C.
e. Bring volume to 100 mL with deionized dH2O.
2. 1 mL Reagent B (50 mM Nα-CBZ-L-Lysine p-Nitrophenyl Ester (LNPE)),
Freshly Prepare
a. Add 21.9 mg of LNPE into 0.2 mL of acetonitrile.
b. Bring volume to 1.0 mL with deionized dH2O.
Bromelain Enzyme Solution
1. Add 100 mg of commercial bromelain (3–7 units/mg) into 1666.7 mL of Reagent
A to obtain 0.2–0.4 units/mL bromelain solution.
Important notes
Allow sample solution to incubate on ice for approximately 2 h.
The LNPE solution should be used as soon as possible as it will hydrolyze while
resting.
Flow of experiment
1. Sample, blank and positive control
3.4.1.4 Results and Discussions
Examples of readings obtained after 5 min follow:

28 A. Amid et al.
Calculation of Enzyme Activity

Units
enzyme
ml
 ∆340nm ∆340nm 
 min − ( 2.8)(dilution factor )
sample min blank 
=
(6.32 milimolar extinction coefficient of p − nitrophenol at 340nm )
(0.1 ml of sample used )
Example of actual case
Blank (Table 3.4)
Table 3.4 Raw data obtained for enzyme activity of blank

Reaction Absorbance at 340 nm
Time (min) 1 2 3 Average Std Dev
0 0.1076 0.9355 1.0387 0.6939 0.5104
1 0.1099 0.9492 1.0518 0.7036 0.5167
2 0.1130 0.9637 1.0658 0.7142 0.5231
3 0.1161 0.9785 1.0795 0.7247 0.5295
4 0.1194 0.9924 1.0955 0.7358 0.5363
5 0.1226 1.0072 1.1088 0.7462 0.5424
Table 3.5 Raw data obtain for enzyme activity of samples

Reaction Absorbance at 340 nm
Time (min) 1 2 3 Average Std Dev
0 1.192 1.217 1.197 1.202 0.013
1 1.222 1.243 1.223 1.229 0.012
2 1.247 1.267 1.245 1.253 0.012
3 1.268 1.290 1.265 1.274 0.014
4 1.288 1.309 1.286 1.294 0.013
5 1.308 1.328 1.303 1.313 0.013
Sample (Table 3.5)
Calculations
Units
⇒ enzyme
ml
 ∆340nm ∆340nm 
 min − ( 2.8)(dilution factor )
sample min blank 
=
=
(0.0221
sample )
− 0.0064blank ( 2.8) (1 dilution factor )
= 0.0696
⇒ σ 2 ( Z ) = σ 2 ( A) + σ 2 ( B )
( )
σ 0.0221sample − 0.0064blank = 0.0007447 2 + 0.000093432 = 0.00075
∴ net accumulative error.
30 A. Amid et al.
In summary, the bromelain activity should be reported as

U
⇒ 0.0696 ± 0.00075
ml
3.4.1.5 Conclusion
The enzyme activity of the recombinant bromelain is 0.0696 ± 0.0075 U/ml.
3.4.2 Enzyme Activity Assay Using Casein as Substrate
3.4.2.1 Principle
This procedure follows Sigma’s non-specific protease activity (Sigma-Aldrich,

Germany). In this assay, casein acts as a substrate for the reaction. When protease
digests casein, the amino acid tyrosine is liberated along with other amino acids and
peptide fragments. Folin and Ciocalteu’s Phenol, or Folin’s reagent, primarily reacts
with free tyrosine to produce a blue-colored chromophore, which is quantifiable and
measured via an absorbance value on a spectrophotometer at 660 nm absorbance.
A greater level of tyrosine released from casein indicates the generation of more
chromophores and stronger protease activity. The absorbance values generated by
the activity of the protease are compared to a standard curve, which is generated by
reacting known quantities of tyrosine with the F-C reagent to correlate changes in
absorbance with the amount of tyrosine in micromoles. Using the standard curve,
the activity of protease samples can be determined in terms of units, which is the
amount in micromoles of tyrosine equivalents released from casein per minute.
3.4.2.2 Objective of Experiment
To measure recombinant bromelain enzyme activity
3.4.2.3 Materials and methods
Consumable item
Microcentrifuge tube (1.5 mL)
Microcentrifuge tube (2.5 mL)
0.4 µm Millipore membrane filter
Equipment
No Equipment Usage
1 Pipette (200 and 1000 µL) To add solution into the microcentrifuge tubes
2 Centrifuge To separate supernatant from precipitate
4 Water bath To regulate the temperature of anything subjected
to heat to obtain the desired temperature
Chemicals and reagents

1 Phosphate dibasic, trihydrate Sigma-Aldrich, St. Louis, USA
2 Casein Sigma-Aldrich, St. Louis, USA
3 Sodium hydroxide Sigma-Aldrich, St. Louis, USA
4 Hydrochloric acid Sigma-Aldrich, St. Louis, USA
5 Trichloroacetic acid Sigma-Aldrich, St. Louis, USA
6 Folin’s Phenol reagent Sigma-Aldrich, St. Louis, USA
7 Anhydrous sodium carbonate Sigma-Aldrich, St. Louis, USA
8 Sodium acetate Sigma-Aldrich, St. Louis, USA
9 Calcium acetate Sigma-Aldrich, St. Louis, USA
11 L-tyrosine Sigma-Aldrich, St. Louis, USA
12 Commercial bromelain Sigma-Aldrich, St. Louis, USA
3.4.2.4 Methodology
Reagent preparation
1. 50 mM Potassium phosphate buffer, pH 7.5 at 37 °C.
a. Add 1140 mg of potassium phosphate dibasic trihydrate into 90 mL of puri-
fied dH2O.
b. Incubate at 37 °C and adjust pH to 7.5 using 1 N NaOH or 1 N HCl.
c. Bring volume to 100 mL with purified dH2O.
2. 0.65 % (w/v) casein solution.
a. Add 65 mg of casein into 9 mL of 50 mM potassium phosphate buffer.
b. Gradually increase the solution temperature with gentle stirring to 80–85 °C
for approximately 10 min until a homogenous dispersion is achieved ( IT IS
VERY IMPORTANT NOT TO BOIL THE SOLUTION).
c. Adjust pH to 7.5 using 1 N NaOH or 1 N HCl.
d. Bring volume to 10 mL with 50 mM potassium phosphate buffer.
3. 110 mM trichloroacetic acid solution
a. Dilute 6.1 N trichloroacetic acid stock with dH2O by adding 1 mL of 6.1 N
trichloroacetic acid stock into 55 mL of dH2O ( TRICHLOROACETIC ACID
IS A STRONG ACID AND SHOULD BE HANDLED WITH CARE).
32 A. Amid et al.
4. 0.5 mM Folin and Ciocalteu’s or Folin’s Phenol Reagent

a. The solution will react with tyrosine to generate a measurable color change
that will is directly related to the activity of proteases.
Folin’s Phenol reagent is an acid and should be handled with care.
5. 500 mM sodium carbonate solution
a. Add 530 mg of anhydrous sodium carbonate to 10 mL of purified water.
6. 10 mM sodium acetate buffer with 5 mM calcium acetate, pH 7.5, at 37 °C
(enzyme diluents solution).
a. This solution is conveniently prepared in a 20X stock solution. To prepare
in 1 liter, add 27.22 g of sodium acetate trihydrate and 15.82 g of calcium
acetate hydrate to 700 ml of distilled water. Let the mixture dissolve with stir-
ring or mild heating. Once completely dissolved, equilibrate the solution first
at 37 °C and then adjust the pH to 7.5. Bring the solution volume to 1 l. For
working solution, simply dilute to 1X concentration.
This solution is to dissolve solid commercial bromelain or to dilute enzyme
solution.
Let the solution be ice chilled before use.
7. Commercial bromelain solution (as a positive control)
a. Immediately before use, dissolve commercial bromelain to 0.1–0.2 unit/ml in
enzyme diluent solution.
b. Commercial bromelain solution serves as a positive control for the quality
control assay and as validation for the calculation that we have to perform to
determine enzyme activity.
8. 1.1 mM L-tyrosine standard stock solution
a. Add 0.2 mg of L-tyrosine into 1 mL of purified water in a tube.
b. Heat the tube gently in a water bath until it dissolves ( DO NOT BOIL THIS
SOLUTION)
c. Allow it to cool to room temperature.
d. This solution will be diluted further to generate the standard curve.
Flow of experiment for Standard Curve

34 A. Amid et al.
1. Sample, blank and positive control (Table 3.6)

Table 3.6 Example of data obtain to generate standard curve

Tube A in B in C in D in E in F in
triplicate triplicate triplicate triplicate triplicate triplicate
Concentration ( µmole) Blank 0.014 0.028 0.055 0.110 0.140
L-tyrosine ( µL) 0 0.013 0.025 0.050 0.100 0.125
dH20 ( µL) 0.500 0.487 0.475 0.450 0.400 0.375
Table 3.7 Raw data for enzyme activity from sample

Run Test sample replicates Average σ Blank replicates reading Average σ
reading
1 0.3180 0.3117 0.2845 0.3047 ± 0.0178 0.0224 0.0211 0.0247 0.0227 ± 0.0018
Calculation of enzyme activity

1. ∆A660 nm = Average ∆A660 nm (sample)—Average ∆A660 nm (blank).
2. Determine the µmole tyrosine equivalent released using the standard curve linear
equation.
Activity unit = (µmole tyrosine equivalent released)(2.2 ml total volume)
ml ( 0.2 ml enzyme solution ) (0.5 ml colorimetric volume)(10 min)
3. For determination of solid protease I enzyme diluent, divide the activity of
enzyme in units/mL by the concentration of solid used in this assay originally in
mg/mL.
4. Therefore, the activity of protease is
Units
enzyme
units mL
= .
mg solid
mg enzyme
mL
3.4.2.5 Results and Discussions (Table 3.7)
Net average absorbance, 660 nm = 0.3047 − 0.0227 = 0.2820

⇒ σ 2 ( Z ) = σ 2 ( A) + σ 2 ( B )
σ ( 0.3047 − 0.0227 ) = 0.01782 + 0.00182 = 0.0179
∴ First net accumulative error.
From the bromelain assay’s standard curve, using a spreadsheet, the following lin-
ear equation is obtained:
y = 5.071x
36 A. Amid et al.
The estimated errors for both the slope and y-intercept are as highlighted below in
the table.
Best-fit values
Slope 5.071 ± 0.04975
Y-intercept when X = 0.0 0.000 ± 0.00000
Goodness of Fit
r² 0.9995
From the linear equation
0.2820 = 5.071x
0.2820
∴= = 0.0556 ( µ mole tyrosine equivalent released )
5.071
 σ (Z )  σ ( A)   σ ( B ) 
2 2 2
⇒  = +
 Z   A   B 
2
  0.2820  
 σ  5.071    σ ( 0.2820)   σ (5.071) 
2 2
  = +
 0.0556   0.2820   5.071 
 
 σ ( 0.2820)   σ (5.071) 
2 2
 0.2820 
σ = 0.0556 ×  +
 5.071   0.2820   5.071 
2 2
 0.0179   0.0498 
= 0.0556 ×  +
 0.2820   5.071 
= 0.0036 ∴ Second net accumulative error.
Thus, from here, bromelain activity can be determined using the following:
 U  ( µ mole tyrosine equivalent released )( 2.2 ml )

⇒ Bromelain activity   =
 ml  (10 min)(0.2 ml )(0.5 ml )
= ( µ mole tyrosine equivalent released ) (2.2)
= 0.0556(2.2)
= 0.1223 U ml
In summary, the bromelain activity should be reported as

U
⇒ 0.1223 ± 0.0036
ml
3.4.2.6 Conclusion
The enzyme activity of the recombinant bromelain is 0.1223 ± 0.0036U / ml.
3.5 Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis (SDS-PAGE)
3.5.1 Principle
SDS-PAGE is a technique to separate proteins using electrophoresis. SDS is an

anionic detergent that is able to destroy most of the complex structure of proteins
and is strongly attracted toward an anode, whereas the polyacrylamide gel is used
as a support medium and restrains larger protein molecules from migrating as fast
as smaller protein molecules. Because the charge-to-mass ratio is nearly the same
among the SDS-denatured polypeptides, the separation of protein is dependent ex-
actly on the differences in the relative molecular mass of the polypeptides.
This experiment aims to separate proteins according to their molecular weight and
is normally used by researchers to obtain a qualitative measurement of the final
product during recombinant enzyme production.
3.5.3 Materials and Methods
Consumable items
Disposable pipette tips (20, 200 and 1000 µL)
Microcentrifuge tubes
Gloves
Equipment
No Equipment Usage
1 Pipettes and dispenser (20, 200 and To add any solution into microcentri-
1000 µL fuge tubes and gel’s wells
2 SDS-PAGE apparatus or electrophoresis set To run the protein sample
3 Water bath To boil the protein sample
4 Rocking platform For use during gel staining
5 Gel imager To document the gel picture
38 A. Amid et al.
Chemicals and Reagents
1 Tris Sigma-Aldrich, St. Louis, USA/Merck, Germany
2 HCl Sigma-Aldrich, St. Louis, USA/Merck, Germany
3 Acrylamide Sigma-Aldrich, St. Louis, USA/Merck, Germany
4 Bis-acrylamide Sigma-Aldrich, St. Louis, USA/Merck, Germany
5 Ammonium persulphate Sigma-Aldrich, St. Louis, USA/Merck, Germany
6 Glycine Sigma-Aldrich, St. Louis, USA/Merck, Germany
7 SDS Sigma-Aldrich, St. Louis, USA/Merck, Germany
8 Coomassie Brilliant Blue Sigma-Aldrich, St. Louis, USA/Merck, Germany
9 Isopropanol Sigma-Aldrich, St. Louis, USA/Merck, Germany
10 Acetic acid Sigma-Aldrich, St. Louis, USA/Merck, Germany
11 TEMED Sigma-Aldrich, St. Louis, USA/Merck, Germany
Methodology
1. Set up the SDS-PAGE apparatus according to manufacturer’s instructions (each
manufacturer has a different apparatus set up).
2. Prepare the 15 % separating gel as shown in the table below
Components 15 % Separating Gel (mL)

30 % Acrylamide, 0.8 % Bis-acrylamide 10
1.875 M Tris-HCl Buffer (pH 8.8) 4
10 % SDS 0.2
Ammonium persulphate 0.1
TEMED 0.0187
Distilled water 5.7
Total 20
3. Transfer the gel mixture into the gel cassette by using a Pasteur pipette. Allow
solidification for 15–20 min.
4. Prepare the 4 % stacking gel as shown in the table below.
Components 4 % Stacking gel (mL)

30 % Acrylamide, 0.8 % Bis-acrylamide 1.35
0.6 M Tris-HCl Buffer (pH 6.8) 1
10 % SDS 0.1
Ammonium Persulfate 0.05
TEMED 0.015
Distilled water 7.5
Total 10
Fig. 3.5 The SDS page

results for protein purifica-
tion under native conditions.
The first lane (M) is the
protein marker (10–150 kDa)
followed by a protein sample
from different purification
steps. Lanes 1 and 2 contain
crude recombinant brome-
lain. Lanes 3 and 4 contain
samples from different wash-
ing steps, and lanes 5 and 6
contain eluted recombinant
bromelain
5. Load the gel mixture on the top of the separating gel, insert a 0.75 mm comb
between the two glass plates and allow to solidify for 20 min.
6. Once solidified, fill in the top reservoir and bottom tank with the electropho-
resis buffer.
7. Mix the protein sample with 5 X sample buffer at a ratio 4:1, and denature the
sample by heating to 90–100 °C for 5 min.
8. Load the denatured sample using a syringe needle just above the bottom of the
well.
9. Use a constant current of 30 mA to run the electrophoresis until the blue marker
reaches the bottom of the gel.
10. Stain the gel with Coomassie blue for 40–60 min. Finally, destain the gel and
document the picture using the gel documentation system.
3.5.4 Results
Figure 3.5 shows the protein bands in different lanes from different samples after
fermentation in a bioreactor and purification using affinity chromatography.
References
1. T
ortora GJ, Funke BR, Case CL (2001) Microbiology: an introduction, 7th edn. Benjamin
Cummings, San Franscisco
2. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of
protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254
3. Karp G (2002) Cell and molecular biology: concepts and experiments, 3rd edn. Wiley, New
York
Chapter 4
Characterization of Recombinant Enzymes
Farah Fadwa Ben Belgasem and Hamzah Mohd. Salleh
Abstract Commercial enzymes can be obtained from natural sources or they can
be produced recombinantly. Once the recombinant enzyme is obtained in purified
form, the identity should be confirmed by various approaches including N-terminal
protein sequencing. Having established the identity, the biochemical and biophysi-
cal characterization of recombinant enzyme is essential for industrial and clinical
applications as well as for research purpose. A number of methods are normally
employed to determine the biophysical properties of enzymes and understand the
biochemical characteristics as well as examine the biological functions and integ-
rity. Recombinant enzyme data are compared with data from enzyme from the natu-
ral source to establish that the recombinant enzyme possesses the desired features
of the naturally occurring counterpart; these include among others the establishment
of the optimum pH and temperature for the enzyme, stability to pH, temperature
and storage, substrate specificity and enzyme kinetics, effects of small molecules
and ions on enzyme activity. A good enzyme characterization is to have the struc-
ture resolved for better understanding of the enzyme function and mechanism of
catalysis.
Keywords Basic local alignment search tool · Catalytic activity · Chromatography

separation · Enzyme active site · Gel-electrophoresis · Halal · Initial velocity ·
Luminescence · Macromolecular modeling database · Michaelis constant · pH
· Post-translational modification · Reaction velocity · Substrate specificity ·
Ultraviolet · -Ray structures
H. M. Salleh ()
Faculty of Engineering, International Institute for Halal Research and Training (INHART),
International Islamic University Malaysia, Ground Floor, Block E0, P.O. Box 10, 50728 Kuala
Lumpur, Malaysia
e-mail: hamzah@iium.edu.my
F. F. B. Belgasem
Department of Biotechnology Engineering, Faculty of Engineering,
International Islamic University Malaysia, P.O. Box 10,
DOI 10.1007/978-3-319-12397-4_4
42 F. F. B. Belgasem and H. M. Salleh
4.1 Introduction
Biological reactions in the absence of enzymes may be too slow to support life.
Many reactions in life depend on the efficient biological catalysts, enzymes, that
work under the mildest conditions—close to ambient temperatures and neutral
pH, as well as at atmospheric pressure—and yet enzymes are able to accomplish
enormous rate enhancements as much as 106–1015 times faster than uncatalyzed
reactions and that enzyme-catalyzed reactions are region-, chemo- and stereo-
selective. Understanding the features of enzyme and how enzyme function has
been one of the most important goals for researchers for academic pursuits and
for commercialization, as interests in the application of enzymes in industry have
grown from traditional food industry to various reactions from the viewpoint of
green chemistry in wide-ranging fields from remediation of the environment, re-
utilizing resources, creating new industrial processes to the creation of new func-
tional foods, contribution to diagnostic screening and research, as well as medical
treatments.
Understanding the biochemical properties, structure and mechanisms of enzymes
allow end users to utilize these enzymes effectively as well as for researchers to
design and produce new enzymes with catalytic efficiencies that can rival those of
natural enzymes and/or with the ability to catalyze reactions that are not found natu-
rally. Enzymes can be obtained from a variety of sources including plants, animals,
humans and microorganisms. Nowadays, many enzymes available commercially
are acquired through biotechnological means employing recombinant DNA tech-
nologies. It is essential that properties such as the catalytic activity and stability are
preserved during production, recovery and handling, formulation, storage and when
the enzyme is put in use. The focus of this chapter shall be the biochemical and
physicochemical characterization of recombinant enzymes. A number of techniques
can be used to determine the biophysical properties of enzymes and to examine their
biochemical and biological integrity. The results of these experiments are compared
with those obtained using naturally occurring enzymes in order to be confident
that the recombinant enzymes have the desired characteristics of the naturally oc-
curring one [1]. In addition to verifying the sequences of gene clones, there is also
the need to characterize the enzymes produced and the processes used to produce
them to ensure consistency in their purity and biological behavior. All recombinant
enzymes produced will be subjected to a regimen of tests and screening procedures
to assess their quality and to provide initial insights into their structures and func-
tions. For each recombinant enzyme, the identity and molecular weight, stability to
pH and temperature, influence of ions and effectors on activity, substrate specificity
should be determined. It is customary that a combination of various spectroscopic,
separation, and imaging techniques will be employed. Where sizable recombinant
enzymes are to be produced and characterized, automated systems are helpful for
simultaneously characterize hundreds of enzymes for purity and, in some cases,
function [2].
4 Characterization of Recombinant Enzymes 43
4.2 Biochemical Characterization
4.2.1 Enzyme Active Site Residues and Catalysis
Enzymes are typically much larger than the substrates they act on. The region where
catalysis occurs—the active site—is usually the largest cleft/pocket on the protein
surface and represents a small portion of the overall volume of the enzyme. The
residues essential for catalysis can be ascertained by chemical labeling experiments
using certain reagents. The resulting covalent attachments of the reagent to the en-
zyme can lead to identification of amino acid residues directly participating in the
enzyme mechanism. For example, diisopropylflouorophosphate forms covalent
linkage to the catalytic nucleophilic serine residue in serine proteases. Site directed
mutagenesis approach can be utilized to scan residues for substitution with a non-
ionizable amino acid residue, e.g. Ala, and the effect on the reaction is measured.
This approach can determine amino acid residues essential for catalysis when en-
zyme activity is abolished by the amino acid substitution. Sometimes the substitu-
tions of the essential catalytic residues lead to an alternative and slower reaction
mechanism [HMS #29].
Enzymes are delicate molecules and are sensitive to changes in the environment
where they exhibit their catalytic activity. Factors that are present in the immediate
environment of the enzyme such as heat, pH and sometimes small molecules (other
than the substrate) influence the activity and stability of enzymes.
4.2.2 Effects of Temperature on Enzyme Activity and Stability
All chemical reactions are affected by temperature. The reaction velocity increases
with temperature increases because more molecules have sufficient energy to over-
come the energy barrier (i.e. activation energy) to enter into the transitions state.
The rate of enzyme-catalyzed reactions also increases with increasing temperature.
However, enzymes are proteins that become denatured at high temperatures. Each
enzyme has an optimum temperature at which it operates at maximal efficiency. Be-
cause enzymes are proteins, optimum temperature values depend on pH and ionic
strength Fig. 4.1 [3].
Enzyme activity increases with temperature up to a certain maximum before the
activity drops due to instability/inactivation of the enzyme at higher temperatures.
The effects of heat on enzyme activity are complex but can be viewed as two forces
exerting simultaneously in counter directions. When temperature is raised, the rate
increases, but there will be inactivation/denaturation occurring progressively and
become more pronounced as the temperatures is raised producing what appears
to be a temperature optimum. The optimum temperature needs qualification with
regard to the enzyme exposure to temperature is noted. The dependence of enzyme
activity on temperature has been described by a model consisting of two processes:
Fig. 4.1 Modest increase in temperature increase the rate of enzyme-catalyzed reactions because
of an increase in the number of collisions between enzyme and substrate. Eventually, increasing
the temperature decrease the reaction velocity. Catalytic activity is lost because heat denatures the
enzyme
the catalytic reaction defined by ΔG‡cat, and irreversible inactivation defined by

ΔG‡inact. Another model known as the Equilibrium Model suggests a new mecha-
nism by which enzymes lose activity at high temperatures. In the latter model, an
inactive form of the enzyme (Einact) is present and it is in reversible equilibrium
with the active form (Eact); it is the Einact that is irreversibly thermal inactivated
to the thermally denatured state. This equilibrium is described by an equilibrium
constant whose temperature-dependence is characterized in terms of the enthalpy
of the equilibrium, ΔHeq, and a new thermal parameter, Teq, which is the tempera-
ture at which the concentrations of Eact and Einact are equal; Teq may therefore be
regarded as the thermal equivalent of KM.
Characterization of an enzyme with respect to its temperature dependent behav-
iour must therefore include a determination of these intrinsic properties. Tradition-
ally, the effect of temperature on enzyme activity has been described by two well-
established thermal parameters: the Arrhenius activation energy, which describes
the effect of temperature on the catalytic rate constant, kcat, and thermal stability,
which describes the effect of temperature on the thermal inactivation rate constant,
kinact. Anomalies arising from this description have been resolved by the develop-
ment [4] and validation [5] of a new model (the Equilibrium Model) that more com-
pletely describes the effect of temperature on enzyme activity by including an addi-
tional mechanism by which enzyme activity decreases as the temperature is raised.
In this model, the active form of the enzyme (Eact) is in reversible equilibrium with
an inactive (but not denatured) form (Einact), and it is the inactive form that un-
dergoes irreversible thermal inactivation to the thermally denatured state (DS) [6]:
The Equilibrium Model has major implications for enzymology, biotechnology and
understanding the evolution of enzymes.
E act  E inact → DS
4.2.3 Effects of pH on Enzyme Activity and Stability
Enzymes are also sensitive to environmental changes. Hydrogen ion concentration

affects enzymes in several ways. First, catalytic activity is related to the ionic state
of the active site. Changes in hydrogen ion concentration can affect the ionization
of active site groups. For example, the catalytic activity of a certain enzyme requires
the protonated form of a side chain amino group. If the pH becomes sufficiently
alkaline that the group loses its proton, the enzyme’s activity may be depressed. In
addition, substrates may also be affected. If a substrate contains an ionizable groups
may change the tertiary structure of the enzyme. Drastic changes in the pH often
lead to denaturation.
Although a few enzymes tolerate large changes in pH, most enzymes are active
only within a narrow pH range. For this reason, living organisms employs buffers
to closely regulate pH. The pH value at which an enzyme’s activity is maximal is
called the pH optimum Fig. 4.2 [3, 7].
Buffers used to formulate proteins should not serve as substrates or inhibitors.
They should exhibit little or no change in pH with temperature, show insignificant
penetration through biological membranes, and have maximum buffer capacity at a
pH where the protein exhibits optimal stability. In conformity with the proposition
that “Nature designs the optimum molecules” buffers should mimic the antidena-
turant properties of nature exhibited by osmolytes [8–12] that are independent of
the evolutionary history of the proteins [13, 14]. Such properties may include pref-
erential exclusion from the protein domain [8] and stabilization without changing
the denaturation Gibbs energy (ΔGd). Change in pH may be a result of buffer salt
crystallization [15].
4.3 Biophysical and Structure Characterization
Hundreds of genomes have been successfully sequenced to date, and the data are
publicly available. At the same time, the advances in large-scale expression and
purification of recombinant proteins have paved the way for structural genomics
efforts. Frequently, however, little is known about newly expressed proteins calling
for large-scale protein characterization to better understand their biochemical roles
and to enable structure–function relationship studies [16].
Fig. 4.2 Effect of pH on two enzymes. Each enzyme has a certain pH at which it is most active.
A change in pH can alter the ionizable groups within the active site or affect the enzyme’s
conformation
Very early ideas about how enzymes worked invoked complementarity between
a reaction’s transition state and the binding surface of the enzyme –what we now
refer to as the enzyme’s active site. Remarkably, these ideas predated knowledge
of the atomic and molecular nature of enzymes [17–19]. This complementarity can
now be visualized from the myriad of enzyme x-ray structures, many with bound
substrates, substrate analogs, or transition state analogs [20].
4.4 Characterizing Post-Translational Modifications
It is important to determine the nature and location of all post-translational modifi-

cations of recombinant enzymes. Among the most problematic are unwanted, non-
specific modifications, including oxidation of Met residues and chance deamidation
of Asn or Gln. These modifications can easily be detected as retention time shifts
when HPLC profiles of peptide maps of the recombinant enzymes are compared to
those of the authentic enzyme. The presence of reducing agents, such as dithioth-
reitol in the isolation buffer often prevents oxidation of Met. There are situations in
which one wants to determine if the correct post-translational modification has been
made in the recombinant enzyme, especially if the modification is important for the
activity of the enzyme. Usually, the expression system has been carefully selected
to provide the appropriate modification. Among the most frequent post-translation-
al modifications are phosphorylation, N-methylation, acetylation, glycosylation,
farnesylation, and sialic acid capping of oligosaccharides. Pinpointing these modifi-
cations can be time-consuming, requiring specific methods for each modification if
traditional approaches are to be used. The usual method is to digest the recombinant
protein, separate the fragments by HPLC, then pick fragments suspected of being
modified for further analysis by N-terminal sequencing using Edman degradation
method. A major disadvantage to this approach is that some of the modifications are
not stable when exposed to the chemicals used in sequencing, and will therefore be
missed. For example, phosphorylated Ser and Thr residues are not easily detected
because they undergo β-elimination of the phosphoryl group [21]. Instead, a system
using radioactive tracers has been developed in which the assignment of the phos-
phorylation sites is based on the release of [32P]Pi during Edman degradation [22].
Newer approaches are being developed that rely on mass spectrometry to pinpoint
the location of the modifications [23]. These approaches offer direct procedures for
identifying the modified residues and should cut the time required for analysis [24].
Many proteins cannot be directly sequenced by Edman degradation because they
have a blocked N-terminal residue. A method is presented for deblocking such pro-
teins when the N-terminal residue is N-acetylserine (which occurs frequently in
eukaryotic proteins) or N-acetylthreonine. The method has been applied success-
fully to the determination of the N-terminal amino acid sequence of human, bovine,
and rat parathymosins. Prothymosin alpha and other blocked proteins and peptides
were also readily deblocked and sequenced by this procedure. It is proposed that the
mechanism of the deblocking reaction involves an acid-catalyzed N–O shift of the
acetyl group followed by a beta-elimination [25].
4.5 Computational Methods and Bioinformatics for

Studying the Structure and Function of Proteins
GeneDoc is a software Created by Karl B. Nicholas [26] and currently maintained

by Hugh B. Nicholas, Jr., of the National Resource for Biomedical Supercomput-
ing (NRBSC; associated with the Pittsburgh Supercomputing Center http://www.
psc.edu). A free download is available at: http://www.nrbsc.org/gfx/genedoc/index.
html. This software permits the editing and alignment of the sequences for proteins
and nucleic acids, the visualization of the secondary structures of proteins, and the
shading of alignment and secondary structure.
Alignment of the sequences of amino acids using the Basic Local Alignment
Search Tool (BLAST) of the NCBI permits to compare the sequences of the amino
acids for enzymes. The BLAST programs are most frequently used for searching
an entire database or a subset of a database. In such searches, a Search Set must be
specified (Table 4.1) [27]. It is necessary to determine a criterion for the minimum
percentage of identity, percentage of positives, or alignment score that is indicative
Table 4.1 Selected definitions for use of the NCBI BLAST

Accession number An identifier for an entry in the database consisting of a combination of
numbers and letters
blastp Program for comparing the sequences of amino acids in proteins
Query sequence The single sequence to be compared to another sequence or sequences
Subject sequence The sequence to be compared to a query sequence
Search set An entire database of sequences of amino acids, or a subset of such a
database
of common ancestry. First, there are only 20 types of amino acids found in proteins,
so some similarity in sequence will occur by chance. Second, a stretch of 50 % iden-
tities is much more significant over 100 amino acids than over 20. There is no magic
number for the percentage of identity, the percentage of positives, or the align-
ment score necessary to conclude that the sequences being compared demonstrate
a common ancestry. The best way to get a feeling for proper data evaluation is to
run alignments on some pairs of sequences that are very unlikely to have common
ancestry and perform different functions [12].
The Macromolecular Modeling Database of the NCBI includes cartoons of the
tertiary structures of the folded polypeptides in the crystallographic molecular mod-
els of enzymes as well as proteins not classified as enzymes. Initially, the arrange-
ment of these cartoons in the database on this Web site may appear a bit ambiguous.
From the menu on the Web site for the NCBI, choose Structure to gain access to
the Macromolecular Modeling Database. The NCBI obtains the coordinates for the
crystallographic molecular models registered in the Protein Data Bank of the Re-
search Collaboratory for Structural Bioinformatics. Each identification number in
the Protein Data Bank is four characters: numbers and uppercase letters. The same
identification number is used on both Web sites. The Macromolecular Modeling
Database also has its own identification system of five numerals [27].
These cartoons of the tertiary structures were drawn from the atomic coordinates
of the actual tertiary structures of molecular models that were constructed from
data obtained from the diffraction of X rays by crystals of these proteins, nuclear
magnetic resonance spectroscopy of soluble molecules, or occasionally both meth-
ods used in combination. In some cases, the cartoon includes one or more small
molecules that are bound to the protein in the crystal and appear in the crystallo-
graphic molecular model. Even though molecules of protein are usually oligomers
of subunits, because of the way in which crystallography is performed, only one or
two subunits of the larger oligomer may appear in the list of atomic coordinates for
a particular protein in the Protein Data Bank. The entire quaternary structure of the
protein can be obtained by applying symmetry operations to the coordinates that are
listed, but this is not consequential to the present exercise [27].
The Cn3D software is an application that helps you with the Web browser. It
animates images of the cartoons by rotating them. It is available on the NCBI site
as a free download. The Vector Alignment Search Tool on this Web site is an ap-
plication that assays for similarity in tertiary structure. This algorithm may discover
Fig. 4.3 tertiary structures

with Cn3D/NCBI, example:
cd00741Lipase
homology among several proteins even if there is no significant similarity in their

sequences of amino acids. Once a set of homologous structures is identified by
the Vector Alignment Search Tool, Cn3D may be used to superpose the two struc-
tures [28]. Examples of Cn3D images of cartoons of the tertiary structure of the lac
repressor [29] from the Macromolecular Modeling Database and a superposition
by the Vector Alignment Search Tool of this structure upon a related protein from
Chromobacterium violaceum, are shown (Fig. 4.3) [27].
The crystallographic molecular model has the identification number 1LBI in the
Protein Data Bank and the scientists that submitted the coordinates also published
a summary [29]. The green cylinders with a cone at one end represent α helices
and the flat, yellow arrows represent strands of β structure. The amino-terminal to
carboxy-terminal orientation is shown by the cones and arrows. The identification
number for the atomic coordinates for the latter molecular model is 3H5O in the
Protein Data Bank, and the coordinates were submitted by [17].
4.6 Physicochemical Characterization
4.6.1 Spectroscopic Method
4.6.1.1 Ultraviolet Absorption Spectroscopy
In the 1950s, it was proposed that structural perturbations of a protein could be cor-
related with changes in its ultraviolet absorbance spectrum [18]. The main drivers
for that correlation are the three aromatic amino acid residues tyrosine, tryptophan,
and phenylalanine that show absorption maxima between 250 and 300 nm. One of
the main applications today is the quantitation of liquid protein solutions using the
Beer–Lambert Law:
A (λ ) = log( I 0 / I ) = ε (λ ) cl
Where:
A is the absorbance intensity at a specific wavelength
I and I0 are the transmitted and the incident light
C is the molar concentration of the sample
l is the pathlength in centimeters
ε is the molar extinction coefficient at that wavelength ( λ).
In addition, it was observed that slight differences in the spectra of protein solutions
can be used to monitor subtle structural changes, for example, the exposure of the
aromatic acid residues to solvent during unfolding processes, as well as to monitor
for impurities in protein solutions such as particles, for example, protein aggre-
gates. For better evaluation of structural protein changes, derivation techniques of
the raw data can be used [19]. Particles whose size begins to approach 1/20 to 1/50
of the wavelength of the incident light scatter this light, hindering it from reaching
the detector and increasing absorbance values [30]. For total protein determination
by ultraviolet (UV) measurement, the scattering effect must be corrected as, for
example, described in the European Pharmacopoeia (EP 2.5.33 [31]). The protein
solution is measured at eight different wavelengths (nm): 280, 320, 325, 330, 335,
340, 345, and 350 [32].
4.6.1.2 Fluorescence Spectroscopy
The emission of light in the wavelength range between 200 and 800 nm by typically
aromatic molecules that are in an electronically excited state is referred to as lu-
minescence. Luminescence can be further categorized into fluorescence and phos-
phorescence depending on the nature of the excited energy states [31]. The primary
molecules in glycoproteins that can be excited and show fluorescence phenomena
are the three aromatic amino acids tryptophan, tyrosin, and phenylalanine. Typical
protein structure analysis uses monochromatic excitation and measurement of the
emission spectrum of the glycoprotein [30].
In practice, fluorescence spectroscopy is often used in pre-formulation and for-
mulation studies to examine the behavior of a protein in different environments,
such as in buffer systems containing additives. If, for example, a protein containing
a single tryptophan is denatured with increasing temperature, a red—shift emission
spectrum will appear when the hydrophobic tryptophan moves from the inner part
of the protein to a more exposed position on the aqueous outside of the protein.
Extrinsic fluorescence measurements use special dyes that bind covalently or via
intermolecular bonds on the protein. These applications are very useful to measure
protein–protein interactions and can also be used in the analysis of product—related
substances and impurities of protein drug substance samples, such as for the analy-
sis of aggregates in combination with a chromatographic method such as SE—
HPLC. The use of Nile Red as a fluorescent dye that binds to hydrophobic macro-
molecules and fluorometric detection with the HPLC device (EP 2.2.21 [31]) can
increase the limit of detection of this HPLC method by a factor of 1000 compared
with UV analysis.
4.6.2 Chromatographic Methods
4.6.2.1 Size—Exclusion Chromatography
Size—exclusion chromatography (SEC) is a chromatographic separation method

that separates molecules based on their size in the diluted state (e.g., hydrodynamic
radius). In glycoprotein analyses, analytical SEC or SE—HPLC columns are used
that possess higher maximal pressure limits compared with the preparative SEC
columns used in purification processes (e.g., downstream polishing processes). The
standard eluent for the analysis of glycoproteins in the native, undenatured state is
phosphate—buffered saline (1xPBS). Size—exclusion chromatography is used in
particular for the analyses of soluble product aggregates, a group of product—re-
lated impurities that are still viewed as one of the critical quality attributes by the
biopharmaceutical industry as well as by the regulatory authorities [33, 34]. Owing
to the fact that soluble aggregate molecules can interact with the column matrix,
due to their relatively high hydrophobicity, eluent additives, such as arginine, are
used to avoid these interactions and to obtain a picture of the true amount of aggre-
gates in the sample [35] Typical detection wavelengths for glycoprotein purity de-
terminations are UV 280 nm, for example, for the determination of proteinogenous
impurities, and UV 254 nm, for example, for the determination of DNA—based
impurities. Nowadays, diode array detection (DAD) is used to obtain the complete
zero—order UV spectrum [36].
4.6.2.2 Reversed—Phase Chromatography
Reversed—phase chromatographic (RPC) methods use, by definition, hydrophobic

stationary phases and hydrophilic eluents, in contrast to normal phase chromato-
graphic methods that use hydrophilic stationary phases and hydrophobic eluents.
Classical RP stationary phases are octyl- and octadecyl- modified silica matrices.
More pH-resistant stationary phases are based on polymeric material, such as
stryrene–divinylbenzene copolymer. Typical RP eluents are aqueous acetonitrile
gradients. RPC characterizes the peptide map of the specific glycoprotein for iden-
tity determination. The method comprises the chemical or enzymatic treatment of
the protein sample to create peptide fragments, in addition to the separation and
identification of the resulting fragments by their retention times (EP 2.2.55 [31]).
RPC is often also used in combination with mass spectrometry (e.g., LC—ESI—
MS) to perform peptide mass fingerprint analyses.
4.6.2.3 Hydrophilic Interaction Chromatography
Hydrophilic interaction chromatography (HILIC is widely used in the analysis of

the glycan moiety of a glycoprotein, especially after separating the glycan part of
a glycoprotein from the protein backbone, and after preparation of the glycans for
chromatographic analysis. Preparation steps in glycosylation analysis often include
enzymatic glycan cleavage or chemical deglycosylation techniques as well as fluo-
rophore labeling techniques, such as 2-aminobenzamide (2-AB) labeling [37].
4.6.2.4 Ion—Exchange Chromatography
Ion—exchange chromatography, especially cation—exchange chromatography

(CEX), is often used in the analysis of C—terminal lysine residues in monoclonal
antibody (mAb) products as well as to detect deamidated product variants that indi-
cate product degradation. Typical CEX resins for the analysis of mAbs are polymer
based and use sulfonate or carboxylate functional groups as well as phosphate buff-
ered aqueous eluents [26].
4.6.3 Electrophoretic Methods
4.6.3.1 Gel Electrophoresis
A special gel electrophoresis for glycoprotein analysis is the isoelectric focusing

technique (IEF, EP 2.2.54 [31]), which uses amphoteric electrolytes as running buf-
fers on a polyacrylamide or agarose gel. Within the electric field the ampholytescre-
ate a pH gradient within the gel matrix. The glycoprotein samples, also amphoteric,
pass through this pH gradient until their characteristic isoelectric points (p I) are
reached and then create stainable bands in the gel. The p I of a glycoprotein is
mainly dependent on its primary structure, but also on post—translational modifica-
tions, such as deamidated amino acid residues and acidic glycan (e.g., sialic acid)
residues [32].
4.6.3.2 Capillary Electrophoresis
CE has become a favorable technique for profiling different glycoprotein iso-

forms with very high resolution. CE also offers unique possibilities in assess-
ing certain important characterization topics, such as characterizing glycoprotein
macroheterogeneity (glycosylation site occupancy) in monoclonal antibody sam-

ples. With CGE (capillary gel electrophoresis) or MEKC (micellar electrokinetic
chromatography), minor amounts of unglycosylated mAb can be elucidated from
major amounts of glycosylated mAb within one electropherogram [38].
4.6.4 Mass Spectrometric Analysis of Biopharmaceutical

Proteins
The quality of recombinant biopharmaceutical product is mainly defined by the

overall product integrity as well as by the correctness of the amino acid sequence of
the product, that is, product identity. Typically, quality attributes for a given product
relate to product integrity issues, such as intrinsic or process—induced aggregation
propensity as well as certain clone or process dependent post—translational modi-
fications, including glycosylation, oxidation, deamidation, and so on. The quality
guideline ICH Q6B requires the verification of the correctness of the amino acid
sequence of the product and that the identity test(s) employed should be highly
specific and based on unique aspects of the product’s molecular structure and other
specific product properties.
4.7 Substrate Specificity, Kinetics and Mechanisms
4.7.1 Substrate Specificity
Enzymes have two extraordinary properties: they are very efficient catalysts, ac-
celerating reactions by as much as 1017-fold while operating in water, at neutral pH
and ambient temperatures [39]; they are exquisitely selective [40], being capable
of discriminating between closely related substrates and controlling reactions to
yield a single product. While it is often convenient to consider these two properties
separately, it is important to realize that they are inextricably intertwined: specific-
ity is expressed in the rate at which a substrate is transformed to product. Substrate
specificity is determined by the accumulation of noncovalent forces: hydrogen
bonding, steric, electrostatic, van der Waals and hydrophobic. These interactions
are reminiscent of the relationship between a lock and key: hydrophobic parts of
the substrate bind in hydrophobic pockets on the enzyme, negative charges of the
substrate interact with positive charges on the enzyme, and so forth [41].
While the lock and key analogy is useful for understanding enzyme–substrate
interactions, it is important to remember that an enzyme active site is not simply
complementary to the substrate. Such an enzyme would merely stabilize the ground
state of the substrate, not accelerate the reaction. Catalysis results from selective sta-
bilization of the transition state. Therefore, the enzyme active site must be comple-
mentary to the transition state of the reaction. This complementarity to the transition
Table 4.2 Symbols and units used in enzyme kinetic studies

Symbol Definition Typical unit
[E]t Total concentration of active sites nM or µM
v Initial rate or initial velocity µMs−1
Vmax Maximum velocity µMs−1
kcat Catalytic constant or turnover number s−1
k Specific rate constant µM−1 s−1
Km Michaelis constant for a particular substrate mM or µM
state produces a corresponding destabilization of the ground state. As stated by J.

B. S. Haldane, ‘The key does not fit the lock quite perfectly, but exercises a certain
strain on it’ [41].
The lock and key analogy falls short in another regard. Whereas locks and keys
have rigid structures, both enzymes and substrates are flexible. Substrate confor-
mation can adapt to fit the enzyme active site. For example, while a peptide may
not be conformationally constrained in solution, it must assume a fixed, extended
conformation when bound to trypsin. Likewise, enzyme conformation can change
in response to substrate binding. Daniel Koshland first proposed this ‘induced fit’
hypothesis: the binding of substrate can convert enzyme from an inactive confor-
mation into an active one, by orienting catalytic residues, structuring a binding
site for a second substrate, or closing the active site to exclude water. The adapta-
tion of the active site to the substrate provides another mechanism of substrate
discrimination [41].
One of the parameters describing the kinetics of an enzyme is the Michaelis
constant, Km, for one of its substrates (Table 4.2) [41–44].
Enzymes, because they are catalysts present in solution in much lower molar
concentration than their substrates, cannot alter the equilibrium constant for the
chemical reactions that they catalyze, because that would violate the second law of
thermodynamics. Consequently, all enzymes necessarily catalyze the reactions for
which they are responsible in both directions. When the kinetics of an enzymatic
reaction are being studied, the observer chooses to follow the rate of the enzymatic
reaction in only one of those two directions. This is accomplished by adding to the
solution the complete set of substrates on only one side of the equilibrium describ-
ing the enzymatic reaction. The substrates in this set are the reactants for the kinetic
study. The experiment is performed by mixing all of the reactants with the enzyme.
One of the reactants or the enzyme itself is chosen to be the last participant to be
added to the mixture. As soon as the last participant is added to initiate the reac-
tion, the concentration of one of the reactants or one of the products is monitored
as a function of time. The rate at which the concentration of the monitored reactant
decreases, or the rate at which the concentration of the monitored product increases,
is the rate of the reaction [27].
As the concentrations of the products increase over the course of the measure-
ment, the reverse reaction catalyzed by the enzyme increases in rate, and the rate
Fig. 4.4 Kinetic data for an

enzyme
being monitored decreases as the concentrations of reactants and products reach

equilibrium. At equilibrium, all measurable changes cease, even though the enzyme
is still catalyzing the reaction rapidly in both directions. No further changes occur
because at equilibrium the enzyme catalyzes the reaction at the same rate in both
directions. To avoid this decrease in the rate of the reaction caused by the increase
in the rate of the back reaction, the overall rate of the reaction is only monitored at
short times to obtain the initial rate of the reaction, or the initial velocity ( v) [27].
Initially, as the measurements are being made, the initial velocity is recorded in
units of micromoles minute−1 milliliter−1. If the concentration of active sites in the
solution being monitored is known, dividing the initial velocity in micromoles min-
ute−1 milliliter−1 by the molar concentration of active sites in micromoles milliliter−1
converts the initial velocity to the units of seconds−1. The choice of these units gives
the number of times an active site on the enzyme converts reactants to products each
second under the conditions of the assay [27].
One would expect the rate of a reaction catalyzed by an enzyme to increase as
concentration of one of the reactants is increased. It is not, however, a simple linear
relationship. It is always the case that as the concentration of one of the reactants in
an enzymatic reaction increases, the initial velocity increases but it increases less
and less until no further increase occurs (Fig. 4.4) or, in some cases, until the rate
begins to decrease. In the former instance, the rate of the reaction reaches satura-
tion; in the latter case, the reactant itself is interfering with the enzymatic reaction
as an inhibitor, and its inhibition increases as its concentration increases. The for-
mer behavior, saturation, is more common and is indicative of enzymatic reactions.
When it occurs, inhibition by a reactant is in addition to the underlying saturation
that is occurring [27].
The data chosen for the example are for the hydrolysis of sucrose by β-
fructofuranosidase. As the concentration of reactant A in the assays is increased,
the initial velocity, v, increases and approaches, as a horizontal asymptote, the
maximum velocity ( V). Initial velocity is typically recorded in units of micromoles
minute−1 milliliter−1 but can be changed to the units of second−1 if the molar concen-
tration of active sites is known. The Michaelis constant for reactant A, Km, has the
same units as those used for the concentration of the reactant A [27].
In the usual, ideal case, the increase in the initial velocity of an enzymatic re-
action with increasing concentrations of one of its reactants is described by the
equation for a rectangular hyperbola. There are two equations that can be used to
describe this particular rectangular hyperbola:
(k0 [E]t ) (kA [E]t [A]0 )
υ= (4.1)
k0 [E]t + kA [E]t [A]0

V [A]0
υ= (4.2)
K mA + [A]0
where kcat is the catalytic constant, kA is the specificity constant for reactant A, [E]
t is the total molar concentration of active sites on the enzyme in the solution, [A]0
is the initial molar concentration of reactant A, Vmax is the maximum velocity, and
KmA is the Michaelis constant for reactant A. These are two ways of writing the
same equation. If the initial velocity of the enzymatic reaction as a function of the
concentration of one or more of the reactants shows saturation and ideal hyperbolic
behavior, the data can be fit by either one of these equations [27].
Aside from the theoretical meaning of KmA, there is an obvious practical mean-
ing of KmA. In the equation defining KmA (Eq. 4.2), KmA is equal to the concen-
tration of reactant A at which the initial velocity of the enzymatic reaction is half of
its initial velocity at saturation, V. Saturation occurs when the initial concentration
of reactant A is infinite. What this means in practice is that in the cell, the concen-
tration of reactant A should be greater than KmA for the enzyme to be operating
efficiently; in other words, for most of the molecules of enzyme to be working at
the same time. Usually, natural selection has adjusted KmA to be in the range of
the normal concentration of reactant A in the cells in which the enzyme is found.
Therefore, KmA has practical consequences [27].
The best way to fit the second equation describing ideal behavior to the data that
you will be gathering, in order to obtain a Michaelis constant, is a nonlinear least
squares fit. This may be accomplished by many applications. For example: Micro-
soft Excel with the Solver add-in [45, 46] or Kaleidagraph from Synergy Software
(www.synergy.com).
4.8 Kinetic Parameters for Native Versus Promiscuous

Functions
Degree of promiscuity—which is enzyme activities other than the activity for which
an enzyme evolved and that are not part of the organism’s physiology- refers to the
level of specificity breach, namely, how diverse are the promiscuous activities of
a given enzyme [47], and how different are the native and promiscuous functions.
The degree of promiscuity can be assessed by examining the type of bonds that are
being formed or broken and by differences in the mechanism between the native
and promiscuous reactions [48].
Magnitude of promiscuity refers to the kinetic parameters for the promiscuous
activity relative to the native one. Whereas most enzymes exhibit kcat/KM values
in the order of 105–108 M−1s−1 for their native substrates, the magnitude of pro-
miscuous activities varies over more orders of magnitude, in absolute terms and
also relative to the native activity. Catalytic proficiency (kcat/KM/kuncat) and rate
acceleration (kcat/kuncat) can provide a measure of the magnitude of catalytic ef-
fects exerted on native versus promiscuous substrates. In many cases, although the
kcat/KM values for the promiscuous substrates are very low, and hence might have
little physiological relevance, the rate accelerations and catalytic proficiencies are
impressively high [47, 49–51].
Differences between the efficiency of promiscuous and native activities can be
manifested in differences in either kcat or KM. Although it is expected that promis-
cuous substrates that bind weakly will exhibit high KM values, many promiscuous
substrates are characterized by low kcat values. Thus, specificity may result not
only from substrate binding interactions per se, but also from appropriate position-
ing relative to the catalytic machinery. For the promiscuous substrates, substrate
binding is driven primarily by nonspecific hydrophobic forces promiscuous sub-
strates are inadequately positioned relative to the catalytic machinery and therefore
exhibit low kcat values binding of the native substrate are typically mediated by
enthalpy-driven interactions, such as hydrogen bonds, whereas for the promiscuous
substrates, hydrophobic and other entropy-driven interactions play a key role.
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Chapter 5
Purification of Recombinant Protein
for Industrial Use
Faridah Yusof
Abstract This article attempts to review the techniques typically used to purify
recombinant protein, intended for industrial usage. The initial part of this review
briefly describes the choices of expression systems available to produce recombi-
nant protein and based on the choices made, techniques used for sample harvest-
ing is deliberated. The method to prepare samples prior to the purification step is
also discussed which includes clarification and retaining the stability of protein of
interest. The detection and quantitation of the protein methods are also pondered
upon, since these two steps are essential for the success of any purification steps.
This is followed by several purification strategies, emphasizing on the purifica-
tion of tagged recombinant protein. Two additional methods of purification are also
suggested, and they are, conventional column chromatography and Aqueous Two-
Phase Systems (ATPS), which can be applied to both, tagged or untagged protein.
ATPS comes with several advantages, including scale-up potential and continuous
operation, which are useful at the industrial scale purification. The common prob-
lems faced in purification of recombinant proteins at higher or industrial scales are
also highlighted. Finally, this article suggests that before any industrial scale protein
purification can be exploited, some issues have to be clarified and resolved which
includes its economic viability, adherence to regulatory requirements and environ-
mental friendliness.
Keywords Affinity tags · Aqueous two-phase separation · Chromatographic ·

Clarification · Desalting · Expression of recombinant protein · Extracellular ·
Glutathione S-transferase · Immunoassays · Intracellular · Maltose binding protein ·
SDS polyacrylamide gel electrophoresis · Sedimentation · Tagged recombinant protein
F. Yusof ()
e-mail: yfaridah@iium.edu.my
DOI 10.1007/978-3-319-12397-4_5
62 F. Yusof
5.1 Introduction
In recent years, the use of recombinant protein in industries has increased tremen-
dously. There are many reasons for this phenomenon. The two most important rea-
sons are; firstly because it is now possible to produce any protein of interest, ir-
respective of its sources and produced it in large amounts and secondly, it is now
comparatively easier to produce these recombinant proteins. Currently, there exists
many techniques and products that can be used for the expression and purification
of recombinant proteins [1]. Recombinant proteins may be produced as tagged or
untagged, whichever is convenient. However tagged recombinant proteins are now
commonly produced, since they have many advantages such as ease in purification,
detection and increased stability and solubility. Before successfully producing re-
combinant proteins, there are many parameters that need to be considered carefully.
Attention has to be given to the choices of hosts, vectors or whether to produce
tagged or untagged protein. Other details that need to be considered are harvesting,
extraction, handling of inclusion bodies, tag removal, and removal of unwanted
salts and small molecules.
Usually laboratory scale purification of recombinant proteins can be performed
manually or automated to save time and effort. The purification can be performed
on many scales, in columns of various sizes, in batch, with gravity flow or centrifu-
gation or with the help of low, medium and high pressure pumps [1]. Proteins are
purified using chromatography techniques that separate them according to differ-
ences in their specific properties [2]. However for industrial purposes, due to its
large scale, purification should be carried out semi- or fully-automatically [3].
If tagged recombinant proteins are produced, purification can be carried out by
affinity chromatography, which is designed to capture the protein based on bio-
recognition of the tag. In that case, several different recombinant proteins can be
purified by the same affinity technique, based on the same tag [1]. Since the same
tag are used, it allows the use of a common detection protocol for different recom-
binant proteins. For many applications, a single purification step, using a commer-
cially available chromatography column, is sufficient to purify tagged recombinant
proteins. On the other hand, untagged recombinant protein can be purified similar
to any other native proteins. Untagged protein maybe purified by a single affin-
ity chromatography step. But when a higher degree of purity is required for either
tagged or untagged recombinant proteins, a multistep purification will be necessary.
This can be carried out by choosing the right combination of purification tech-
niques, based on the properties of the protein of interest [2].
Laboratory scale and industrial scale protein purification usually exploit the
same separation principles, but methods developed in the laboratory are not al-
ways directly applicable to large scale processes [3]. For the industrial production,
extreme focus should be paid on quality of the end product and its adherence to
regulatory requirements, whereby these issues are easily overlooked in laboratory
scale separations.
This article attempts to review the techniques typically used to purify recom-
binant protein, intended for industrial usage. The first part of this review briefly
5 Purification of Recombinant Protein for Industrial Use 63
describes the choices of expression systems available to produce recombinant pro-

tein. Based on the choices made on the types of expression system, techniques used
for sample harvesting and preparation are deliberated, followed by several purifica-
tion procedures, emphasizing on the purification of tagged recombinant protein.
Finally this article tries to discuss the common problem faced in purification of
recombinant proteins at industrial scales.
5.2 Expression of Recombinant Protein
In expressing recombinant protein, there are many parameters that need to be con-
sidered seriously, namely host, vector and whether to tag or untag protein [1]. The
choices of these parameters will affect the downstream processing, including har-
vesting and extraction and purification of protein.
There are many host system available commercially nowadays, which includes
bacteria, yeast, plants, filamentous fungi, insect or mammalian cells and transgenic
animals or plants. Each host system has its own advantages and disadvantages, and
it is important to consider these before the final selection of host. The choice of host
affects not only the expression of the protein but also the way in which the product
can be subsequently purified. In order to decide which host is most suitable, the
amount and the degree of purity of the product, as well as its biological integrity and
potential toxicity, should be considered [1]. For example, bacterial expression sys-
tems are not suitable if posttranslational modification is required to produce a fully
functional recombinant protein. The location of product within the host will affect
the choice of methods for isolation and purification of the product. For example, in
addition to expressing the protein cytoplasmically, a bacterial host may secrete the
protein into the growth medium, transport it to the periplasmic space, or store it as
insoluble inclusion bodies within the cytoplasm. Expression in different parts of the
cell will lead to varying amounts of cellular (contaminant) proteins that will need to
be removed to obtain a pure target protein.
A protein expression system usually includes a vector with an appropriate pro-
moter and other regulatory sequences, along with the gene encoding the recombi-
nant protein of interest. Vectors are available commercially for the expression of
recombinant proteins either fused to a tag or untagged. The selection of a suitable
expression system depends on the desired scale of production, the time and re-
sources available, and the intended use of the recombinant protein. The choice of
vector family is largely governed by the host. Once the host has been selected, many
different vectors are available for consideration, from simple expression vectors to
those that contain specialized sequences needed to secrete the recombinant proteins.
The expression of a recombinant protein fused to a tag of known size and biological
function can greatly simplify subsequent purification and detection. In some cases,
the protein yield can also be increased. Sources of recombinant protein to be puri-
fied can be soluble or expressed as inclusion bodies.
Inclusion of affinity tags in the expression system was carried out for several
reasons. Besides simplifying the purification process, these tags can also have roles
64 F. Yusof
in stabilizing, solubilizing and detection of the protein [1]. The most common tag,
the Histidine (His) tag, is often a 6-His, but other poly-His tags consisting of be-
tween four and 10 His residues have been used [4], where the latter provides for
the strongest affinity for the chromatography medium. There are many other tags,
in which the DNA sequence are very well known, such as Glutathione S-transferase
(GST) [5] and Maltose Binding Protein (MBP) tags [6], both of which are proteins.
5.3 Detection and Quantitation of Recombinant Protein
As being mentioned by Yusof [2], before conducting any expression and purifica-
tion of recombinant protein, a protocol or an assay to detect and quantitate the target
protein must be ready. Detection and quantitation of the target protein are needed
at various stages:
1. During sample preparation where the crude lysate from many clones are
screened so that optimal expression levels and growth conditions can be readily
determined.
2. When optimizing the purification protocols, and this may require functional
assays to assess the intactness of the target protein.
There are many ways to detect and quantitate the target proteins [1], such as:
1. For over-expressed proteins, the high concentration in itself can be used for
detection of the target protein fraction in a chromatogram, but in such a case
verification of the identity of the protein in the final preparation is needed.
2. Specific detection of tagged proteins can often be accomplished by analyzing the
presence of the tag by activity or immunoassay, or simply by the spectral proper-
ties of the tag. This may be especially important when multiple constructs with
the same tag are prepared in high-throughput platforms.
3. Immunoassays of the target protein by Western blot, ELISA, immunoprecipita-
tion, etc. can be used for quantitation if a suitable standard curve can be pro-
duced. In this case, it is not necessary to purify the tagged protein so long as
a purified standard is available. Therefore, these techniques may be used for
quantitation during protocol development. The immunoassay technique is also
particularly suitable for screening large numbers of samples when a simple yes/
no answer is required (e.g., when testing fractions from a chromatographic run).
4. Specific functional assay can be used to determine the activity of the target pro-
tein/ enzyme.
5. SDS polyacrylamide gel electrophoresis (SDS-PAGE) can be used to check the
purity of proteins. The target protein band can often be identified using the appar-
ent molecular weight obtained by including standard molecular weight markers
in the analysis.
6. The yield of protein may also be determined by standard chromogenic methods
(e.g., Lowry, BCA™ protein assay, Bradford method, etc.).
7. Absorbance of the relative yield of tagged protein can often be determined at

280 nm. The extinction coefficient of the target protein will be needed. A good
estimation may be obtained by theoretical calculation from the amino acid com-
position of the protein.
5.4 Recombinant Protein Production
Before expression of intended recombinant proteins can be carried out, the expres-
sion levels and the growth conditions has to be determined [1]. Once conditions for
optimizing the expression of recombinant proteins are established, preparation of
large-scale cultures of the desired clones can starts followed by subjecting sample
to downstream processing which includes purification. There are many methods
available for the downstream processing, depending on the expression system and
the tag used. Recombinant proteins can be expressed intracellularly (target proteins
can be insoluble in cytoplasm, soluble in cytoplasm or at the periplasmic space) or
expressed extracellular (target proteins in the culture medium). Expression of both,
extracellularly or intracellularly recombinant proteins have their own advantages
and disadvantages in terms of its purification processes. Yield of recombinant pro-
teins is highly variable and is affected by the nature of the recombinant protein, the
host cell, and the culture conditions. Recombinant protein yields can range from 0
to 10 mg/l [1].
Cell harvesting and extraction procedures should be selected according to the
source of the protein, such as bacterial, plant, or mammalian, intracellular or extra-
cellular [1]. Harvesting, in which the cells are separated from the cell culture media,
generally involves either centrifugation or filtration. Selection of an extraction tech-
nique depends as much on the equipment available and scale of operation as on the
type of sample. There are many extraction processes for recombinant proteins and
usually in order to achieve optimal results, it is common to select a combination of
these methods.
5.5 Preparation of Sample for Purification
Before sample is submitted to the purification steps it is utmost important to main-

tain the followings [1]:
• Stability of the target protein
• Clarifying of sample from particulate matter
• Desalting and buffer exchange
The target protein needs to be stabilized not only before the purification steps
but the stability has to be maintained all throughout the purification techniques.
66 F. Yusof
Stability includes protecting the target protein from denaturation and undergoing
proteolytic degradation. As for the clarification of sample, many easy and sample
steps are carried out before beginning purification to avoid clogging the column.
The clarification of sample may reduce the need for stringent washing procedures
and this can extend the life of the chromatographic medium. Protein samples can
be cleared by centrifugation or filtration. Desalting and buffer exchange are impor-
tant in cases where small contaminating entities including salts need to be omitted,
whereas buffer exchanges are needed when sample are prepared prior to loading in
the chromatographic column.
5.5.1 Protein Stability
In the majority of cases, biological activity of the target protein needs to be retained
during and after purification. Retaining the activity of the target molecule is also
an advantage when following the progress of the purification, because detection of
the target molecule often relies on its biological activity. It is advisable to perform
stability tests before beginning to develop a purification protocol. The list below
may be used as a basis for such test:
• To test pH stability in steps of one pH unit between pH 2 and pH 9.
• To test salt stability with 0–2 M NaCl and 0–2 M (NH4)2SO4 in steps of 0.5 M
• To test the buffering salts or agents
• To test the temperature stability in 10 °C steps from 4 to 44 °C, including in cold
room (4–10 °C) and at ambient temperature (22 °C).
• To test for protein stability and proteolytic activity by leaving an aliquot of the
sample at room temperature overnight.
In stabilizing the target protein, the followings may be added to maintain the stabil-
ity; protease inhibitor, reducing agents (mercaptoethanol or dithiothreitol) or stabi-
lizing additives.
5.5.2 Sample Clarification
Centrifugation and filtration are standard laboratory techniques for sample clari-
fication and are used routinely when handling small samples. It is highly recom-
mended to centrifuge and filter any sample immediately before chromatographic
purification. Centrifugation removes most particulate matter, such as cell debris. If
the sample is still not clear after centrifugation, use filter paper or a 5 μm filter as a
first step and one of the fine pored filter as a second step. For small sample volumes
or proteins that adsorb to filters, centrifuge at 10,000 × g for 15 min. For cell lysates,
centrifuge at 40,000–50,000 × g for 30 min. Serum samples can be filtered through
glass wool after centrifugation to remove any remaining lipids. For large volume
of sample, a continuous flow disc stack centrifugation can be carried out which
removes a slurry with a fairly high solids ratio.
Filtration removes particulate matter. Membrane filters that give the least
amount of nonspecific binding of proteins are composed of cellulose acetate or
polyvinylidene fluoride (PVDF). For sample preparation before chromatography,
select a filter pore size in relation to the bead size of the chromatographic medium.
Filters become “saturated”, that is, they have a certain capacity. It may be necessary
to check the capacity when setting up a protocol. Ultrafiltration can also be used,
which is a pressure-driven, membrane-based separation process. In fact, ultrafiltra-
tion is used for protein concentration, desalting, clarification and fractionation (i.e.
protein-protein separation).
Precipitation technique can also be used to recover the target protein. Precipita-
tion maybe induced by the following methods, such as, addition of salt (e.g. ammo-
nium sulfate), addition of organic solvent, changing the pH, addition of non-ionic
polymer or addition of metal ions.
5.5.3 Desalting and Buffer Exchange
In many cases, desalting or buffer exchange is needed before the submission of

sample to column purification. Desalting columns are suitable for many different
sample volumes and will rapidly remove low-molecular-weight contaminants in a
single step at the same time as transferring the sample into the correct buffer con-
ditions. However for buffer exchanges, the following can be considered, such as,
dialysis, dilution to reduce ionic strength, addition of salt to increase the concentra-
tion or titration to adjust pH.
5.6 Purification of Recombinant Protein
Purification of protein via column chromatography is very commonly employed at

laboratory scale as well as in the industry. As a rule of thumb, all proteins can be
purified by column chromatography to a satisfactory degree of purity, whether it is
recombinant or not, tagged or untagged. The purification can be done satisfactorily
by a single-step or multi step. In fact, single step purification saves time and re-
sources and reduces both the risk of denaturation of the target protein and the loss
of essential molecules that are weakly attached to the protein.
Column chromatography is the most common physical configuration, in which
the stationary phase is packed into a tube, a column, through which the mobile
phase, the eluent, is pumped [2]. The degree to which the molecule adsorbs or in-
teracts with the stationary phase will determine how fast it will be carried by the
mobile phase. Chromatographic separation of protein mixtures has become one of
the most effective and widely used means of purifying individual proteins. Besides
column chromatography, Aqueous Two-Phase Separation (ATPS) have been gain-
ing interest to be used for the recovery and purification of many biological products
68 F. Yusof
including proteins [7–10]. This is due to some advantages of this technique, in-
cluding scale-up potential. In the following sections, purification of untagged and
tagged recombinant protein using column chromatography will be deliberated, fol-
lowed with purifications of proteins using ATPS.
5.6.1 Purification of Untagged Recombinant Protein by Column

Chromatography
Methods normally adopted to purify untagged recombinant protein will be very

similar to any other native protein. Individual protein possesses a variety of char-
acteristics that distinguish them from the other protein molecules and such charac-
teristics include size, shape, overall charge, the presence of surface hydrophobic
groups and the ability to bind various ligands. Quite a number of proteins molecules
maybe similar to one another if compared on the basis of one such characteristics,
however all proteins has their own unique combination of characteristics. Various
chromatographic techniques have been developed which separates protein from
each other on the basis of the differences in such characteristics. Chromatographic
techniques most commonly used and their basis of separations are as follows [2]:
• Ion exchange chromatography separates proteins base on the differences in pro-
tein surface charge at a given pH.
• Hydrophobic interaction chromatography separates proteins based on the differ-
ences in surface hydrophobicity of the protein
• Gel filtration chromatography separates proteins base on the differences in mass
or shape of the different protein.
• Chromatofocusing separates proteins based on their isoelectric points.
• Affinity chromatography separates proteins based upon biospecific interaction
between a protein and an appropriate ligand.
The details of purification of proteins using all the above techniques have been
reported in Yusof [2]. It is important to note here that the same basic principle of
the above-mentioned ‘affinity chromatography’ is used in purifying recombinant
tagged protein. This is based on the interaction between the purposely added recom-
binant protein ‘tag’ and the special ligand specially attached to the stationary sup-
port that occupies the column chromatography. The details of tagged recombinant
protein purification are elaborated in the next section.
5.6.2 Purification of Tagged Recombinant Protein by Column

Chromatography
Tagged recombinant protein will normally undergo a single step protein purifica-
tion. This can be done via affinity chromatography which isolates a specific protein
or a group of proteins with similar characteristics. The technique separates proteins
on the basis of a reversible interaction between the protein and a specific ligand
attached to a chromatographic matrix. Whenever a suitable ligand is available for
the protein of interest, a single affinity purification step offers high selectivity, and
usually high capacity for the target protein [1]. The technique is well-suited for a
capture or as an intermediate purification step and can be used whenever a suitable
ligand is available for the protein of interest. Affinity chromatography offers high
selectivity and usually is frequently used as the first step (capture step) of a two-
step purification protocol, followed by a second chromatographic step (polishing
step) to remove remaining impurities. The target protein(s) is/are specifically and
reversibly bound by a complementary binding substance (ligand). The sample is ap-
plied under conditions that favor specific binding to the ligand. Unbound material is
washed away, and bound target protein is recovered by changing conditions to those
favoring desorption. Desorption is performed specifically, using a competitive li-
gand, or nonspecifically, by changing the pH, ionic strength, or polarity. Samples
are concentrated during binding, and the target protein is collected in purified and
concentrated form.
Recombinant protein expression may allow production of large amounts of an
affinity-tagged protein so that a single purification step using affinity chromatogra-
phy is sufficient to achieve the desired level of purity [1, 11]. However, the purifi-
cation obtained after a single step is frequently not sufficient, and affinity tags may
sometimes interfere with the post-purification use of the protein. In these instances,
multistep purification will be necessary. A significant advantage when working with
recombinant proteins is that there is often considerable information available about
the product (amino acid sequence, molecular weight, pI, functional properties) and
contaminants (the expression host may be well known). With this information, de-
tection assays and sample preparation and extraction procedures can be much less
complicated, to ensure faster method development, a shorter time to pure product,
and good economy.
Affinity tags can be defined as exogenous amino acid sequences with a high
affinity for a specific biological or chemical ligand. A major group of affinity tags
consists of a peptide or protein that binds a small ligand linked on a solid support.
Table 5.1 shows the list of affinity and solubility tags for recombinant proteins [11].
Affinity tags are highly efficient tools for protein purification. They allow the
purification of virtually any protein without any prior knowledge of its biochemical
properties. The inclusion of an affinity tag might be attractive for a number of ad-
ditional reasons besides aiding in purification, such as, to improve protein yield, to
prevent proteolysis, to facilitate protein refolding, to protect the antigenicity of the
fusion protein and to increase solubility and also to increase the sensitivity of bind-
ing assays [4, 12–19]. On the other hand, adding a tag can also have negative effect
such as changing the protein conformation, lower protein yield, inhibition of en-
zyme activity and alteration in biological activity and undesired flexibility in struc-
tural studies and toxicity [20–27]. Thus it is usually desirable to remove the tag,
especially if it is intended for human used. And consequently both the enzymes used
to cleave the tag and the cleave tag need to be removed from the purified protein.
70 F. Yusof
Table 5.1 Affinity and solubility tags for recombinant protein [11]
Tags Size of amino acid Comments
His-tag 5–15 Purification under native or denaturing
conditions
FLAG 8 Calcium-dependent, mAb-based purification
Streptag II 8 Modified streptavidin, elution with biotin
analog
HA-tag 9 Influenza virus hemagglutinin tag, Ab-based
purification
Softag1, Softag 3 13, 8 Recognized by polyol-responsive mAb
c-myc 10 mAb-based purification
T7-tag 11–16 mAb-based purification
S-tag 15 S-protein resin affinity purification
Elastin-like peptides 18–320 Protein aggregation by temperature shift,
intein-based
Chitin-binding domain 52 Binds only insoluble chitin
Thioredoxin 109 Affinity purification with modified resin
Xylanase 10A 163 Cellulose based capture, elution with glucose
Glutathione S-transferase 201 Glutathione or GST-Ab affinity
Maltose binding protein 396 Amylose affinity purification
NusA 495 Increased solubility in E. coli. Affinity tag
needed for purification
With the advance of DNA recombinant technology, it has even made it easier to
purify any recombinant proteins of interest. The latest technique is by the introduc-
tion of ‘Inteins’ tag to the expressed protein [28–32]. Intein is a segment of a protein
that is able to excise itself and rejoin the remaining portions (the exteins) with a
peptide bond. To aid purification, a system that uses inteins are able to purify the
target protein in a column chromatography without the use of protease to remove
the tag. Inteins are self-cleavable proteases. For example, the intein-based IMPACT
system uses a protein fusion consisting e.g., of an N-terminal chitin-binding domain
(affinity tag), a central intein and a C-terminal target protein [29]. Binding to a chi-
tin matrix is followed by on-column cleavage using either a thiol reagent or pH and
temperature shift to yield intein cleavage and elution of the target protein [30, 31].
5.6.3 Purification of Recombinant Protein by Aqueous

Two-Phase Systems (Atps)
Another purification method that can be used in purifying proteins is ‘Aqueous

Two-Phase System’ (ATPS). ATPS have been developed for the recovery and puri-
fication of many biological products including proteins [7–9]. This method can be
used to purify non recombinant as well as recombinant proteins. The advantages
of this technique include scale-up potential, continuous operation, ease of process

integration, low toxicity of phase forming chemicals and biocompatibility. ATPS
exploits the incompatibility between aqueous solutions of two polymers, or a poly-
mer and a salt at high ionic strength. Hence, as the polymers are mixed, large ag-
gregates form and the two polymers will tend to separate into two different phases
due to steric exclusion. The most commonly used polymers are polyethylene glycol
(PEG) and dextran. A similar exclusion phenomenon can be observed between a
polymer and a high concentration of salt (e.g. PEG and phosphate, sulphate, citrate)
since the salt will capture a large amount of the water present. Separation of proteins
from one another is achieved by manipulating the partition coefficients, K. The pro-
tein of interest will equilibrate between the two phases, according to its, K, such as:
CT
K= (5.1)
CB
where CT and CB represent the equilibrium concentrations of the partitioned pro-
tein in the top and bottom phases, respectively. The two-phase partitioning system
can be used to separate proteins from cell debris or to purify proteins from other
proteins. Most soluble and particulate matter will partition to the lower, more polar
phase, whilst proteins will partition to the top, less polar and more hydrophobic
phase, usually PEG [33]. Separation of proteins from one another is achieved by
manipulating the partition coefficient by altering the average molecular weight of
the polymers, the type of ions in the system, the ionic strength of the salt phase or
by adding an additional salt such as NaCl. Affinity partitioning can also be used
to increase the degree of purification; in this case affinity ligand, such as triazine
dyes (e.g. Cibracon blue) are covalently attached to one polymer. Alternatively, one
polymer may be modified with hydrophobic group to alter the partitioning of the
protein.
Two-phase aqueous partitioning is a very mild method of protein purification,
and denaturation and loss of biological activity are not usually seen. This is due to
the high water content and low interfacial tension of the system which will protect
the protein. The polymers themselves may also have a stabilizing effect. ATPS has
a unique phase diagram under a particular set of conditions such as pH and tempera-
ture [10]. The phase diagram provides information about concentration of phase
forming components required to form a two-phase, the concentration of phase com-
ponents in the top and bottom phases, and the ratio of phase volumes. In Fig. 5.1 [9],
the binodal curve TCB divides a region of component concentrations that will form
two immiscible aqueous phases (above the curve) from those that will form one
phase (below the curve). The three systems X, Y and Z differ in their initial com-
positions and in the volume ratios. However, they all have the same top phase equi-
librium composition (TPEG, TSalt) and the same bottom phase equilibrium composi-
tion (BPEG, BSalt). This is because they are lying on the same tie-line (TB), whose
end points determine the equilibrium phase compositions and lie in a convex curve
called as the binodal curve. This curve represents the separation between the two
immiscible phases. The binodal data is required for the design of ATPS extraction
processes and development of models that predict partitioning of any biomolecules.
72 F. Yusof
Fig. 5.1 Binodal curve. In the figure, TCB = Binodal Curve, C = critical point, TB = Tie line,
T = Composition of the top phase, B = Composition of the bottom phase, and X, Y and Z = Total
composition of ATPS [9]
The tie line length (TLL) has units of %w/w, same as the component concentra-
tions. The length of the tie line is related to the mass of the phases by the equation:
Vt ρt XB
=
Vb ρb XT (5.2)
where V and ρ are the volumes and densities of the top ( t) and bottom ( b) phases and XB
and XT are the segment lengths of the tie line as shown in Fig. 5.1. TLL and the slope
of the tie-line (STL) can be related to the equilibrium phase composition as follows:
TLL = [B salt − Tsalt ] + [TPEG − BPEG ]
2 2
(5.3)
Tie lines are commonly parallel and hence the STL can be calculated by the follow-
ing formula thus facilitating the construction of further tie lines.
STL =
[T PEG − BPEG ]
=
∆PEG
[B salt − Tsalt ] ∆salt (5.4)
As tie-lines decrease in length, they ultimately approach a critical point (C) on the
binodal curve, where the TLL = 0. At this point the composition and volume of the
two phases theoretically become equal.
A protein interacts with the surrounding molecules with a phase via various
bonds, such as hydrogen, ionic and hydrophobic interactions, together with other
weak forces. The net effect of these interactions is likely to be different in the two
phases and therefore the protein will partition into one phase. The following prop-
erties of partitioning can be exploited individually or in conjunction to achieve an
effective separation of particular protein [33].
• Hydrophobicity, where the hydrophobic properties of a phase system are used
for separation according to the hydrophobicity of the protein.
• Electrochemical, where the electrical potential between the phases is used to
separate the molecules or particles according to their charge.
• Size-dependent partitioning where molecular size of the proteins or the surface
area of the molecules (protein) or particles is the dominating factor.
• Biospecific affinity, where the affinity between sits on the proteins and ligands
attached to one of the phase polymers is exploited for separation.
• Conformation-dependent, where the conformation of the proteins is the deter-
mining factor.
Partition coefficients (K) of biomolecule are important in the design of an extrac-
tion process employing ATPS. Several approaches have been explored to assess
the most important parameters determining partitioning behavior using simplified
expressions obtained by grouping the various contributing factors. According to
Albertsson, [34], the partition coefficient K is a function of several interacting prop-
erties and can be expressed by the equation:
K = K o ∗ K elec ∗ K hphob ∗ K size ∗ K conf (5.5)
where subscripts ‘elec’, ‘hfob’, ‘size’ and ‘conf’ refer to the electrochemical, hy-
drophobic, size, and K includes other environmental factors such as salt type and
concentration, pH and temperature. Some of these factors are discussed in the fol-
lowing section.
5.6.3.1 Molecular Weight of Polymer
The molecular weight (MW) of the polymer used influences the partitioning of
proteins [33]. The higher the molecular weight of the polymers, the lower is the
polymer concentration required for phase separation. As polymer concentration in-
creases, differences in density, refractive index, and viscosity between the phases
increase. Binodal curves shift towards the origin with the increase in PEG molar
mass. In PEG/Salt system, the partitioning of biomolecules is governed by volume
exclusion effect (polymer-rich) and salting-out effect (salt-rich). The systems with
high concentration or high molecular weight polymer and high salt concentration
will result in partitioning of biomolecules at the inter phase due to the influence of
both volume exclusion and salting out effect. In PEG/Salt systems, the increase in
K may be because of the following:
• If the MW of PEG is lower, the interfacial tension is lower between the two
phases which increases K.
74 F. Yusof
• If salt concentration is high, the ionic strength increases in the bottom phase
which improves biomolecule partition to the top phase.
• If the PEG concentration is high, number of polymer units involved in the bio-
molecular partitioning also increases and hence more biomolecules partition into
the PEG phase due to hydrophobic interaction between the biomolecule and PEG.
5.6.3.2 pH
The pH of the system affects the partitioning because it may alter the charge of
the solute or it may alter the ratio of the charged molecules [33]. The net charge of
the protein depends on whether the pH is greater than pI (negative), lesser than pI
(positive), or equal to pI (zero). Several researchers reported that at higher pH, the
negatively charged biomolecule prefers the top phase and partition coefficient in-
creases. It may be because of the electrostatic interactions between the biomolecule
and PEG units. Moreover, the change in pH affects the phase composition which in
turn affects the partitioning behavior. The two phase area expands with an increase
in temperature and pH. The binodal curves become more asymmetric and close to
origin with an increase in molecular weight.
5.6.3.3 Presence of Neutral Salts
The presence of neutral salts such as NaCl does not drastically affect the liquid-
liquid equilibrium data of ATPS [33]. But high salt concentration (greater than 1M)
alters the phase diagram. The presence of NaCl in ATPS alters partition coefficient
because of the differential distribution of the salt ions between the phases. The add-
ed salt contains ions with different hydrophobicities. The hydrophobic ions force
the partitioning of their counter ions to the more hydrophobic phase and vice versa.
The salting-out effect forces the biomolecules to move from salt-rich phase to the
PEG-rich phase.
5.6.3.4 Surface Properties of Biomolecules
A linear relationship was developed between the hydrophobicity of the proteins and
partition coefficient by:
 P
log K = Rlog   (5.6)
 Po 
where P is the protein hydrophobicity in solution measured by precipitation and log
Po represents the intrinsic hydrophobicity of the given ATPS. The surface charges
of proteins play a major role in partition coefficient. Most proteins have a large
number of charged groups with different pK values. At the interface of the two
phases the different affinities of the salt ions results in an electrical potential differ-
ence. Albertsson [34] derived the following thermodynamic principles relating the
partitioning of salt ions and protein partitioning in ATPS:
 F 
InK p = InK po + InK elec = InK po +  Z p
 RT  ψ (5.7)

where, ψ is the interfacial potential and is given by,
RT − K
ψ= In (5.8)
(Z +
+Z −
)F K+
where, Zp = the net charge of the protein of interest and Z + & Z − are the number of
net charges of the cations and anions. K + and K- are the charge-independent parti-
tion coefficients of the cation and anion of the salt. Kp is the partition coefficient
of the protein, R, the gas constant, T, the absolute temperature, and F, the Faraday
constant. Kpo is the partition coefficient of the protein in the system at zero interfa-
cial potential i.e. Zp is zero. There is no ψ when the salt ions have the same charge-
independent partition coefficients and, under these conditions, Kp is the same as Kpo.
Generally the higher the molecular weight of the proteins, the lower the con-
centration needed for the formation of two phases, and the larger the difference in
molecular weights between the polymers, the more asymmetrical is the curve of the
phase diagram. Also the larger the molecular weight of the PEG, the lower the value
of K. The practical application of ATPS has been demonstrated in many cases in-
cluding a number of industrial applications with excellent levels of purity and yield.
5.7 Purification Process Scale-up
The term ‘scale-up’ is used to describe a transition of size, volume or output for any
given process, or sequence of operational steps. It is generally used when transfer-
ring a process from the development to pilot or manufacturing scale but it can be
equally applied to any change that is intended to increase the quantity of the final
product produced in a given time frame. The requirement of process scale up is to
allow more product to be made in any given time period, the simplest way is to in-
crease the output. This can be achieved by automation of the laboratory techniques
or process or increasing the equipment number [3]: Both ways have pros and cons.
The process of automation in most laboratories can simply be achieved by using
computer controlled equipment. The later can be achieved provided the equipment
used are identical to the original operations but the system is limited in that there is
very little improvement in process scale economies. However, automation and in-
creasing equipment numbers can be combined to yield increased throughput many
times the original capacity of a laboratory scale process.
The first step in scale-up for any protein purification process is to determine the
target of the scale up. The various techniques that are available at laboratory scale
have several analog or direct equivalents at larger scale and the selection is depen-
dent on the goal and the scale of operation. Table 5.2 provides the list of common
laboratory scale techniques and some analogs for these larger scales.
76 F. Yusof
Table 5.2 List of laboratory Lab-scale techniques Larger Scale Analogs

scale techniques and its larger
scale analogs Ultracentrifugation Low speed centrifugation
Depth filtration
Membrane filtration
Protein Concentration Cross Flow filtration
Liquid-liquid extraction
Stirred cell system
Precipitation (PEG or salt)
Dialysis (buffer exchange) Size-exclusion chromatography
Tangential flow filtration
Column chromatography Column chromatography
Batch adsorption
Filter chromatography
In scaling up process from laboratory scale to larger scale, it is common to expe-

rience product loss and such losses can happen during the following steps:
• During clarification and concentration and losses can be the result of binding of
product to filter media, as a result from shear in pumping/separation system, incom-
plete product recovery from raw material and solubility effects in two-phase systems.
• During column chromatography and losses can be due to incomplete column
equilibration, elution or washing, overloading of product, incompatible buffer
composition for product elution, resin fouling, product aggregation, incompat-
ible flow rate distribution along the column system and dilution of product.
• During ultrafiltration/diafiltration and normal flow filtration and losses can be as a re-
sult of binding of product to filter media, as a result from shear in pumping/separation
systems, incomplete mixing in retentate vessels or product aggregation or precipitation.
The techniques required for scale-up of a purification process are varied and in
some cases, conflicting. There is a need to balance all the conflicting choices and
options to provide the most cost-effective, efficient method of production in a short
as possible timescales. Although scientifically it can be modelled and predicted,
there is a tendency when it will fall foul of the fiscal demands placed on the system.
The processes involved need to be continuously improved and renewed to gain that
extra percent recovery or reduce operating time and cost.
5.8 Problems in Industrial Purification of Recombinant

Protein
Efficient strategies for the production and the downstream purification of recom-
binant proteins are gaining increasing importance, as more applications that re-
quire high amount of high-quality proteins reach the market. Higher production
efficiencies and, consequently, lower cost of the final product are needed for ob-
taining a commercially viable process. Many of the problems faced in large scale
industrial purification steps is normally not a problem when it was first developed at
the laboratory scales [3]. Some of the issues are discussed in the following sections.
5.8.1 Temperature
This is often neglected process parameter in the scale-up of chromatography, and it

represents a true variable in making life difficult at the large scale, especially larger
than 10 L. Temperature can dramatically affects the viscosity of buffer and process
solutions to the point that processes become inoperable because of excessive pres-
sure drops at even moderately larger scales than laboratory column. Incorporation
of temperature controlled chromatography (such as jacketed columns) will incur
cost to the final process.
5.8.2 Mobile Phase Degassing
Dissolved gasses in the running buffer can seriously interfere with the chromato-
graphic separation. At the laboratory scale, these dissolved gasses are easily re-
moved by vacuum degassing with filtration. If degassing is not performed well,
bubbles can be observed to accumulate in the column body, usually at the column
wall and may, if unchecked over time, result in channeling and poor column flow
dynamics. In larger scale, such as more than 10 L, degassing with vacuum filtration
is becoming unpractical. Other alternative ways need to be carried out to overcome
this problem, for example, the effect of gassing may be minimized by allowing buf-
fer to reach the same temperature as the column prior to loading.
5.8.3 Product Loss
This is a common phenomenon in any exercise of up-scaling process [3]. All puri-
fication processes involve a certain loss in product at each step. It is normal not to
expect 100 % recovery, for example, at the ultrafiltration, column chromatography
and so on. The total of these losses is actually the product of them all and so each
step should be investigated for the reason of decreased recovery. When addressing
issues of recovery, the four questions should be asked are:
• Is the product truly lost or are assay variation affecting measurement? Different
assay techniques are sometimes used at different phase of downstream process-
ing to assess product recovery.
• Is the lost product of equivalent quality to the desired product? If isomers are
involved, characteristics should be assessed before returning the product to the
pool.
78 F. Yusof
• Where in the process is the loss occurring? It is better to focus on the latest steps
that incur the largest loss in a process to provide yield improvement.
• What process validation is required prior to implementing the changes? It is
often required to address the impact of process changes first, with scaled-down
experiments, before implementing in scale-up processes.
Only when these questions can be answered satisfactorily, then the processes can
be investigated and made in the scale-up to yield and improve processes. The steps
and the ways in which product can be lost has been given in the previous section
(Section 5.7).
5.9 Conclusion
Laboratory scale and industrial scale protein purification exploit the same separation
principles, but methods developed in the laboratory may not always directly appli-
cable to large scale or industrial processes. Industrial production typically demands
extreme focus on end product quality and adherence to regulatory requirements
that are easily overlooked in laboratory scale separations. Efficient development
of industrial purification makes demands on throughput. The cost of purification at
industrial scale is of major concern. The prospect of using an expensive buffer or
other media is more daunting if we are talking about 10,000 L of the stuff compared
to one or two L at the laboratory scale processes. Anything that can be done in the
laboratory can be carried out at large scale, but it is a matter of time, cost and some-
times environmental/disposal considerations. There are various techniques that are
conducted at laboratory scale that have several analogs or direct equivalent at larger
scale. The selection and choice of the up-scaling techniques is dependent on the
type of protein and desired purity or specific activity. In order to obtain a commer-
cially viable process, the production has to reach a high level of efficiency to lower
the cost of the final product.
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Chapter 6
Recombinant-Enzyme Fermentation
Abstract This chapter presents an overview of the issues involved in the fermenta-
tion of E. coli harboring a transgene. The successful production of a heterologous
enzyme requires the thoughtful integration and optimization of media formula-
tions, physical factors associated with fermentation, host specificities and induction
strategies.
Keywords Aeration rate · ANOVA · Carbon flux · Dissolved oxygen · Fermentation ·

Metabolic loads · Plackett-Burman design · Plasmid stability · Response surface design
6.1 Introduction
The production of a recombinant enzyme through fermentation of recombinant E.

coli harboring the appropriate transgene requires the detailed design of each pro-
cessing unit. The processing unit involves fermentation, cell disruption, purifica-
tion and product formulation. Fermentation is the first step in recombinant-enzyme
production and is critical because it determines productivity. Many factors affect
fermentation, including media formulation and the physical properties of the strain
used to express the recombinant enzyme. Different host strains require different
media compositions and fermentation conditions and detailed experiments were de-
signed to identify optimal conditions. Normally, these experiments start with media
formulation.
A. Amid () · M. J. A. Jamaluddin
M. J. A. Jamaluddin
N. A. Ismail
University Technology Mara, Campus Puncak Alam, Shah Alam, Selangor, Malaysia
DOI 10.1007/978-3-319-12397-4_6
82 A. Amid et al.
6.2 Media Formulation
Successful medium formulation requires knowledge of the main factors that sup-
port bacteria growth. The media must be formulated carefully because they may
have significant effects on cell biomass and recombinant-enzyme production [1].
There are two main types of media: chemically defined growth media and complex
growth media. A chemically defined growth medium is a medium for which the
exact chemical composition is known, while complex growth media are formulated
by adding nutrients such as yeast, and meat or plant extract, so the exact chemical
composition varies slightly between batches [2]. Slower growth and low protein
production are normally observed in chemically defined media [3]. Commonly, the
main required growth factors are carbon, nitrogen, sulfur, phosphorus, vitamins and
other organic growth factors. Researchers may begin with any medium to determine
the type that most suitable for biomass and recombinant-enzyme production. Ex-
amples of media tested in this chapter include Luria broth (LB), terrific Broth (TB),
2x yeast extract and tryptone (YT), auto-induction broth, super broth (SB), and TY
broth. The detailed composition of each medium is listed in Table 6.1. Researchers
are advised to formulate media using three main steps: (1) screening of important
component, (2) identification of suitable concentration ranges of each component
and (3) optimization of the new formulation. However, preliminary experiments
must be conducted for each product to identify the most important factors for pro-
duction. For example, the effect of growth medium on biomass and recombinant-
bromelain production is shown in Fig. 6.1 (A and B). For this recombinant enzyme,
the auto-induction medium maximizes biomass and recombinant-enzyme produc-
tion. Carbon, nitrogen and other required elements are all present in concentrations
sufficient to support cell growth and expression of recombinant bromelain. There-
fore, the next optimization experiment should focus on auto-induction media.
6.2.1 Screening of Important Media Components
Traditionally, to increase both cell density and productivity, growth was optimized
by optimizing growth medium composition, physical parameters and induction con-
ditions [4, 5], but recent aims have included the optimization of critical nutrients
for growth, prevention of product degradation and decreasing the production of
toxic products [6]. As auto-induction media are complex, an experiment is needed
to select the most important elements. There are few examples of media optimiza-
tion studies in the literature [7–9]. Among the many suitable designs for screening
of media the most common one is the Plackett-Burman design. This design is used
to identify and improve the factors that affect a process or product. It is suitable for
ruggedness testing in which the study hopes to find little or no effect on the response
(Deign Expert software, Stat-Ease, Inc. Minneapolis). An example of such a design
and data used to screen for important media components is shown in Tables 6.2, 6.3
6 Recombinant-Enzyme Fermentation 83
Table 6.1 Compositions of different media

LB TB 2x YT SB TY Autoinduction
1 % peptone 1.2 % peptone 1.6 % 3.2 % 8 % tryptone 2.5 % tryptone
peptone peptone
0.5 % yeast 2.4 % yeast 1 % yeast 2 % yeast 5 % yeast 1.25 % yeast
extract extract extract extract extract extract
1 % NaCl 72 mM K2HPO4 0.5 % NaCls 0.5 % NaCl 2.5 % NaCl 37.5 mM
Na2HPO4
17 mM KH2PO4 37.5 % KH2PO4
0.4 % glycerol 7.5 mM NH2Cl
8 mM Na2SO4
1.25 % glycerol
0.13 % glucose
0.1 lactose
0.1 L-arabinose
1 mM MgSo4
5 µM FeCl2
2 µM CaCl2
1 µM MnCl2
1 µM ZnSO4
0.2 µM CoCl2
0.2 µM CuCl2
0.2 µM NiCl2
0.2 µM Na2MoO4
0.2 µM Na2SeO2
0.2 µM H3BO3
Fig. 6.1 a Effect of medium type on the biomass of recombinant E.coli, b Effect of medium type
on the specific activity of recombinant bromelain
84
Table 6.2 27 Runs of experiments designed by DesignExpert software to screen 23 parameters using a Plackett-Burman design
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A. Amid et al.
Table 6.3 Results of screening of significant parameters in the formulation of a medium used
to culture E. coli harboring recombinant bromelain. The enzyme specific activity is the response
variable
Run Enzyme specific activity (U/mg)
1 1.68 ± 0.0603
2 1.35 ± 0.0389
3 3.45 ± 0.0675
4 1.18 ± 0.0515
5 1.69 ± 0.0660
6 0.78 ± 0.0752
7 2.78 ± 0.0794
8 1.74 ± 0.1452
9 3.27 ± 0.1695
10 2.35 ± 0.0516
11 1.14 ± 0.0553
12 2.28 ± 0.0403
13 1.82 ± 0.0630
14 1.89 ± 0.1414
15 1.53 ± 0.1071
16 1.43 ± 0.0789
17 3.41 ± 0.0659
18 3.66 ± 0.1058
19 1.57 ± 0.0403
20 1.75 ± 0.0763
21 1.14 ± 0.0539
22 1.65 ± 0.0459
23 1.73 ± 0.0451
24 0.60 ± 0.0342
25 2.06 ± 0.0421
26 1.41 ± 0.0913
27 0.70 ± 0.0358
and 6.4 Researchers should pay attention to significant model terms, R2, predicted
R2 and adjusted R2 (Table 6.4) in optimizing the new formulation.
The example shown in Table 6.2 reveals that maximum production of recombi-
nant bromelain is in the range of 3.2–3.6 U/mg and that the most significant param-
eters are tryptone, NH4Cl and CoCl 2 (Fig. 6.2). Therefore, an experiment using
an OFAT design should be carried out to find a suitable range to optimize concen-
tration of these components. Based on the result seen in Fig. 6.3 A, B and C, the
optimization experiments should test the following concentration range: tryptone
(4–16 %), NH4Cl (100–260 mM) and CoCl2 (0.4–1.2 µM), with the other param-
eter held constant as shown in Table 6.1.
86 A. Amid et al.
Table 6.4 Results of the ANOVA test used to identify a significant model and parameters for
growth medium formulation
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the red line. Tryptone is the most significant factor
6.2.2 Optimization of Media Formulation
Response surface (RS) design is the most suitable method to optimize performance
[7, 8]. Therefore, 20 experimental runs were designed to optimize media formula-
tion based on 3 parameters with 3 levels using RS design. The details of the experi-
mental design and results are shown in Table 6.4. The highest specific recombinant-
enzyme activity (in this case, bromelain) was observed in medium with 16 % tryp-
tone, 180 mM NH4Cl and CoCl2 added Table 6.5.
6.2.3 Summary of Media Formulation Strategy
Based on the above discussion, the media-formulation strategies can be summa-

rized into the flow chart below (Fig. 6.4); however, this chart only outlines a general
strategy. Normally, the strategy for each recombinant enzyme will be unique and
based on the expression host (Fig. 6.4).
88 A. Amid et al.
Fig. 6.3 a result from experiment using OFAT design to identify the suitable range for tryptone; b
result from experiment using OFAT design to identify the suitable range for NH4Cl; c result from
experiment using OFAT design to identify the suitable range for CoCl2
Table 6.5 Results from an experiment designed using the RS method under center-composite
design to optimize media formulation. The center-point experiments for this design are highlighted
in yellow
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6.2.4 Fermentation Physical Factors
The parameters commonly tested to optimize recombinant-enzyme activity include

temperature, agitation speed and dissolved oxygen.
6.2.5 Effect of Temperature on Recombinant-Enzyme Specific

Activity
Two types of temperature determine the specific activity of a recombinant enzyme

which are; (1) culture temperature and (2) post-induction temperature. Culture tem-
perature is usually determined by the expression host. Most E. coli strain grow well
at 37 °C, while different Bacillus spp. grow well at 37 °C or at 35 °C [10]. Therefore,
researchers must identify a suitable culture temperature for each host. To obtain
soluble and active protein, low temperatures are recommended during and after in-
duction because lower rates of protein production allow newly transcribed recombi-
nant proteins time to fold properly. An example of the effect of post-induction tem-
perature on a recombinant enzyme is shown in Fig. 6.5. The target protein achieved
its highest specific activity (1.65 ± 0.13 U/mg) at a post-induction temperature of
25 °C. The specific activity increased as the post-induction temperature was raised
from 20 to 25 °C but declined from 30 to 37 °C.
6.2.6 Effect of Dissolved Oxygen on Recombinant-Enzyme

Production
According to Tripathy et al. [1], fluctuations in oxygen content during fermenta-

tion can cause oxidative stress limiting amino acid production and causing plasmid
90 A. Amid et al.
Fig. 6.5 The highest specific activity is observed at a post-induction temperature of 25 °C
instability and oxidation of proteins. These affects reduce the quality of the recom-
binant protein. The aeration rate and agitation speed directly affect the dissolved
oxygen content. An increase in agitation speed will increase the dissolved oxygen
content of the medium. For recombinant bromelain expressed by E. coli BL21AI,
35 % dissolved oxygen is the optimal for good cell growth and high specific activity
(Table 6.6).
6.2.7 Effect of Induction Time (Cell Concentration)

on Recombinant-Enzyme Production
In most cases, the expression of recombinant protein imposes a metabolic burden

that results in a reduced growth rate and cell density [11]. When induction occurs
very early in fermentation, low cell density and low productivity result. Therefore,
one of the main factors affecting success in recombinant-enzyme production is op-
timization of incubation time. In the case of recombinant bromelain, the gene was
inserted into pDES17, with L-arabinose as the inducer. Productivity is sensitive to
the timing of L-arabinose addition [12]. Figure 6.6 describes the result of the induc-
tion-time experiments. The activity of recombinant bromelain at different induc-
tion times varied widely, from 0.6 to 1.2 U/mg. The maximum amount of recombi-
nant bromelain was obtained when the induction was carried out at approximately
OD600 nm of 0.6, suggesting that induction during the middle log phase (during
maximum growth) is optimal for recombinant-bromelain production.
Table 6.6 Design and results of experiments to optimize conditions for fermentation in a bioreactor
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6.2.8 O
ptimizing Physical Parameter For Recombinant Enzyme
Specific Activity.
After an optimal medium has been identified, the next step in pre-commercialization
of a recombinant enzyme involves optimization of fermentation. As the ultimate
goal is commercialization, growth in a bioreactor is an appropriate choice because
conditions in bioreactor can be controlled and monitored throughout fermentation.
The common parameters tested for optimum fermentation in bioreactors include
dissolved oxygen, pH, aeration and temperature reduction after induction. To fa-
cilitate optimization full factorial design should be carefully implemented (Design-
Expert, Stat-Ease, Inc., Minneapolis) to ensure that enough experiments will be
carried out. The experimental design to optimize the fermentation of recombinant
bromelain involved 19 runs. The results were used to estimate the effects of each
factor and their interactions. Example results are shown in Table 6.6. Run 14 (DO—
35 %; aeration—0.5 vvm; pH—7.4; temperature reduction—30 °C) produced the
92 A. Amid et al.
Fig. 6.6 Effect of different cell concentration on the specific activity of bromelain produced by
E. coli BL21-AI
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Fig. 6.7 Half-normal plot to select significant factors
highest specific activity at 0.187 U/mg-protein. Centre point replications (3 runs

17–19, DO—25 %; aeration—1.25 vvm; pH—7.0; temperature reduction—25 °C)
show consistent values for specific activity, indicating good reproducibility. The
half-normal plots analysis in Fig. 6.7 indicates the significant factors and interac-
tions. Temperature reduction was the most significant factor, followed by the in-
teraction between dissolved oxygen and pH. Aeration and dissolved oxygen, were
ranked third and fourth, respectively, in significance
6.3 Challenges in E. coli Fermentation for Recombinant-

Enzyme Production in a Bioreactor
The challenges that affect the production of recombinant enzyme in a bioreactor

include plasmid stability, metabolic loads and fermentation respiration. Plasmid
stability is related to the agitation speed, and the concentrations of carbon sources
supplied in the fermentation medium affect metabolic loads and fermentative res-
piration.
6.3.1 Plasmid Stability
In the large-scale production of recombinant enzyme, high-copy number DNA plas-

mids in E. coli are needed to produce high levels of the desired product. The relative
growth rates of plasmid-harboring and plasmid-free cells are the most important
factors in the design of a bioreactor-fermentation strategy for recombinant organ-
isms [13]. Generally, plasmid–harboring cells grow slower than plasmid-free cells
because the former must synthesize more protein. Therefore, stable maintenance of
a recombinant plasmid in a growing cell, requires that the rate of replication of the
plasmid be concordant with the growth rate of the cell and that plasmid segregate
equally between daughter cells [11, 14].
Normally, a system for E. coli growth will force the maximum number of bacte-
ria to retain the plasmid but keep the copy number low to restrict energy and nutri-
ent consumption for plasmid maintenance. Several approaches have been tested to
ensure these results. The incorporation of an antibiotic as selective agent for plas-
mid instability is one common strategy, thus, bacteria that do not carry the plasmid
are not able to survive in a selective medium [15].
Several systems use auxotrophic markers based on complementation of a muta-
tion or deletion in the host chromosome for stable plasmid selection based on au-
totrophies in the biosynthetic pathway for the production of a heterologous protein,
as in Lactococcus lactis [16]; however, many auxotrophic strains show low growth
rates, and thus are not well suited to commercial production.
There is some evidence that defined media support higher plasmid titers and in
particular tend to reduce segregation-rate-associated plasmid loss, while complex
media seem to reduce growth-rate-associated plasmid instability [17, 18]; however,
an increase in plasmid titer not generally observed during slow growth. Instead,
titer depends strongly on the method used to decrease the growth, such as nutrient
limitation [19] or exploitation of pH or temperature effects. As shown in Fig. 6.8,
an E. coli culture carrying pDEST17/Bromelain has good plasmid stability until 10
hours of cultivation, even in a complex medium.
Another strategy is the operator-repressor titration system reported by Garmory
and co-workers [20], which utilizes E. coli strains carrying genes essential for host
survival under the control of the lac operator or promoter system. This system allows
the growth of the modified host strain only when the lac promoter is induced by the
94 A. Amid et al.
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Fig. 6.8 The pDEST17/Bromelain plasmid is stably maintained throughout fermentation
addition of β-galactose or IPTG or when the cell is transformed with a multi-copy

plasmid containing the short lac-operator sequence. This system maintains plasmid
stability at high cell densities and during expression of a recombinant protein [21].
6.3.2 Metabolic Loads
The term “metabolic burden” is defined as the amount of resources in term of raw
materials and energy that are taken from the host-cell metabolism for the mainte-
nance and replication of foreign DNA [22]. According to the review by Carneiro
et al. [23] of Glick works [24], the most important effects of metabolic stress are
growth arrest and reduced protein synthesis, but many other effects have been de-
scribed including the accumulation of undesired by-products such as acetate [25].
During high cell-density culture, growth-inhibiting metabolites such as acetate are
commonly problematic. Thus, many attempts have been made to avoid accumula-
tion of acetate in high cell-density cultures [26, 27]. Glucose is the usual carbon
source for E. coli grown for the production of foreign proteins; however, growth of
E. coli in excess glucose may cause the accumulation of acetate when the carbon
flux through glycolysis exceeds the capacity of the tricarboxylic acid (TCA) cycle
[28]. The physiological background for acetate formation is the result of imbalance
between carbon flux into the central metabolic pathway and biosynthetic demands
within the cell [29].
Many strategies have been tested to limit acetate [26, 30, 31] including replace-
ment of glucose by other carbon sources, such as lactose [32], which also serves as
an inducer. Lactose shows some similarities to IPTGs, including rapid protein ex-
pression, lower metabolic burden, and decline in the specific growth rate; however,
that same study also revealed that under conditions of poor oxygen supply, glycerol
and lactose concentrations can be reduced. These results suggest that media refor-
mulation alone is not sufficient; physical conditions must also be optimized.
6.3.3 Fermentative Respiration
The dissolved-oxygen (DO) concentration becomes a rate-limiting factor in many

aerobic fermentation processes due to poor solubility of the oxygen in the cultivat-
ing medium or high oxygen-uptake rate (OUR) in fast growing microorganisms.
The oxygen-transfer rate (OTR) is the most important parameter. This factor de-
pends on the agitation speed and aeration rate in bioreactors and plays a significant
role in determining the productivity of fermentation [33, 34]. The OTR can be af-
fected by several factor, such as the geometry and characteristics of the vessel,
liquid properties, energy dissipation in the fluid, biocatalyst properties, medium
concentration and microorganism morphology [35].
Oxygen transfer into the cells during aerobic fermentation strongly affects prod-
uct formation by influencing metabolic pathways and changing metabolic fluxes
[36]. Insufficient oxygen supply results in the excretion of several metabolites
from mixed-acid metabolism, such as succinate, formate, acetate, lactate, ethanol
and hydrogen gas [37]. Excretion of such metabolic by-products is undesirable, as
the productivity of the bioreactor and the production of the recombinant protein
might be affected [37–40]. The accumulation of acetate under aerobic conditions
generally occurs under high growth rates and/or low oxygen concentrations [41].
As discussed in the previous section, over-loading the tricarboxylic acid (TCA)
cycle by rapid metabolic flux through glycolysis is the primary cause of acetate
accumulation.
Carbon dioxide is also reported to cause growth inhibition and toxic effects [42,
43], but species vary in sensitivity. At elevated CO2 concentrations, carboxylation
alters metabolite pools and amino-acid production. Previous efforts to use oxygen-
enriched air for penicillin production have failed due to increased carbon dioxide
concentrations [44]. E. coli is less sensitive than molds to carbon dioxide concentra-
tion [45]. The impact of elevated oxygen and carbon dioxide concentration on E.
coli metabolism and production rate must be investigated for each product. Previous
results suggest that proper mixing is crucial for maximum production of the micro-
bial product and biomass. Mixing could be achieved by optimizing the aeration and
agitation rates for better oxygen transfer and product formation [36, 46].
96 A. Amid et al.
Fig. 6.9 Recombinant bromelain concentration is reduced when speed is increased, suggesting
that E. coli might experience shear stress at high mixing speed
Most previous studies showed that the optimum oxygen-transfer rate can be
determined by adjusting the rate of agitation in the bioreactor. Agitation rate is a
crucial parameter for proper oxygen transfer (through air-bubble mixing) and ho-
mogeneous mixing of nutrients during fermentation [47]. Higher agitation rates
(600–700 rpm) might reduce cell growth and productivity, by causing mechanical
damage to microbial cells through high sheer stress; such damage would ultimately
affect the protease yield (Fig. 6.9). Higher aeration rates also inhibit cell growth by
creating air-dispersion problems in the fermentation system, which, in turn, affect
the cell biomass in the broth. Low agitation speeds, however, may cause improper
mixing of nutrients, resulting in decreased cell biomass and productivity [36, 46].
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Chapter 7
Scaling-Up Recombinant Enzyme Fermentation
Azlin Suhaida Azmi, Sarina Sulaiman, Nor Fadhillah Mohamed Amin

and Fathilah Ali
Abstract Bioreactor or fermenter is the heart of a process. Scaling up a bioreactor

and maintaining the same optimized process in a recombinant enzyme production in a
higher scale is an engineering challenge. Scale-up is about mixing and mass transfer,
a combination of art and science. It is more to methodological approach with some
combination of rules of thumb, experience and/or trial and error. This chapter presented
several bioreactor or fermenter modes and types before encompasses four different
scale-up strategies. The scale-up strategies related to mass transfer involving constants
scale-up of; (i) volumetric transfer coefficient ( Kla), (ii) power input per liquid volume
( P/V), (iii) impeller tip speed ( Vi), and (iv) mixing time ( tm) are described and discussed.
Keywords Airlift bioreactor · Bubble column · Fluidized bed bioreactor · Impeller

tip speed · Mixing time · Power input per liquid volume · Stirred tank bioreactor
7.1 Introduction
The bioreactor is the heart of a fermentation process. Anything coming out of it will
subsequently pass to next unit operation. The bioreactor governs the product quality
throughout the process lines and dictates the production cost. Thus, it is important
A. S. Azmi () · N. F. M. Amin · F. Ali

e-mail: azlinsu76@iium.edu.my
N. F. M. Amin
e-mail: norfadhillah@iium.edu.my
F. Ali
e-mail: fathilah@iium.edu.my
S. Sulaiman
Department of Biotechnology Engineering, Bioenvironmental Engineering Research Centre,
Faculty of Engineering, International Islamic University Malaysia, P.O. Box 10, 50728 Kuala
Lumpur, Malaysia
e-mail: sarina@iium.edu.my
A. Amid (ed.), Recombinant Enzymes—From Basic Science to Commercialization,
DOI 10.1007/978-3-319-12397-4_7
100 A. S. Azmi et al.
to optimize the production yield in the bioreactor. Once optimized process param-
eters in a bench-scale bioreactor are obtained, a scale-up is required for larger scale
production to maintain an almost identical environment as achieved during the op-
timized small-scale production process.
Scale-up is an engineering challenge to increase the power and magnitude from
laboratory scale to pilot scale and from pilot scale to industrial scale. It is about mix-
ing and mass transfer, a combination of art and science. Two rules of thumb in the
scaling-up procedure are usually followed: (i) geometry and configuration similar-
ity of fermenter and (ii) minimum of three or four stages of increment in volumes.
In biotechnological processes, the typical scale-up size is ten increment stages.
However, five increment stages are also used to reduce the risk of the unexpected
results or fermenter performance. Typical production volumes from recombinant E.
coli and yeast cultures with high specific productivities are usually not more than
5000 L [1].
The process of scaling up recombinant enzyme production is essential for higher
scale production. The objective of scaling up is to develop the same fermentation
efficiency, maintaining similar product yield and ensure consistent product quality
as obtained in the small-scale fermenter at attractive economical values. Scaling-
up from a procedure to process requires a well-planned strategy. Several scale-up
parameters have been described and used with varying degrees of success. A com-
bination of different methods generally yields a good result [2]. There are some
parameters that have proven their usefulness in scale-up procedures: geometric
similarity, impeller design, tip speed, power input volume (P/V), gassing strategy
and KLa value.
This chapter discusses the types of bioreactors, along with their advantages and
disadvantages. Four scale-up strategies related to mass transfer are described and
also discussed.
7.2 Bioreactor Types
The choice of suitable bioreactor is the major step in the scaling-up process. A
bioreactor is the heart of any biochemical process in which the desired products are
produced by living microorganisms such as recombinant enzymes. The microor-
ganism in the bioreactor will attempt to adapt to the environment for their optimal
growth to produce metabolites. Thus, the main task of the bioreactor is to ensure the
means of growth are well supplied [3]. A good bioreactor performance is of para-
mount importance in determining the economic viability of the overall design and
fundamentally important to the environmental impact of the process [4].
There are several modes and types of bioreactors available for recombinant
enzyme production. The fermentation can be performed in batch, fed-batch or
7 Scaling-Up Recombinant Enzyme Fermentation 101
continuous mode bioreactors. Batch bioreactors or fermenters are closed systems

in which the contents are added at the beginning of operation and contain initial,
limited nutrient amounts. The contents are subject to perfect mixing for a cer-
tain period, after which the product is discharged [4]. Several advantages can be
obtained from batch operation. It is easy to operate and has closer control over
bacterial and wild yeasts contamination, which can invite host problems and sub-
sequently affect productivity [5, 6]. Furthermore, adequate knowledge related to
the design and operation of large-scale batch bioreactors are available, which has
made the measurement and control of such systems much easier and also more
manageable compared with other types of operations. Because of these reasons,
many manufacturers and researchers select batch operations when dealing with
submerged fermentation [5].
Semi-batch or fed-batch operations are an intermediary operation mode between
batch and continuous fermentation. In this operation mode, the nutrient is fed in-
termittently or continuously to supplement the reactor contents and provide control
over the substrate concentration [7]. If the substrate (with or without nutrient) is
added, it is fed at, or over, particular intervals of time during the operation, and the
product is withdrawn only at the end of the batch [8].
The advantages of using fed-batch operation are the same as batch operation. In
addition, excessive substrate feed, which inhibits microorganism growth, can be
avoided in this case. The sterility can be maintained as products from both opera-
tions are withdrawn at the end of the process. The disadvantages of batch opera-
tion also apply to fed-batch. In addition, both of the modes are personnel intensive
because of the need to sterilize the equipment for every batch. Furthermore, there
are difficulties in operation and control such as maintaining the specific growth
conditions for the microorganism in the face of variations in medium properties,
the batch-to-batch system variability, and the inherent nonlinear dynamic nature of
fed-batch bioreactors [8].
Continuous fermentation provides advantages over batch fermentations, in-
cluding optimized process conditions for maximal product productivity, long-term
continuous productivity, higher volumetric productivity, reduced labor costs once
steady state is reached, reduced vessel down time for cleaning, filling and sanitiz-
ing, and easier process control and operation than batch fermentation during steady
state operation [9]. Unfortunately, continuous fermentations are more susceptible
to long-term bacteriological problems, as they do not allow more frequent cleaning
because of their continuous operational nature [5, 9]. The addition of antibiotics
to address the problem incurs more cost to the process and can result in mutation.
Recombinant enzymes are generally overproduced by two group of species:
bacteria (i.e., Escherichia coli) and yeast (i.e., Pichia pastoris and Saccharomyces
cerevisiae). These microorganisms require oxygen usually from the air. An aerobic
bioreactor is normally used for the high recombinant cell densities that are expected
Table 7.1 Types of bioreactors used for production of recombinant proteins from E. coli
Type of bioreactor Capacity(L) Microorganism Production Reference
Batch run-bubble column 60 Recombinant Fusion protein [11]
Fed batch run-airlift tower E. coli
loop reactor
pH-stat fed batch control 2, 20, 200 Recombinant Recombinant [12]
E. coli protein vaccine
Stirred tank reactor 1 Recombinant Recombinant [13]
E. coli aspartase
Bioreactor (Virtis) equipped 1.5 Recombinant Penicillin [14]
with two six-blade Rushton E. coli acylase
turbines
Rocking-motion-type 1 E.coli Recombinant [15]
bioreactors protein
Fed-batch reactor 5, 70 Recombinant CYP3A4 [16]
E. coli enzyme
to provide high levels of enzyme production. In general, depending on how the gas
is distributed, an aerobic bioreactor can be classified into following categories:
a. Stirred tank bioreactor—Most common type of bioreactor using mechanical
agitation.
b. Bubble column—Cylindrical vessels with height greater than twice the diameter.
Compressed air is sparging into the column. Column normally has no internal
structure.
c. Airlift bioreactor—A pneumatic reactor without any mechanical stirring. The
gas is circulated by means of pressurized air/ liquid in upward motion and the
results of circulatory flow in the entire reactor.
d. Fluidized bed bioreactor—A packed bed column operated in upflow mode.
Table 7.1 lists different types of biorecators used for production of recombinant
proteins from E. coli. Bubble column, stirred-tank, fed-batch and air-lift bioreactors
are commonly applied for microbial fermentation, specifically recombinant produc-
tion. There are also other types of bioreactors, e.g., rocking-motion-type bioreac-
tors. Common conditions for choosing a suitable bioreactor and bioreactor design
must consider low shear stress to cells, acceptable oxygen mass transfer to cells,
adequate mass transfer between nutrient supply to cells and product and byproduct
removal from cells [10].
7.3 Scale-up Strategies
There are several methods or strategies that can be applied to fermentation process.
However in this chapter only four strategies are described and discussed. This in-
volve scale-up of following constants;
i. Volumetric transfer coefficient (KLa)

ii. Power input per liquid volume (P/V)
iii. Impeller tip speed (Vi)
iv. Mixing time (tm).
7.3.1 Volumetric Transfer Coefficient (KLa)
Cells in aerobic culture take up oxygen from liquid medium, but often, the oxygen
supply is insufficient and becomes the limiting factor. This is due to low solubility
in the medium. The rate of oxygen transfer from the gas to liquid phase is governed
by the size of the air-liquid interface, its resistance to mass transfer and residence
time of air bubble distribute in fermenter. These factors, in turn, depend on the gas
flow rate and agitation characteristics. The volumetric mass transfer coefficient,
kLa, is mostly used for scaling-up the fermenter to establish its aeration efficiency
and quantify the effects of operating variables on oxygen supply.
The oxygen transfer rate (OTR) from air bubbles to liquid phase in fermenter/
bioreactor can be described by Eq. (7.1).

dCL (7.1)
= k L a (CL* − CL )
dt
where dCL/dt is the change in oxygen concentration over a period of time (i.e., the
OT); CL is the concentration of dissolved oxygen in the fermentation broth; kL is the
mass transfer coefficient; a is the gas/liquid interface area per liquid volume, and C*
is the saturated dissolved oxygen concentration.
Mass transfer coefficient, kL, is the sum of the reciprocal of resistances in liquid
phase and gas phase mass transfer as shown in Eq. (7.2).
1 1 1
= + (7.2)
k L HK g K L
where H is the Henry’s law constant; Kg and KL are the local mass transfer coeffi-
cient of gas and liquid, respectively. Taking into consideration that oxygen is slight-
ly insoluble, the rate of diffusion is controlled in the liquid phase, and consequently,
H is large (i.e., 4.2 × 104 bar mol−1 fraction for the solution of oxygen in water).
Thus, the overall mass transfer coefficient is equal to the local coefficient of kL = KL.
It is difficult to assess kL and a separately. Thus, the combination of both terms, kLa,
is always measured together, and the term is called volumetric mass-transfer coef-
ficient. The coefficient indicates the aeration capacity of the fermenter. The larger
the value is, the higher the aeration capacity of the system is.
Several factors affect kLa value, including the air-flow rate employed in the ves-
sel, degree of agitation, impeller design, rheological of fermentation broth and the
presence of antifoam. Several methods are used to determine the coefficient value
chemically and physically and have been discussed by many researchers [17]. The
methods can be divided into chemical and physical methods. Chemicals methods
include the sodium sulfite oxidation method and absorption of CO2 [17]. Physical
methods include the static gassing-out method and dynamic gassing out method.
Physical methods are mostly adopted for determining the bioreactor kLa value and
subsequent use in scaling-up because chemical methods change the physiochemical
properties of the liquid by the addition of chemicals and indicate higher kLa values
than the true value.
Of the two primary physical methods, dynamic out-gassing is the most suit-
able during fermentation containing respiratory activity of a growing culture in a
fermenter. In the presence of respiring organisms, the unsteady state of the mass
balance in the liquid phase can be described as in Eq. (7.3).
O2 transferred from gas phase—O2 consumed by cells = accumulation of O2 in
liquid.
(7.3) dCL
k L a (CL* − CL )·V − qo 2 X ·V = ·V
dt
The equation can be simplified as Eq. (7.4) at constant liquid phase volume.

dCL (7.4)
= k L a (CL* − CL ) − qo 2 X
dt
where X is the biomass concentration, and qo2 is the specific oxygen consumption
rate (mmols O2/g biomass·s). Considering final steady-state of dissolved oxygen
where CL = CL , and dCL/dt = 0, Eq. (7.4) reduces to Eq. (7.5):
 _

k L a  CL* − C L  = qo 2 X (7.5)
 
where CL is the final steady-state oxygen concentration at the time of the experi-
ment. Substituting Eq. (7.5) into Eq. (7.4) results in Eq. (7.6):

dC AL
dt
( ) (
= k L a C *L − CL − k L a C *L − C L ) (7.6)
Equation (7.6) can be rearranged to the following Eq. (7.7):

dC AL
dt
(
= k L a CL − CL ) (7.7)
Integrating Eq. (7.7)

 C −C 
L1
ln  L  = k L a (t2 − t1 ) (7.8)
 C − C 
L L2
Table 7.2 Agitation rates and corresponding values of volumetric oxygen transfer coefficient
( KLa) at constant aeration of 2.5 vvm [20]
Agitation rate (rpm) KLa (h−1) (Uninduced) KLa (h−1) (Induced)
250 42 48
500 56 65
650 59 71
750 61 84
1000 84 104
1200 102 128
 C −C 
L1
Therefore, a plot of ln  L  vs t should result in a straight line of slope kLa.
 C − C 
L L2
In this method, aeration efficiency is usually measured in the optimized produc-
tion small-scale system. At larger scale, the optimized conditions, such as tempera-
ture, pH and media formulation, are maintained similar to that at the small scale,
and the higher-scale aeration-agitation is varied to meet the same or at least be in the
range of aeration efficiency as the small-scale process.
As explained earlier, the kLa value is the measurement of aeration capacity and a
yardstick of fermenter performance [18]. Bhattacharya et al. [19] found that higher
aeration capacity is required by transformed or recombinant E. coli (58.2 h−1) com-
pared with untransformed E. coli (56.4 h−1) in the production of penicillin acylase.
In addition, the production of recombinant enzyme requires an induction at the right
time, and this increases the oxygen demand. Fermenter systems having induced
microorganisms require more aeration capacity than those containing uninduced
organisms, as shown in Table 7.2. The table shows the KLa values at different agita-
tion rates for uninduced and induced systems produced by Bhattacharya and Dubey
[20]. They observed that a significant drop of dissolved oxygen occurred after in-
duction of culture was introduced, implying that oxygen demand and consumption
were higher and affected the cell growth and target gene expression. However, this
is not the case in a Kapat et al. [21] study. The production of glucose oxidase by
recombinant S. cerevisiae by Kapat et al. [21] demonstrates that the production of
glucose oxidase by recombinant S. cerevisiae does not depend on kLa. Instead, a
combination of impeller and aeration are the two important parameters in the en-
hancement of specific glucose oxidase production.
Mel et al. [22], in their study on the effect of air flow rate and agitation on the
kLa value, obtained a maximum cell density at an optimum kLa of 1.686 h−1. It is
considered a very low oxygen requirement for recombinant E. coli when producing
β-glucuronidase enzyme. In a scale-up study conducted by Amid [23], they success-
fully scaled-up production of recombinant bromelain from a 2 to 30 L bioreactor
using a constant kLa method at range of 75.6–120.6 h−1 at a specific growth rate of
0.29 h−1 and specific enzyme activity of 0.174 U/mg. Another large-scale produc-
tion study of recombinant Streptoverticillium platensis transglutaminase was con-
ducted by Lin et al. [24] in 5, 30 and 250 L fermenters. The scale-up from 5 to 30 L
resulted in higher enzyme activity; however, the activity was decreased at the 250 L
scale. They found that dissolved oxygen is the limitation to the low activity of the
recombinant enzyme at their largest scale. In a scale-up study conducted by Anane
et al. [25], they successfully scaled-up α-gluconidase expressed by recombinant
S. cerevisiae using a fed-batch bioreactor. The scale-up was performed in 100 L
based on a constant kLa value of 102.34 h−1 from an optimized 14 L bioreactor at a
specific growth rate of 0.12 h−1.
7.3.2 Constant Power Consumption per Unit Volume (P/V)
During fermentation, various physical, biochemical and physical reactions take

place in the medium. The nature and composition of the fermentation medium will
affect the efficiency of the fermentation process. The mass transfer is complex and
requires agitation to maintain uniform conditions within the fermenter. Greater
mass transfer requires a higher degree of agitation; however, this will result in high-
er power consumption and shear rate.
The ungassed power input, Po, is expressed as
(7.9)
Po = N P N 3 Di5 ρ
The power number, NP, is the ratio of the external force to the inertial force exerted
by the fluid. It is based on the property of the fluid and geometry parameters of the
impeller, Di. N and ρ are the agitation rate and broth density, respectively. If the
same impeller type is used, NP can be considered as constant and is represented in
Eq. (7.10):
(7.10)
N P = C1 Re x Fr y
where C1 is a dimensionless shape factor that represents the structural properties of

the system, whereas the Reynolds number, Re, and the Froude number, Fr, are each
defined as in Eqs. (7.11) and (7.12), respectively:
(7.11)
Fr = N 2 D / g
Re = ρ ND 2 / µ
(7.12)
For vessel, i, the constant ungassed power input per liquid volume, ( P0 / VL )i , is
defined as
( P0 / VL ) 2 / ( P0 / VL )1 = (VL1 / VL 2 )c or P0 / VL = f1VLc (7.13)
Based on a survey of industrial plants of various scales and processes, c is equal to

− 0.37 [26]. When the scaling up is performed on the basis of the constant P0 / VL , it
Table 7.3 Value of exponent Pg/V [28, 29]

Scale (L) X y
5 0.95 0.67
500 0.67 0.67
23,000–50,000 0.50 0.50
can result in a larger motor size. At a large scale, it is hard to achieve high power per
unit volume because of the practical limitations in motor size. Note that for certain
type of enzymes, a different scale-up approach is needed. For example, a constant
Pg / VL is not useful in the scale-up of toyocamycin production by shear-sensitive
Streptomyces chrestomyceticus mutants [27].
Scaling up on the basis of constant power input per unit volume for ungassed and
gassed system can also be reduced to Eq. (7.14).

V2  N 23 DT5 2  (7.14)
=
V1  N13 DT51 
For geometrically similar vessels,

1/ 3
DT 2  V2  (7.15)
=
DT 1  V1 
Substituting for DT2/DT1 in Eq. (7.14) and solving for N2 and simplifies into

2/9 1/3
N2 V  V  (7.16)
= T1 =  T1 
N1  VT 2  V  T2
Equation (7.13) can also be applied for gassed power inputs per unit liquid vol-
ume, Pg / VL . However, gassed systems differ slightly from the ungassed systems in
that the sparged bubbles reduce density and thereby decrease power consumption.
This results in a lower gassed power, Pg, compared with ungassed power, Po. There
are correlations that relate power consumption, kLa and gas velocity, as given in
Eq. (7.17):
x
 Pg 
k L a = C   vsy (7.17)
V 
where C is a constant; Pg/V is the impeller passed power per unit volume; vs is
the superficial gas velocity, and x and y are exponents. The exponential constants
have been reported to have a dependent correlation with vessel size, as shown in
Table 7.3.
For highly viscous fermentation broths, viscosity is introduced in Eq. (7.17) as

shown in Eq. (7.18) [30]:
x
 Pg  (7.18)
k L a = C   vsy η − o.86
V 
7.3.3 Tip Speed
A constant impeller tip speed is often used as a scale-up criterion in viscous mycelia
fermentations because of the shear sensitivity of the used micro-organisms [30].
The tip speed produced by the stirrer is important in bioprocess because it deter-
mines the maximum shear stress in the tank, determining the possible cell damage,
and also influences the stable size of flocks and gas bubbles [17]. The tip speed is
related to the shear rate produced by the impellers moving through the cell culture
media.
Usually during the scaling-up of the bioreactor, the impeller tip speed is main-
tained at a constant value. This is supported by Imamoglu and Sukan [31], who
stated that scale-up could be conducted based on a constant impeller tip speed when
working with recombinant organisms. However, scale-up based on a constant mix-
ing time or tip speed usually has a higher success rate when the scale-up factor is
small (4–40 L, scale-up factor 10).
The tip speed is calculated based on equations below [1, 16, 32, 33]:
where
(7.19)
Vi1 = Vi 2
where Vi is impeller tip speed and subscript 1 and 2, respectively, refer to small and
large scales.
(7.20)
Vi1 = π NDi1
So substitute Eq. (7.20) to (7.19) obtains the following:

(7.21)
π N1 Di1 = π N 2 Di 2
Rearranging Eq. (7.21), the following is obtained:

D 
N 2 = N1  i1  (7.22)
 Di 2 
Equation (7.22) also could be written as follows:

1/3
N 2  Di1   V1 
= =  (7.23)
N1  Di 2  V2 
Table 7.4 Effects of tip speed in the scaling up of production

Production Microorganism Tip speed, m/s Content/yield Reference
Cyanophycin Recombinant Strains 1.3–5.0 30 L [35]
of E. coli 21–24 % (wt/wt)
500 L
21 % (wt/wt)
Enterokinase Recombinant S. Constant tip 5 L [34]
cerevisiae speed 3.8 mg/l
30 L
3.7 mg/l
300 L
3.5 mg/l
CYP3A4 Recombinant E.coli 7.065 Shake flask [16]
enzyme 80 %
5 L-not stated
70 L- not stated
Vaccine Any microorganism 4.36 20 L [32]
100 %
4.39–4.95 200 L
25–50 %
where
N2 agitation speed in scale-up
N1 agitation speed in scale-down
Di1 impeller diameter of scale-down
Di2 impeller diameter of scale-up
Equation (7.23) is similar to Eq. (7.15) derived for constant power input per liquid
volume. Table 7.4 describes the tip speed used, scale-up yield and microorganism
used. The scaling up of enterokinase fermentation using recombinant S. cerevisiae
from a 5 L jar to a 300 L jar was successfully achieved by maintaining constant im-
peller tip speed and a minimum DOT level at 60 % or more [34]. The yield produced
3.8, 3.7 and 3.5 mg/L for 5, 30 and 300 L reveals that there were no significant
changes during the scale-up process. Dubey et al. [16] reported that during scale-up
from 5 to 30 L for the production of 3-DCM by recombinant E. coli, an intermedi-
ate tip speed at 7.065 m s−1 obtains the maximum yield compared with the higher
tip speed. Lee [32] reported that for vaccine production, the yield achieved at the
production scale, 200 L (25–50 %), is only one-third of that compared with pilot
scale, 20 L (100 %). They concluded that the oxygen transfer within the production
fermenter may be the limiting factor.
Frey et al. [35] reported that by using a tip speed between 1.3–5.0 m s−1, the yield
of cyanophycin achieved was 21–24 and 21 % (wt/wt) for 30 and 500 L, respective-
ly. They also stated that for a 500-L scale, the airflow was kept at 0.15 vvm to avoid
foam formation and stirrer speed has to be adjusted manually to avoid insufficient
supply of oxygen at the exponential growth phase.
A rough rule of thumb is that cell damage can occur at tip speeds above 3.2 m/s,
but the exact value is influenced by many factors such as broth rheology [1, 32, 34,
36, 37]. Junker [26] also reported that calculated tip speeds usually are greater than
3 m s−1 for production fermenters, in the range of 5–7 m s−1 for scale-up of antibi-
otic fermentations and ranged from 5–7 m s−1 according to a survey of industrial
fermenters.
The maximum shear occurs in a fermenter at the tip of the impellers. The cell can
be physically damaged if the impeller speed is too fast, and low speeds may not mix
the broth well. Thus, it is essential to use a reasonable impeller tip speed throughout
the fermentation process. Therefore, scale-up based on a constant impeller tip speed
is capable of yielding reproducible results at different scales.
7.3.4 Mixing Time (tm)
Another factor that can affect the scaling-up process is mixing time. For geometri-
cally similar vessels in the region of turbulent flows [29], the following is true:

tm N 2/3 Di−1/6 = constant (7.24)
During scale-up, on the basis of constant mixing time, Eq. (7.24) then becomes

tm1 N12/3 Di−11/6 = tm 2 N 22/3 Di−21/6 (7.25)
Solving Eq. (7.25) at constant mixing time gives Eq. (7.26):

1/ 4
 
N 2 = N1  D 2  (7.26)
 D1
The corresponding power input thus given by Eq. (7.27):

(
( P0 / VL ) 2 / ( P0 / VL )1 = N 23 D22 / N1 D12 ) (7.27)
Mixing time in stirred tank depends on vessel size, impeller, broth viscosity and stir
speed. In larger vessels, the mixing time increases because of the larger volumes to
be stirred, which cannot be ignored. Therefore, in increased volumes, the mixing
time can be kept constant by increasing the stirrer speeds and energy inputs. During
scale-up based on constant mixing time, the power consumption per unit volume
will increase proportionally to the scale. During scale-up, the maximum shear rate
is increased (28 %) at constant P/V and constant Re number, which is not preferred
during the scale-up. This is because of the low value of P/V ( P/V = 0.05), which
will cause improper mixing. Moreover, high shear stress during mixing could cause
cell damage and unstable gas bubble concentrations. However, decreases of oxygen
transport are also caused by reduced of power input per unit volume and stirrer
speed.
Cascaval has reported that the mixing time increased at the scale of 2.4–4.3 in
4–40 L bioreactors [37]. In their work, they used aerobic stirred reactor using yeasts
( S. cerevisiae) and Penicillium chrysogenum free mycelia and pellets. They report-
ed that in a large bioreactor, the mixing time differs at different positions in the
vessel because of the distances of the stirrer in the bioreactor. Whereas, the mixing
time for cell cultures used in recombinant protein production displayed an increase
in mixing time from 5 to 10 s at a 10 L scale to 60 s at a 100 L scale [38]. In the
scale up of pneumocandin production from the filamentous fungus Glarea lozoyen-
sis from pilot scale to production scale (57 m3), the mixing time at production scale
was faster than at pilot scale because of the large liquid height/tank diameter ratio
[39]. These studies indicate that mixing time is among the crucial parameters for
the successful scale-up of recombinant enzymes and thus should not be ignored or
eliminated during the process.
Nomenclature
a gas/liquid interface area per liquid volume (cm2/cm3)
C constant
C1 constant dependent on vessel geometry
C* saturated dissolved oxygen concentration (mmoles O2 dm−3)
CL concentration of dissolved oxygen in fermentation broth (mmoles O2 dm−3)
dCL/dt change in oxygen concentration over a period of time (mmoles O2 dm−3 h−1).
Di impeller diameter (m)
Di1 impeller diameter of scale—down (m)
Di2 impeller diameter of scale-up (m)
Fr Froude number
kL mass transfer coefficient (cm/h)
N agitation rate, rev/min (rpm)
N1 agitation speed in scale-down, rev/min (rpm)
N2 agitation speed in scale-up, rev/min (rpm)
NP power number
P power consumption (watt or hp)
Pg gassed power consumption (watt or hp)
Po ungassed power consumption (watt or hp)
qo2 specific oxygen consumption rate (mmols O2/ g biomass·s)
Re Reynolds number
t time (h)
tm mixing time (h)
VL liquid volume (L or m3)
V1 vessel volume in scale-down (L or m3)
V2 vessel volume in scale-up (L or m3)
Vs superficial velocity (m/h)
X biomass concentration (g/mL)
x exponent
y exponent
η viscosity (g/cm-s or poise)
ρ broth density (g/cm3)
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Chapter 8
Downstream Processing of Recombinant
Enzymes for Commercialization
Fadzilah Adibah Abdul Majid
Abstract Recovering recombinant enzyme from the production system is a chal-

lenging task. A series of unit operations are needed to ensure the recovery of the
enzyme and of higher purity. The commercialized processes involved should be
optimized for higher yield and quality and at the same time adopt a simplified and
cost effective processing systems. Once the manufacturing facility being endorsed
by the regulatory authorities, the operation must adhere to the already approved pro-
cessing systems. Any in house process optimizations are usually confined to physi-
cochemical parameters of the systems. Future downstream processing research is
focusing on innovative approaches such as integration of the physical processing
systems, modification of cellular host systems, as well as optimization of physi-
cochemical parameters and upstream. In this chapter, the conventional and basic
unit operations as well as the innovative approaches for downstream processing are
explained and discussed.
Keywords Centrifugation · Crystallization · Depyrogenation · Desiccation ·

Downstream processing · Filtration · Flocculation · Lyophilization · Polishing ·
Precipitation · Settling
8.1 Introduction
Downstream processing is an essential processing procedure in the recovery and

purification of proteins from various biological sources, including recombinant en-
zymes. This processing section requires several stages to facilitate the recovery of
the final protein. In general, the unit operations involved are solid-liquid separation
and concentration. Many different approaches, including mechanical design and
physicochemical optimizations, are applied to each unit operation in downstream
processing to facilitate protein recovery. The level of purity and activity of the pro-
tein are also determined during downstream processing. Furthermore, the creative
F. A. A. Majid ()
Department of Bioprocess Engineering, Faculty of Chemical Engineering, University
Technology Malaysia, Johor, Malaysia
e-mail: adibah@cheme.utm.my
DOI 10.1007/978-3-319-12397-4_8
116 F. A. A. Majid
and innovative design of recombinant proteins in suitable hosts through genetic and
protein engineering offer potential ease of protein recovery in downstream process-
ing as well as selection of simple medium, which could offer significant economical
alternatives. The commercialization process demands intensive communication and
teamwork between the scientists and engineers to develop an optimum and work-
able protein recovery process. This chapter will review the development in the area
of downstream processing of recombinant enzymes in view of future commercial-
ization. The recombinant enzyme, as mentioned in the title, will be represented by
the more general term ‘protein’.
8.2 The Sources of Recombinant Enzymes
The advancement in biotechnology has resulted in many biological compounds

being produced outside their original sources. Many biopharmaceuticals, once
isolated and extracted from plants and animals, are now being produced in mi-
croorganisms. The most common example is the insulin previously extracted from
animals for human usage is now is being produced in recombinant bacterial system,
whereas animal or plant sources vary from lot to lot depending on nutrient or other
environmental factors, recombinant microorganisms offer high reproducibility with
minimal production variation with strict nutrient and environmental control. En-
zymes or other proteins, once extracted from higher organisms such as animals
and plants, are now able to be produced by recombinant microorganisms, mainly
bacteria ( Escherichia coli), yeast ( Saccharomyces cerevisiae) and mammalian cells
(CHO, hybridoma).
The DNA recombinant technology, as well as fermentation process, are well
developed, thus allowing us to simply produce different proteins through gene
transfer in different host systems, especially the microorganisms. Many commer-
cial enzymes available in the market today are being produced in recombinant mi-
croorganisms, such as chymosin in non-pathogenic strains of E. coli (K-12) [1],
thermo-stable alpha-amylase in Pichia pastoris [2], and lipase in Aspergillus ni-
ger[3]. The enzymes produced through fermentation of the recombinant hosts must
be separated from the fermentation broth and purified for further use. A unique sys-
tem combining several unit operations to separate and purify the enzymes from the
broth is called downstream processing. The processing stages and equipment used
in downstream processing are discussed in the following sections.
8.3 The Process Stages in Downstream Processing
The stages involved in downstream processing depend on the sources of the re-
combinant enzymes. In general, four steps are involved as described below http://
en.wikipedia.org/wiki/Downstream_processing:
8 Downstream Processing of Recombinant Enzymes for Commercialization 117
8.3.1 Removal of Insoluble Material
Prior to the separation step, the enzyme of interest must be made soluble in the
clear broth. If the enzyme is secreted outside the host cell, any insoluble material,
including the cells in the broth must be discarded. If the enzyme remains in the host
cells after fermentation is completed, cells must be separated from the broth and
subsequently broken up to release the enzyme. Then, the enzyme in the solute will
be separated from the cell debris and other insoluble matters. Typical unit opera-
tions involved are filtration, sedimentation, centrifugation, flocculation, precipita-
tion or gravity settling. For cell break up, additional operations include grinding or
homogenization prior to separation of the insoluble material in the homogenized
broth using any of the above mentioned unit operations.
8.3.2 Product Isolation
The main component in clear broth containing the enzyme of interest is water. This
element needs to be removed. In doing so, the crude enzyme becomes more con-
centrated. The typical unit operations involved are absorption, ultrafiltration and
precipitation.
8.3.3 Product Purification
This stage separates the enzyme of interest from the contaminants in the concen-
trated broth. Most of the contaminants could potentially have physical and chemical
properties that closely resemble those of the enzyme of interest. More sophisti-
cated, sensitive and expensive equipment are needed to purify the enzyme of in-
terest at this stage. The typical unit operations involved include chromatographic
techniques, especially affinity, size exclusion and reverse phase, depending on the
nature of the enzyme with respect to the contaminants. Fractional precipitation and
crystallization can also be considered at this stage.
8.3.4 Product Polishing
Polishing is the final step in downstream processing of biologics prior to packaging.

Polishing will make the product more stable, easy to handle and transportable. De-
pending on the end user and costumer requirement, purified enzymes will be dried
into powder form using a non-destructive method or dissolved in an appropriate
buffer at high concentration. Lyophilization, crystallization and desiccation are the
proposed unit operations at the polishing stage. If the enzyme is required to be free
118 F. A. A. Majid
from any possible contaminations, such as viruses and bacteria, depyrogenation or

non-destructive sterilization is used, respectively.
Downstream processing is facilitated with series of equipment. The main equip-
ment used in this solid liquid separation stages is discussed in the next section.
8.4 Equipment for Recombinant Enzyme Concentration

and Purification
The equipment used in downstream processing depends on the processing stage.

Simple centrifugation or membrane filtration should be able to separate the insol-
uble material in the suspension broth. This section will divide the equipment use
depending on the processing stages. The same unit operation may be used in differ-
ent processing stages. The chromatographic method can be used in protein isolation
as well as polishing. Centrifugation is used in separation of insoluble material as
well as in protein isolation, where it is needed to sediment the insoluble precipitate.
8.4.1 Unit Operations for Separation of Insoluble Material
The separation of insoluble material in the broth involves unit operations such fil-
tration, sedimentation, centrifugation, flocculation, precipitation or gravity settling.
a. Filtration (http://en.wikipedia.org/wiki/Filtration) is a common mechanical or
physical operation used for the separation of insoluble from fluids. A porous
membrane is used to separate insoluble material from the liquid. Insoluble mate-
rial of a larger size than the membrane pores will be retained, and the filtrate will
pass through. The membrane material can be made from cellulose acetate, glass
fiber fleeces or polyurethane. The pore size can be as small as 0.2 μm, which is
usually used to sterilize liquid. The recovery of the insoluble or liquid is depen-
dent on where the protein of interest resides.
b. Sedimentation (http://en.wikipedia.org/wiki/Sedimentation) is the process to
allow the insoluble material in the suspension to settle out of the fluid. Insoluble
material will settle through gravitational force in the bottom of the container that
holds the suspension. Centrifugal and electromagnetism forces are other possible
forces acting on the insoluble materials forcing them to settle. The equipment
for sedimentation is generally a holding tank or the bioreactor itself. Once the
insoluble material is totally settled, the supernatant can be withdrawn for the
next operation.
c. Centrifugation (http://en.wikipedia.org/wiki/Centrifugation) is a process that
involves the use of the centrifugal force to sediment the insoluble material in the
suspension. Centrifuges are used in laboratories or industry to assist insoluble
material separation. Similar methods can be used to separate two immiscible liq-
uids. The concepts rely on the migration of more-dense insoluble material away
from the axis of the centrifuge, whereas the less-dense insoluble material in the
mixture migrates toward the axis. Precipitated insoluble material (aka the pellet)
will be gathered at the bottom of the centrifuge tube, and the supernatant can be
easily decanted without disturbing the pellet. Again, the collection of the pellet
or supernatant depends on the presence of the protein of interest.
d. Flocculation (http://en.wikipedia.org/wiki/Flocculation) is a process where
insoluble material merely suspended in the liquid separates out to form flocs or
flakes. The process can be either spontaneous or due to the addition of a clarify-
ing agent. A holding tank can be used for the flocculation process.
e. Precipitation (http://en.wikipedia.org/wiki/Precipitation_(chemistry)) is the pro-
cess in which a solid is form in a solution containing insoluble material. The
precipitant (chemical) is used to cause precipitation of the insoluble material.
Precipitates (solid) will remain in suspension until another separation process is
applied. In this case, centrifugation, filtration or sedimentation could be applied
to separate the precipitates from the suspension. The precipitate free liquid can
be easily separated once the precipitate is turned into a pellet. A holding tank
with a stirrer is need for the precipitation process. If the precipitation requires
low temperature and extended time to complete, a refrigeration system is needed,
or the unit can be placed into cold room.
f. Settling (http://en.wikipedia.org/wiki/Settling) is the process by which insoluble
material settles to the bottom of liquid and forms sediment. Insoluble material
that experiences a force, either due to gravity or due to centrifugal motion, will
tend to move in a uniform manner in the direction exerted by that force. For
gravity settling, this means that the insoluble material will tend to fall to the bot-
tom of the vessel, forming a slurry at the vessel base. Settling by gravity is time
consuming. Thus, sterilization can be an issue. This operation can be performed
in sterile holding tank with no agitation needed.
8.4.2 Unit operations for Protein Isolation
Protein isolation involves the use of a single or several types of equipment for ab-
sorption, ultrafiltration and precipitation purposes. Its main function is to reduce the
amount of water and concentrate the protein of interest. A popular and commonly
used type of equipment is the ultrafiltration system.
a. Ultrafiltration (UF) (http://en.wikipedia.org/wiki/Ultrafiltration) is a process in
which clear suspension containing suspended solids such as protein of interest
will be passed through a series of semipermeable membranes with the aid of
forces such as pressure or concentration gradients. Suspended solids and solutes
of high molecular weight are retained in the so-called ‘retentate’, whereas water
and low molecular weight solutes pass through the membrane in the ‘perme-
ate.’ This separation process is used in industry and research for purifying and
concentrating macromolecular (103–106 Da) solutions, especially protein solu-
tions. Ultrafiltration is not much different from microfiltration, nanofiltration or
120 F. A. A. Majid
membrane gas separation, except in terms of the size of the molecules it retains.
The size of molecules retained is dependent on the Molecular Weight Cut Off
(MWCO) of the membrane used. Two modes of ultrafiltration commonly used
are cross-flow or dead-end.
b. Precipitation (http://en.wikipedia.org/wiki/Precipitation_(chemistry)) (refer
8.4.1 above) is often used to isolate the insoluble protein in the clear broth.
Chemical methods, such as ammonium sulfate precipitation, are commonly
used. The size of protein being precipitated depends on the concentration of the
chemical added to the clear broth. Temperature, agitation and time will also con-
tribute to the amount of protein being precipitated. This process will require the
centrifugation to facilitate the separation of the precipitate from the liquid.
8.4.3 Unit Operations for Protein Purification
In the protein purification sections, the typical unit operations involved are chro-
matographic techniques, especially affinity, size exclusion and reverse phase. In
some other situations, fractional precipitation and crystallization are used. Many
manufacturers produce various types of columns for the purification of specific
proteins.
a. Affinity chromatography (http://en.wikipedia.org/wiki/Affinity_chromatography)
is a method of separating protein mixtures based on a highly specific interaction.
This method works if the protein to be separated has a strong interaction with the
other protein embedded to the column bed, for example, antigen and antibody
binding, enzyme and substrate binding, or receptor and ligand binding. Protein A
with strong affinity to antibody is usually used to bind immunoglobulin G (IgG) in
the supernatant. Affinity chromatography can be used to
1. Purify and concentrate a substance from a mixture into a buffering solution
2. Reduce the amount of a substance in a mixture
3. Discern which biological compounds bind to a particular substance
4. Purify and concentrate an enzyme
b. Size-Exclusion Chromatography (SEC) (http://en.wikipedia.org/wiki/Size-
exclusion_chromatography) is the separation technique based on the molecu-
lar size of the components. Separation is achieved by the differential exclusion
from the pores of the packing material of the sample molecules as they pass
through a bed of porous particles. The principle feature of SEC is its gentle
non-adsorptive interaction with the sample, enabling high retention of bio-
molecular activity. Size-exclusion chromatography is used primarily for ana-
lytical assays and semi-preparative purifications. It is not commonly used
for process scale work because of to the low capacity of the size exclusion
technique.
c. Reversed-phase chromatography (RPC) (http://en.wikipedia.org/wiki/Reversed-
phase_chromatography), sometimes called hydrophobic chromatography, is
a liquid chromatography technique that uses a hydrophobic stationary phase.

Thus, the hydrophilic molecules in the mobile phase tend to bind to the column,
whereas the hydrophobic molecules pass through the column and are eluted first.
Hydrophobic molecules can be eluted from the column by decreasing the polarity
of the mobile phase using an organic (non-polar) solvent, which reduces hydro-
phobic interactions. The more hydrophobic the molecule is, the more strongly
it will bind to the stationary phase, and the higher the concentration of organic
solvent that will be required to elute the molecule.
d. Fractional precipitation (http://www.rpi.edu/dept/chem-eng/Biotech-Environ/
PRECIP/precpph.html) can be achieved by varying the pH of the medium. At
low pH values, proteins have a net positive charge because the amide gains an
extra proton. At high pH values, proteins have a net negative charge because
of the carboxyl on the protein backbone losing its proton. At their pI value, a
protein has no net charge. This leads to reduced solubility because the protein
is unable to interact with the medium, and thus it will then fall out of solution.
The solubility of a particular protein in aqueous solution depends on the solvent
composition and on the pH. Thus, variation in these parameters provides a way
of purifying proteins by fractional precipitation.
8.4.4 Unit Operations in Protein Polishing
The final form of the protein products depends on end users, storage conditions
and mode of transportation. Product polishing techniques, such as lyophilization,
crystallization and desiccation, are employed to achieve the final form of the prod-
uct. When higher quality is required for the final product, depyrogenation or non-
destructive sterilization are used for viral and bacterial elimination. Proteins are
heat sensitive. Thus, drying or sterilization of protein requires non-destructive tech-
niques. Freeze drying is commonly used to dry proteins without compromising their
activity.
a. Lyophilization (http://en.wikipedia.org/wiki/Freeze-drying) is a process in which
water is extracted from the solution leaving the protein dry. The protein can then
be stored at room temperature (ambient air temperature). Lyophilization is con-
ducted using a simple principle of physics called sublimation. Sublimation is the
transition of a substance from the solid to the vapor state without first passing
through an intermediate liquid phase. To eliminate water from solution, it has
to be first frozen and under a vacuum. The ice is sublimated directly into water
vapor and released, leaving freeze-dried protein in concentrated form.
b. Crystallization (http://en.wikipedia.org/wiki/Protein_crystallization)—Protein
crystallization is the process of formation of a protein crystal. Two of the most
commonly used methods for protein crystallization fall under the category of
vapor diffusion known as the hanging drop and sitting drop methods. Most
biological macromolecules such as proteins can be prompted to form crystals
when the solution in which they are dissolved becomes supersaturated. Some of
122 F. A. A. Majid
the factors influencing the formation of protein crystals include protein purity,
pH, concentration of protein, temperature, precipitants and additives. The more
homogenous a protein in solution is, the better the chances are for it to form a
crystal. Typical standards require the protein solution to be at least 97 % pure.
pH conditions are very important because of the fact that different pH values
can result in different packing orientations.Buffers, such as Tris-HCl, are often
necessary for the maintenance of a particular pH.Precipitants, such as ammo-
nium sulfate or polyethylene glycol, are compounds that cause the protein to
precipitate out of solution.
c. Desiccation [(http://en.wikipedia.org/wiki/Desiccation)] is the state of extreme
dryness or the process of extreme drying. A desiccant is a hygroscopic (attracts
and holds water) substance that induces or sustains such a state in its local vicin-
ity in a moderately sealed container.
d. Depyrogenation (http://en.wikipedia.org/wiki/Depyrogenation) refers to the
removal of pyrogens from solution, most commonly from injectable pharmaceu-
ticals. Pyrogens can often be difficult to remove from solutions because of the
high variability of their molecular weight. Pyrogens are also relatively thermally
stable and insensitive to pH changes. However, several removal techniques exist,
including ion-exchange chromatography, ultrafiltration and distillation
e. Sterilization (http://en.wikipedia.org/wiki/Sterilization_(microbiology)) is a term
referring to any process that eliminates (removes) or kills all forms of microbial
life, including transmissible agents (such as fungi, bacteria, viruses, spore forms,
etc.) present in the polished products.Sterilization can be achieved by applying
heat, chemicals, irradiation, high pressure, and filtration or combinations thereof.
The term has evolved to include the destruction of infectious proteins such as
prions related to Transmissible Spongiform Encephalopathies (TSE).
There are many innovative approaches in downstream processing to save time and
reduce costs. For example, affinity chromatography can be applied to purify as well
as to polish the enzyme simultaneously. Many researchers are interested in finding
the fastest, simplest and cost effective ways to separate and purify proteins right
from the early design of the host metabolic and excreting pathways and the up-
stream processing section. Several innovative approaches for effective downstream
processing are discussed in the following sections.
8.5 Continuous Innovations in Downstream Processing
Physical, chemical, enzymatic, and physicochemical approaches are continuously

being adopted in the recovery process of biological products, including recombi-
nant enzymes. Many research teams are working on different approaches to ensure
high yield and quality of the end product recovered. When recovering the intra-
cellular enzymes trapped in the cell body, mechanical and enzyme methods are
commonly used to disrupt cell membrane. Homogenizers are one type of common
Table 8.1 Summary of the recovered antibody in fractions obtained from affinity chromatography
after application of different pre-treatment methods. (Loh and Abdul Majid, [5])
Protein recovery Pre-treatment
Control Dilution with PBS NH2SO4 Ultrafiltration
precipitation
(ug) (%) (ug) (%) (ug) (%) (ug) (%)
Crude sample 2438 100 2678 100 2526 100 2228 100
After pre-treatment 2438 100 2678 100 441 22.3 1801 80.8
After affinity 1338 54.9 1745 65.2 273 10.8 1285 57.7
chromatography
equipment used. Ho and co-workers [4] developed an efficient mechanical cell dis-
ruption method using ultrasonication with a combination of centrifugation, precipi-
tation, dialysis and sucrose gradient ultracentrifugation for the release of HBcAg
from E. coli cells. The team found that the system used managed to recover HBcAg
at 22-fold higher levels than that of the enzymatic method.
Loh and Abdul Majid [5] studied different pre-treatment techniques to isolate
and purify monoclonal IgG from culture broth. Ammonium sulfate precipitation, di-
lution of the antibody in the binding buffer of affinity chromatography and ultrafil-
tration through an Amicon Ultra-15 filter with molecular weight cut-off at 100 kDa
were applied (Table 8.1). Their report indicated that all of the methods used are
suitable for sample preparation and concentration of the cell supernatant prior load-
ing onto an affinity column. The diluted sample supernatant, which has its pH al-
tered before affinity column loading, results in a significant improvement in product
recovery. Although ammonium sulfate precipitation offers significant recovery, it
must be avoided to avoid contamination of the precipitate with unwanted proteins.
The application of ultrafiltration is an extremely effective method for concentration
of the supernatant at 4 ºC because of its ease of preparation and time saving.
[6] Chromatographic separation processes, such as simulated moving bed
(SMB), are widely used in the petrochemical, fine chemical, sugar, and pharma-
ceutical industries. Their separation efficiency can be improved by optimizing the
components’ adsorptivity in the different unit sections through, e.g., the use of sol-
vent, temperature, or PH gradients. Wu and co-workers [6] used a salt-gradient
ion-exchange SMB to separate two proteins, β-lactoglobulins A and B. Two model-
based sequential optimization studies have been performed for both closed loop
isocratic SMB as well as closed and open loop gradient ion-exchange SMB. The
results indicate that the gradient SMB is more efficient than the isocratic SMB in
terms of startup time, throughput, and desorbent-to-throughput ratio. The use of
open-loop gradient ion-exchange SMB to separate proteins has been found to be
more effective than using closed-loop gradient SMB.
Preservation of enzyme activity is the most vital aspect of any processing steps.
Enzymes are heat sensitive. Freeze drying is usually an attractive option to trans-
form an enzyme in solution into powder form. However, the freeze-drying con-
ditions and the type of substrate added to the enzyme solution require intensive
124 F. A. A. Majid
optimization. Pisano and co-workers [7] combined several substrates with acid
phosphatase enzyme to facilitate the powder formation of the enzyme. Their study
indicated that temperature and drying time are as vital as the type of substrate in
ensuring the lowest enzyme destruction.
The innovative and integrative approaches in downstream processing demonstrate
that isolation and purification is rather unique for each protein of interest. Although
generally downstream processing uses almost similar process flow for protein puri-
fication, optimization at every unit operation is considered vital to ensure optimized
recovery. Overall innovation to facilitate downstream processing is not just limited
to optimization of at each unit operation. Upstream processing and well as molecular
intervention of the host cells also contribute to facilitating downstream processing.
The following sections discuss the contribution of upstream processing and molecu-
lar modification to improve the recovery and quality of the protein of interest.
A few intelligent innovations in the upstream processing indeed assist the ease
of downstream processing. In the earlier days of bioprocessing, bioreactors were
modified to retain cells in the bioreactor, whereas the used supernatant was continu-
ously withdrawn and fed into the downstream section. This method eliminates the
solid liquid separation stage and thus minimizes the unit operations in downstream
processing flow. Another innovation is to grow cells in a high-density culture sys-
tem by cell immobilization onto microporous beads/membrane discs coupled with
packed bed units in which cell biomass is packed in a porous membrane with low
molecular weight cut off enough for nutrients to pass through. The approach not
only increases the concentration of extracellular protein but, at the same time, re-
leases the clear concentrated broth into the downstream section without the need
for solid liquid separation and supernatant concentration. A few companies offer
different types of bioreactor systems with in-house downstream stages such as the
CelliGen Bioreactor range. Warnock and Al-Rubeai [8] reviewed various types of
bioreactor systems in production of various biological products from mammalian
cell systems.
Another novel approach to facilitate downstream processing is to grow cells in
protein-free medium. The usage of chemically defined medium will eliminate the
risk of foreign protein contaminants from other sources than from the host, espe-
cially for mammalian cell systems. Microbial cultures producing human proteins
should be ideally free of foreign proteins, especially of the same molecular weight
as the protein of interest, as it will make the purification difficult. Recently, Peck
and co-workers [9] demonstrated that EnBase (BioSilta, Finland), a microbial culti-
vation system that replicates fed-batch systems through sustained release of glucose
by enzymatic degradation of a polymeric substrate was able to increase, eightfold
higher, the cell densities of BL21-AI E. coli (expressing capripoxvirus proteins)
when grown in EnBase media compared with standard media. Greater yields of
capripoxvirus proteins were obtained using EnBase media, either through increases
in the amount of expressed protein per cell in conjunction with higher cell density or
through the increase in cell density alone. Purified capripoxvirus proteins expressed
using EnBase or standard media were demonstrated to be equally capable of spe-
cifically binding capripoxvirus antibodies.
8.6 Molecular Approaches to Facilitate Downstream

Processing
Host systems used to produce recombinant proteins are not perfect. Modifications
of hosts through molecular approaches have been in place ever since the establish-
ment of the recombinant DNA technology. E. coli has been successfully used for
the production of various recombinant proteins with some limitations influencing
the extent of recombinant protein expression. Heterologous protein accumulation
is often observed in inactive inclusion bodies either in the cytoplasm or periplasm,
or lytic activity of recombinant proteins, which causes cell lysis, could hinder high
production yield. Lee and co-workers [10] developed a novel strategy for the ef-
ficient production of aggregation-prone proteins and lytic enzymes in the E. coli
host. They used an anchored periplasmic expression (APEx) system, in which target
proteins are produced in the periplasm and tethered on the inner membrane. Protein
aggregation and lytic activity can be prevented through anchoring of individual pro-
teins to the inner membrane. The study demonstrated that two model proteins (ag-
gregation-prone human leptin and lytic Pseudomonas fluorescens SIK W1 lipase)
were successfully produced and anchored to the inner membrane under optimized
culture conditions. The proteins were confirmed to have high purity and bioactivity
after simple purification procedures.
Another simpler method to increase the expression of a protein of interest in the
host system is by increasing the cell density as well as exposing the recombinant
host to stress inducer. Restaino and co-workers [11] produced mammalian 6-O-
sulfotransferase (6-OST-1) in recombinant E. coli. In this study, high cell density
cultivation techniques were exploited to obtain recombinant 6-OST-1. Physiologi-
cal studies were performed in shake flasks to establish optimized growth and pro-
duction conditions. The induction strategies were tested in fed-batch experiments to
improve yield and productivity. The results revealed that high cell-density cultiva-
tion in 7 L culture, together with a coupled inducer strategy using isopropyl β-d-1-
thiogalactopyranoside and galactose, afforded 482 mg l−1 of enzyme with a biomass
yield of 16.2 mg gcdw−1 and a productivity of 10.5 mg l−1 h−1.
Another multiple-part strategy at the cellular level was elaborated in the work
of Jonet and co-workers [12]. A heterologous signal peptide (SP) from Bacillus
sp. G1 was optimized for secretion of recombinant cyclodextrin glucanotransferase
(CGTase) to the periplasmic and, eventually, extracellular space of Escherichia coli.
Eight mutant SPs were constructed using site-directed mutagenesis to improve the
secretion of recombinant CGTase. M5 is a mutated SP in which replacement of an
isoleucine residue in the h-region to glycine creates a helix-breaking or G-turn motif
with decreased hydrophobicity. The mutant SP resulted in 110 and 94 % increases in
periplasmic and extracellular recombinant CGTase, respectively, compared with the
wild-type SP at a similar level of cell lysis. The formation of intracellular inclusion
bodies was also reduced, as determined by sodium dodecyl sulfate-polyacrilamide
gel electrophoresis when this mutated SP was used. The addition of as low as 0.08 %
glycine at the beginning ofcell growth improved cell viability of the E. coli host.
126 F. A. A. Majid
The secretory production of other proteins, such as mannosidase, also displayed

similar improvement, as demonstrated by CGTase production, suggesting that the
combination of an optimized SP and a suitable chemical additive leads to significant
improvements of extracellular recombinant protein production and cell viability.
These findings will be valuable for the extracellular production of recombinant pro-
teins in E. coli.
Indeed, molecular intervention, media optimization and stress induction offer
advantages at downstream processing through high enzyme concentration in the fer-
mentation broth of recombinant E. coli. Similar approaches can be applied for other
recombinant host systems, such as hybridoma TBC3.bcl2, in which the bcl-2 onco-
gene was inserted to prolong the shelf life of cells in stressful culture environment
thus allowing more protein of interest to be secreted in the culture medium [13].
It is obvious that an integrated approach toward facilitating the downstream pro-
cessing of protein facilitates achieving significant purity and activity of the protein
of interest. Although the unit operations used are standard for downstream process-
ing flow, physical, chemical and physicochemical optimizations are important to
establish for each product to be purified. The molecular and cellular modifications
of the host system, as well as the protein itself, can help accommodate downstream
processing in novel and cost effective ways.
References
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acterisation of extracellular lipase from Aspergillus niger. Biotechnol Lett 18:547–552
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disruption of Escherichia coli by an ultrasonicator and recovery of intracellular hepatitis B core
antigen. Process Biochem 41:1829–1834
5. Loh MPS, Abdul Majid FA (2006) The performance of different pre-treatment techniques in
the isolation and purification of monoclonal IgG antibody. 1st International Conference on
Natural Resources Engineering & Technology 2006. Putrajaya, Malaysia 2006
6. Wu XD, Arellano-Garcia H, Hong WR, Wozny G (2013) Improving the operating conditions
of gradient ion-exchange simulated moving bed for protein separation. Ind Eng Chem Res
52:5407–5417
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enzymes in case of water-binding and non-water-binding substrates. Eur J Pharm Biopharm
85:974–983
8. Warnock JN, Al-Rubeai M (2006) Bioreactor systems for the production of biopharmaceuticals
from ani mal cells. Biotechnol Appl Biochem 45:1–12
9. Peck GR, Bowden TR, Shiell BJ, Michalski WP (2014) Increased bacterial cell density and
recombinant protein yield using a commercial microbial cultivation system. Prep Biochem
10. Lee JH, Velmurugan N, Jeong KJ (2013) Novel strategy for production of aggregation-prone
proteins and lytic enzymes in Escherichia coli based on an anchored periplasmic expression
system. J Biosci Bioeng 116:638–643
11. Restaino OF, Bhaskar U, Paul P, Li NY, De Rosa M, Dordick JS, et al (2013) High cell
density cultivation of a recombinant E. coli strain expressing a key enzyme in bioengineered
heparin production. Appl Microbiol Biotechnol 97:3893–3900
12. Jonet MA, Mahadi NM, Murad AMA, Rabu A, Abu Bakar FD, Rahim RA, et al (2012)
Optimization of a heterologous signal peptide by site-directed mutagenesis for improved
secretion of recombinant proteins in Escherichia coli. J Mol Microbiol Biotechnol 22:48–58
13. Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M (1998) In hybridoma cultures,
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Chapter 9
Economic and Environmental Evaluation
of Recombinant Enzyme Production
Mohd Jamil Aizat Jamaluddin, Azlin Suhaida Azmi, Sarina Sulaiman,

Dzun Noraini Jimat, Muhd. Ezza Faiez Othman and Azura Amid
Abstract Economic and environmental evaluation on three different through-

puts and comparison between these throughputs were presented. Comparisons
are based on simulation of 10, 100 and 1000 kg/batch of recBromelain using
SuperPro Designer v8.5. The economic analysis shows that 100 kg/batch gives
the most competitive price and maintaining a sizeable profit gross margin of
28.21 %. A positive ROI of 18.41 % and minimal payback time of 5.43 years is
predicted from 100 kg/batch of production. Environment analysis showed that
total waste and energy consumption increased as production size increased. This
simulation information will assist to predict the cost and environment impact at
different size of productions.
Keywords Economic analysis · Environmental assessment · Oral toxicity · Process

simulation · Product isolation · Quality control · SuperPro designer
A. S. Azmi () · D. N. Jimat · A. Amid

e-mail: azlinsu76@iium.edu.my
M. J. A. Jamaluddin · M. E. F. Othman
M. E. F. Othman
D. N. Jimat
e-mail: jnoraini@iium.edu.my
A. Amid
S. Sulaiman
Bioenvironmental Engineering Research Centre, Department of Biotechnology Engineering,
Faculty of Engineering, International Islamic University Malaysia, P.O. Box 10, 50728 Kuala
Lumpur, Malaysia
e-mail: sarina@iium.edu.my
DOI 10.1007/978-3-319-12397-4_9
130 M. J. A. Jamaluddin et al.
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Fig. 9.1 Main production process for recBromelain enzyme production
9.1 Introduction
One of the most important enzyme applications is in medicine. Currently, approxi-

mately 7 billion people depend on therapeutic enzymes. In addition to high produc-
tion costs, conventional enzyme production approaches cannot sustain the surge in
demand. Recombinant technology has been proven to be a potentially useful alter-
native for therapeutic enzyme production because it is generally regarded as safe,
environmentally friendly, cost efficient and has a high throughput.
Many laborious attempts have been pursued by scientists or engineers to produce
recombinant therapeutic proteins in the laboratory. However, few of these enzymes
progress to the commercialization stage. In our case, a gene for a proteolytic en-
zyme, bromelain, was successfully cloned and expressed in Escherichia coli. The
enzyme was successfully purified and spray dried during the final production stage.
Using the current optimized bioprocess technology applied in our lab, the final re-
combinant bromelain products > 80 % pure and remained active. There are currently
no published reports on the economic and environment perspectives for recBrome-
lain production that can be used as a benchmark. Hence, it is unknown whether our
current recBromelain production scheme will translate into a profitable endeavor
with and a low environment impact.
The transition from the lab scale to the development stage is vital to the commer-
cial success of recombinant therapeutic protein production. Several reports have ob-
served that decisions early in the development process often are difficult to change
at later stages due to cost and regulatory constraints on process modification [1].
Thus, process simulation has become a standard guidance tool to plan, design, and
predict the economics and the environment impact from production.
9 Economic and Environmental Evaluation of Recombinant Enzyme Production 131
9.1.1 Process Simulation
recBromelain production in engineered E. coli requires three main processes as

shown in Fig. 9.1. The processes are fermentation, product recovery and product
formulation. These general processes are similar to those required by most enzyme
production processes utilizing bacteria, yeast or fungi as discussed by Nielsen [2].
An integrated bioprocess flow simulation was performed using the software
package, SuperPro Designer v8.5 (Intelligen, NJ, USA) for a 16 liter fed-batch fer-
mentation of recombinant E. coli. The data process was obtained from our experi-
mental runs at the 1 and 16 l scales. The production layout is shown in Fig. 9.2.
Fermentation is the heart of the process where the product is formed. In this case,
E. coli expressing recBromelain from pineapple crown are grown in the bioreactor.
For optimum growth, carbon sources, mineral salt and vitamins are supplied to the
bioreactor. This aerobic fermentation requires oxygen, which is supplied at the op-
timal percentage. Aeration and mixing are necessary to maintain the dissolved oxy-
gen percent during fermentation. Temperature and pH are also controlled at optimal
set points. Fermentation in bioreactors requires 16 hours for optimal growth. Subse-
quently, the fermentation broth is centrifuged to separate liquid broth from cellular
biomass. Because recBromelain is expressed intracellularly, the cells are then lysed
to recover the crude enzyme prior to an additional centrifugation step to separate
cellular debris from the crude enzyme. The crude enzyme further separated using an
aqueous two phase system (ATPS). ATPS has been used to separate proteases from
fermentation broth such as bromelain from pineapple fruit [3] using PEG-potassium
phosphate. A similar system is used for recBromelain crude enzyme separation.
Finally, excipient is added in a mixer for product formulation before it is subjected
to spray drying and packaging.
9.2 Economic Analysis
The economic analysis is based on a report generated by SuperPro Designer v8.5.

The fixed capital investment was estimated from the equipment purchase cost.
Equipment purchase cost and associated installation, piping and instrumentation
costs were obtained from equipment vendors, and our own data. The other fixed
capital components (electrical, buildings, yard improvements, engineering and con-
struction, etc.) were estimated using SuperPro Designer default values. Operation
labor was estimated by allocating operator hours required per equipment operation,
preparation and cleaning time. Other labor-dependent cost items such as shift super-
visors, quality control (QC), and laboratory services were estimated as a percentage
of operation labor. The annual cost of raw materials and consumables were calcu-
lated on the basis of process requirements for each batch. The cost of chemicals and
raw materials were obtained based on the quotations provided by suppliers.
The highest selling price in current market for bromelain is around USD
3000.00/kg (Sigma-Aldrich, USA). This is based on bromelain with high proteolytic
Fig. 9.2 Process flow diagram of the integrated process of recombinant bromelain
activity (> 10,000 GDU/g). However, the majority of bromelain available commer-

cially produce bromelain with activity at 1000–3000 GDU/g with an approximate
average price of USD 1000.00/kg (2014 market report). Therefore, the simulation
focused on 3000 GDU/g grade production using the process data from pilot plant-
scale production of ~1 kg/batch and a current production-scale of ~10 kg/batch.
Table 9.1 Key economic indicators for recBromelain competitive selling prices (in USD) for the
current ~ 10 kg/batch
Selling Annual oper- Annual Gross ROI (%) Payback Net present
price ating cost revenues margin time (year) value—NPV
(USD/kg) (USD/year) (USD/year) (%) (USD)
1000.00 4,791,000.00 4,500,000.00 − 6.47 4.12 24.29 − 6,200,000.00
2000.00 8,999,000.00 46.76 47.78 2.09 13,801,000.00
3000.00 13,499,000.00 64.51 89.63 1.12 33,186,000.00
4000.00 17,999,000.00 73.38 131.48 0.76 52,571,000.00
Table 9.2 Key economic indicators for recBromelain competitive selling prices (in USD) for a
100kg/batch
price ating cost revenues margin time (year) value—NPV
(USD/kg) (USD/year) (USD/year) (%) (USD)
500.00 4,673,000.00 5,426,000.00 13.87 12.78 7.83 − 1,479,000.00
600.00 6,511,000.00 28.22 18.41 5.43 2,850,000.00
700.00 7,596,000.00 38.48 24.04 4.16 7,529,000.00
800.00 8,681,000.00 46.17 29.67 3.37 12,204,000.00
Table 9.3 Key economic indicator against recBromelain competitive selling price (in USD) for
~ 1000 kg/batch
price ating cost revenues margin time value—NPV
(USD/kg) (USD/year) (USD/year) (%) (year) (USD)
700.00 5,860,000.00 7,209,000.00 18.70 12.62 7.92 − 2,008,000.00
800.00 8,239,000.00 28.87 15.41 6.49 1,988,000.00
900.00 9,268,000.00 36.77 18.19 5.50 6,425,000.00
1000.00 10,298,000.00 43.09 20.97 4.77 10,861,000.00
The effect of selling price per kg of recombinant bromelain on several economic

parameters are tabulated in Tables 9.1, 9.2 and 9.3 and illustrated in Figs. 9.3, 9.4,
9.5 and 9.6. The minimum competitive selling price was highest (USD 2000.00) at
the current 10 kg/batch production-scale. The selling price of USD 2000.00 is not as
competitive as USD 600.00 and USD 800.00 obtained at the 100 and 1000 kg/batch
production-scales, respectively. Production at 100 kg/batch allows the most com-
petitive price while maintaining a sizeable profit gross margin of 28.21 %. This value
also translated into a positive ROI (18.41 %) and minimal payback time (5.43 years).
As the direct production costs are dominated primarily by labor, reagents, consum-
ables and utilities, an insignificant effect was observed by increasing the scale from
10 to 100 kg/batch, whereas, there was an approximately 25 % cost increment when
increasing the scale from 100 to 1000 kg/year.

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Fig. 9.3 Comparison of minimum competitive selling prices (USD) of recBromelain for 10, 100
and 1000 kg/batch

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Fig. 9.4 Annual production costs (USD) comparing three different throughputs of recBromelain
production

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Fig. 9.5 Annual production revenues (USD) comparing three different throughputs of recBrome-
lain production

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Fig. 9.6 Gross margins and return of investment comparing three different throughputs of rec-
Bromelain production
Costs associated with recBromelain production will largely depend on the final
product; whether it is intended for an oral or topical therapeutic, a nutraceutical, or
an industrial application. It will cost significantly less to prepare a functional food
compared with a high-purity pharmaceutical. The simulation study demonstrates
that it would cost approximately USD 0.98 to generate 1 g of recombinant brome-
lain in a cGMP facility operating at a scale of 100 kg/year. This low cost is possible
because of the following purification and formulation systems. Reasons: (1) Stable
expression levels of recombinant bromelain in E. coli. (2) Low cost protein induc-
tion agent. (3) Low cost of purification and formulation systems.
9.3 Environmental Assessment
The environmental assessment process provides a mechanism for reviewing major

projects to assess their potential environmental impacts. Physical and biochemical
properties as well as toxicity and side effects of recBromelain are discussed in the
next subsections prior to the discussion of environment impacts from the recombi-
nant enzyme production process.
9.3.1 Physical and Biochemical Properties
The source of recombinant bromelain is pineapple fruit. Bromelain or bromelin,

a pineapple extract, is derived from the fruits and stems. Fruit-bromelain (EC.
3.4.22.33) is different from stem-bromelain (EC. 3.4.22.32), and they contain
different enzyme compositions, such as thiol-endopeptidases, and other not yet
completely characterized components, such as phosphatases, glucosidases, peroxi-
dases, cellulases, glycoproteins and carbohydrates, among others [4, 5]. Bromelain
contains a complex mixture of protease and non-protease components. Stem bro-

melain contains cysteine proteinases in bromelain preparations derived from pine-
apple stems. Fruit bromelain is a glucoprotein proteinase present in pineapple juice
that enzymatically cleaves the internal polypeptide bonds of proteins with a broad
substrate specificity [4]. Muntari and co-workers reported that there are other mi-
nor cysteine endopeptidases (ananain, comosain) present in pineapple stem brome-
lain [6]. Corzo and co-workers reported that many bromelain manufacturers do not
declare whether bromelain extracts were obtained solely from pineapple fruit or
stems because bromelain preparations may contain a combination of fruit brome-
lain and stem bromelain and these products may contain two or more proteolytic
enzymes [4].
Bromelain extract solution is usually dried using a spray dryer or freeze dryer,
producing a creamy white to yellow-brown crystalline powder [7]. Maurer reported
that enzyme activity is determined using different substrates such as casein (FIP
units), gelatin (gelatin digestion units) or chromogenic tripeptides.
Table 9.4 summarizes the composition, pH, and enzyme activity of natural bro-
melain, commercial bromelain and recombinant bromelain. The pH for natural bro-
melain enzymatic activity is within the range of 5.5–8.0 [5, 8, 9]. Nadzirah and
co-workers reported that bromelain activity is stable at pH 3.0–6.5 [7]. Amid and
co-workers reported that purified recombinant bromelain exhibited optimal activity
at pH 4.6 and 45 °C. In summary, Corzo and co-workers, stated that little research
has been performed to identify the optimal temperature for bromelain activity, but
studies have reported that the optimal temperature is between 30–60 °C dependent
on the pH [4, 10]. It has also been stated that the high sucrose content in pineapple
crown extracts may effect bromelain activity [7].
In aqueous solutions, bromelain rapidly deteriorates via self-digestion which can
be prevented by the addition of serum containing α-macroglobulin [5]. Amid and
co-workers also reported recombinant bromelain activity was moderate at tempera-
tures between 15 and 35 °C and had reduced activity at 65 °C. These results are in
agreement with natural bromelain characteristics, which is active between 40 and
60 °C and has an optimal pH between 4.5 to 5.5 [10]. They also reported that com-
mercial bromelain exhibited higher activity at 30, 45, and 60 °C compared with
recombinant bromelain. When the temperature reached 100 °C, the activity of com-
mercially available bromelain decreased more rapidly than recombinant bromelain.
The activity of recombinant bromelain and commercial bromelain was 2 U/mg and
reached 0 U/mg after a 30 min incubation at 100 °C, respectively. Ketnawa also re-
ported that the activity of natural bromelain decreased its lowest point at 90 °C [9].
9.3.2 Toxicity and Side Effect
The oral toxicity (LD50) for pineapple bromelain is reportedly greater than 10 g/kg,
which is considered a very low toxicity [11]. The latest test report by SIRIM Ber-
had (2014) on recombinant bromelain stated that the recombinant bromelain LD50
Table 9.4 Physio-chemical properties of pineapple extract
Physico-chemical Pineapple Peel[9] Stem[9] Core[9] Commercial Stem[10]
properties crown, [7] (Merck, Ger-
many) [10]
Extract Bromelain Bromelain Bromelain Bromelain Bromelain Recombinant
bromelain
% Pulp 2.41 ± 0.08 50 50 50 – –
Total soluble solid 1.6 ± 0.0 4.33 ± 0.58ab 4.73 ± 0.87a 4.73 ± 0.87a – –
% Acid 0.30 ± 0.00 – – – – –
% Fructose 0.83 ± 0.04 – – – – –
% Glucose 0.51 ± 0.01 – – – – –
% Protein – 132.4 ± 1.40b 29.8 ± 0.76d 45.4 ± 0.87c 39 ± 0.5(mg/mL) 39 ± 0.5 (mg/mL)
pH 3.94 ± 0.00 4.02 ± 0.30ab 4.76 ± 0.16b 4.27 ± 0.24b 4- 4.6

Total enzyme 426.49d ± 8.76 90,653 ± 1.08(U/100 mg) 14,435 ± 2.26 36,111 ± 1.62 33 ± 0.6 (U/mg) 48 ± 0.7 (U/mg)
activity (casein) CDU/mg (U/100 mg) (U/100 mg)
Temperature,°C – 50 °C 50 °C 50 °C 30–60 45
9 Economic and Environmental Evaluation of Recombinant Enzyme Production
137
is greater than 2000 mg/kg body weight. Therefore, it is classified as a Category 5

compound according to the Globally Harmonized System for the classification of
chemicals [12].
However, recent studies investigating bromelain side effects are lacking. It has
been reported that in human clinical tests, side effects are generally not observed;
however, caution is advised when administering bromelain to individuals with hy-
pertension because one report indicated individuals with pre-existing hypertension
may experience tachycardia following high doses of bromelain [13].
Other studies by Pillai and co-workers investigated the antitumor effect of bro-
melain on MUC1-expressing malignant peritoneal mesothelioma (MPM) cell lines.
This study indicated that bromelain alone may be used as a therapeutic agent for
the treatment of MPM; they observed that bromelain was capable of inhibiting cell
proliferation by 90–95 % when dosed at 400 and 100 mg/ml [14]. Muller and co-
workers investigated the effects of bromelain on immune cell activities after oral in-
gestion by healthy volunteers. They observed significant changes in the response of
physiologically stimulated immune cells after a single oral dose of bromelain (3000
FIP units). This is the first study demonstrating bromelain can modulate the cel-
lular responses of lymphocytes after oral administration [15]. Moreover, Aiyegbusi
and co-workers investigated the effects of bromelain on tenocyte proliferation and
tendon malondialdehyde (MDA) levels during the early stage of healing after Achil-
les tendon crush injuries in Sprague-Dawley rats. They observed that 600 GDU
bromelain administered once daily at a dosage of 7 mg/kg promoted healing after
acute tendon injury by stimulating tenocyte proliferation. These results indicated
bromelain may be effective for reducing pain and swelling following acute injuries
[16]. Secor Jr and co-workers determined the effects of orally administered brome-
lain in an ovalbumin (OVA)-induced murine model of acute allergic airway disease
(AAD). They observed that methacholine sensitivity and bronchoalveolar lavage
(BAL) cellular differentials were decreased. These findings demonstrated that oral
treatment with bromelain has a beneficial therapeutic effect in the murine model of
asthma and bromelain may be effective in human conditions [17].
9.3.3 Environment Assessment
The environmental impact report was generated by SuperPro Designer v8.5. Three
types of wastes are generated: gaseous emissions, aqueous waste and solid waste.
All three size product outputs generate a similar percent waste. The general waste
breakdown is shown in Fig. 9.7, 9.8 and 9.9 for 10, 100 and 1000 kg/batch, respec-
tively. Total waste is presented in Fig. 9.10. The highest fraction of waste is gaseous
emissions with the emission size increasing as the production size increases. The
most pressing environmental issue with gaseous emissions is the carbon dioxide
accumulation due to global warming concerns. However, in this study, carbon di-
oxide (a greenhouse gas) generated approximately only 0.11 % of total emissions.
The remaining gaseous wastes are nitrogen, oxygen and water vapor. Practically all
carbon dioxide waste was produced during fermentation.

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Fig. 9.7 Waste generation proportions by type at 10 kg/batch throughput

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Fig. 9.10 Total waste generated by the three different recBromelain production throughputs

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Fig. 9.11 Power demand proportions for a 10 kg/batch throughput

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Aqueous waste is the second largest generated waste at all three throughputs.
The aqueous waste portion is 88–95 % water followed by phosphate at less than
0.5 %. Phosphate waste is mostly generated by the ATPS system. Solid waste is
the least generated, consisting biomass residue, cellular debris, denatured protein,
EDTA and water.
The breakdown report of power consumption generated by the software is shown
in Figs. 9.11, 9.12 and 9.13. Figure 9.11 shows that the highest power or energy
consumption is from the product recovery process for a 10 kg/batch of recBrome-
lain. Product recovery consists of centrifugation, sonication and the ATPS system.
The most power intensive process is sonication, used for cellular disintegration to
release intracellular recBromelain. Sonication consumes 71.13 % of total power
consumed. Mechanical cell disruption such as sonication is known to require high
energy utilization. However, sonication has been proved to significantly improve

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Fig. 9.14 Energy consumption comparing the three different batch throughputs for recBromelain
production
crude enzyme release. Furthermore, using sonication avoids the use of expen-
sive detergents and chemical post-treatments. Fermentation has the lowest power
consumption, consisting only 0.4 % of total power consumption. The majority of
power consumption is from the use of a gas compressor. The energy consumption
amounts for all three batch throughputs are shown in Fig. 9.14.
Interestingly, as the product throughput is increased 10-fold, the fermentation
process the percentage energy consumption increases and the product recovery per-
centage decreases (Figs. 9.11, 9.12 and 9.13). As the size of fermenter increases, the
gas compressor consumes 100-fold more energy, increasing the power consumption
percentage. This increase is due to a higher quantity of air supplied to the fermenter
by the compressor.
9.4 Conclusion
Economic and environment analyses for three different batch throughputs were
discussed in this chapter. Production at 100 kg/batch or higher allows the most
competitive price while maintaining a sizeable profit gross margin. Costs associ-
ated with processing recBromelain are largely dependent on the grade of the final
product. It will cost much less to prepare a functional food product compared with
a high-purity pharmaceutical. Production size dictates the total waste generation
and the proportions of waste types. The gaseous emission percentages increase as
the production batch size increases. Energy consumption is similar, increasing as
the production batch size increases. However, the energy consumption percentage
increases at the fermentation stage and decreases at the product recovery stage when
the production batch size increases. This information can be used to address the
environmental impact of several batch sizes of recBromelain production.
References
1. Ernst S, Garro OA, Winkler S, Venkataraman G, Langer R, Cooney CL et al (1997) Process
simulation for recombinant protein production: cost estimation and sensitivity analysis for
heparinase I expressed in Escherichia coli. Biotechnol Bioeng 53:575–582
2. Nielsen PH, Oxenbøll KM, Wenzel H (2007) Cradle-to-gate environmental assessment of
enzyme products produced industrially in Denmark by novozymes A/S. Int J Life Cycle As-
sess 12:432–438
3. Ketnawa S, Rawdkuen S, Chaiwut P (2010) Two phase partitioning and collagen hydrolysis
of bromelain from pineapple peel Nang Lae cultivar. Biochem Eng J 52:205–211
4. Corzo CA, Waliszewski KN, Welti-Chanes J (2012) Pineapple fruit bromelain affinity to dif-
ferent protein substrates. Food Chem 133:631–635
5. Maurer HR (2001) Bromelain: biochemistry, pharmacology and medical use. Cell Mol Life
Sci 58:1234–1245
6. Muntari Bala, Maizirwan M, Mohamed Saedi Jami, Hamzah Mohd. Salleh AA (2012) Bro-
melain production: current trends and perspective. Arch Des Sci 65:369–399
7. Nadzirah KZ, Zainal S, Noriham A, Normah I, Roha AMS (2012) Physico-chemical proper-
ties of pineapple crown extract variety N36 and bromelain activity in different forms. AP-
CBEE Procedia 4:130–134
8. Pavan R, Jain S, Shraddha, Kumar A (2012) Properties and therapeutic application of brome-
lain: a review. Biotechnol Res Int 2012:6
9. Ketnawa S, Chaiwut P, Rawdkuen S (2012) Pineapple wastes: a potential source for brome-
lain extraction. Food Bioprod Process 90:385–391
10. Amid A, Ismail NA, Yusof F, Salleh HM (2011) Expression, purification, and characterization
of a recombinant stem bromelain from Ananas comosus. Process Biochem 46:2232–2239
11. (2010) Bromelain. Monograph. Alterna Med Rev J Clin Ther 15:361–368
12. Shahab N, Zainudin N, Badrudin S (2013) Evaluation of recombinant bromelain in the
BALB/c 3T3 NRU cytotoxicity study. Standard and Industrial Research Institute of Malaysia
(SIRIM) Berhad
13. Gutfreund A, Taussig S, Morris A (1978) Effect of oral bromelain on blood pressure and heart
rate of hypertensive patients. Hawaii Med J 37:143
14. Pillai K, Ehteda A, Akhter J, Chua TC, Morris DL (2014) Anticancer effect of bromelain
with and without cisplatin or 5-FU on malignant peritoneal mesothelioma cells. Anti-cancer
Drugs 25:150–160
15. Müller S, März R, Schmolz M, Drewelow B, Eschmann K, Meiser P (2013) Placebo–con-
trolled randomized clinical trial on the immunomodulating activities of low–and high–dose
bromelain after oral administration–new evidence on the antiinflammatory mode of action of
bromelain. Phytother Res 27:199–204
16. Aiyegbusi AI, Olabiyi OO, Duru FI, Noronha CC, Okanlawon AO (2011) A comparative
study of the effects of bromelain and fresh pineapple juice on the early phase of healing in
acute crush achilles tendon injury. J Med Food 14:348–352
17. Secor ER Jr, Carson IV WF, Cloutier MM, Guernsey LA, Schramm CM, Wu CA et al (2005)
Bromelain exerts anti-inflammatory effects in an ovalbumin-induced murine model of aller-
gic airway disease. Cell Immunol 237:68–75
Chapter 10
Case Study: Recombinant Bromelain Selection
Azura Amid, Nurul Azira Ismail and Zatul Iffah Mohd Arshad
Abstract This chapter presents an investigation that we performed prior to the

decision to proceed cloning and producing recombinant bromelain. The criteria that
we examined were the following: (1) easy access to a DNA source; (2) broad appli-
cation; (3) an enzyme size amenable to the cloning strategy; (4) available data in
free online databases; (5) broad industrial application.
Keywords Animal feed industry · Bread industry · Brewing industry · Bromelain ·

Bromelain mechanism of action · Cysteine protease · Diarrhea treatment · Fish
industry · Food industry · Formulation · Meat industry · Media formulation ·
Nutraceutical and pharmaceutical industries · OFAT design · Optimizing · Personal
care industries · Soy source industry
10.1 Why Bromelain?
Bromelain is an enzyme that is extracted from pineapples. Two types of bromelain,

fruit and stem, exist. As indicated by the names, fruit bromelain is extracted from
pineapple juice and stem bromelain is extracted from pineapple stems. Bromelain is
classified as a proteolytic enzyme or protease because it exhibits proteolytic activity
in nature, which indicates that it acts on a protein substrate. It is used in many in-
dustrial applications including the food, pharmaceutical, nutraceutical, and cosmetic
industries as well as in the preparation of animal feed and human food supplements.
A. Amid ()
Biomolecular and Bioprocess Engineering Research Unit,
Department of Biotechnology Engineering, Faculty of Engineering,
International Islamic University Malaysia
P.O. Box 10, 50728, Kuala Lumpur, Malaysia
Z. I. M. Arshad
Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang,
Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia
e-mail: zatul84@yahoo.com
N. A. Ismail
University Technology Mara, Campus Puncak Alam, Shah Alam, Selangor, Malaysia
DOI 10.1007/978-3-319-12397-4_10
144 A. Amid et al.
Bromelain is a group 3 (hydrolase) and subgroup 4 (peptide bond hydrolase) enzyme,

based on the International Union of Biochemistry and Molecular Biology (IUBMB)
enzyme classifications. Bromelains are further subdivided into endo-peptidases or
exo-peptidases, depending on their ability to hydrolyze internal or terminally local-
ized peptide bonds. Additionally, proteases can be classified based on their mecha-
nism of action. There are six mechanistic classes based on their catalytic sites [1], and
bromelain is identified as a cysteine endopeptidase (EC 3.4.22). Cysteine protease
(CP) activity depends on a catalytic dyad consisting of cysteine and histidine and the
preferred arrangement of Cys and His (Cys-His or His-Cys) residues differ among
family members [2]. The protease structure exhibits an α-helix and β-barrel-like mo-
tif, separated by a groove containing the active site which is formed by the Cys-25
and His-159 residues that are located at each side of the groove and are evolutionarily
conserved among all family members [3]. Therefore, this unique enzyme and its bio-
chemical properties potentiate its various applications in multiple industries.
10.1.1 Biochemical Properties of Bromelain
Several diverse properties of bromelain have been well studied such as stability, pH,
optimum temperature and molecular weight. The enzymatic activity of bromelain
on various substrates including casein, gelatin and other synthetic substrates can be
determined under optimal pH and temperature conditions. Numerous publications
have reported the molecular weight, as well as the optimal pH and temperature
for bromelain activity. For example, Suh and co-workers estimated the molecular
weights of the stem and fruit bromelains at approximately 37 and 32.5 kDa, respec-
tively [1]. These researchers observed the maximum activity for the stem and fruit
bromelains at pH 7.0, 60 °C and pH 8.0, 70 °C, respectively. Both enzymes were
completely inhibited by sulfhydryl reagents and the Ki values for the stem and fruit
bromelains for p-chloromercuribenzoate were 0.10 and 0.18 mM, respectively. In
addition, the optimal pH for stem bromelain activity was between 6 and 7 for the
two substrates studied (Z-Arg-Arg-NH-Mec and Bz-Phe-Val-Arg-NH-Mec). Ad-
ditionally, stem bromelain had a molecular weight of 28 kDa and was inhibited
by E-64 [2]. Similarly, Ketnawa and co-workers revealed that stem bromelain had
the highest relative activity at pH 7.0 and 55 °C [3], but they later reported that the
molecular weight of bromelain was 29 kDa and had the highest relative activity at
pH 8.0 and 60 °C [4]. In another study, crude bromelain extracts from pineapple
cultivars displayed caseinolytic activity over a broad pH range of 3–9 [5]. Other
researchers have also reported molecular weights of 25, 24.5, 26 and 30 kDa for
bromelain [6–8]. Table 10.1 summarizes results from different studies that assessed
bromelain’s molecular weight and the optimal pH and temperature for its activity.
10.1.2 Molecular Structure of Bromelain
The amino acid composition of bromelain was compared with two other types of
pineapple proteases (ananain and comosain) extracted from the pineapple stem. The
10 Case Study: Recombinant Bromelain Selection 145
Table 10.1 Bromelain molecular weight and optimum pH and temperature for its activity
Type of Optimum pH Optimum temperature Molecular weight Reference
bromelain (oC) (kDa)
Stem 7.0 60 37 [1]
Fruit 8.0 70 32.5
Stem 6–7 – 28 [2]
Fruit 7.0 55 – [3]
Fruit 8.0 60 29 [5]
Fruit 3–9 50–60 – [4]
Fruit 2.9–7.7 37–59 – [12]
Stem – – 29 [7]
Fruit 24.5 [13]
Stem – – 26 [8]
Fruit – 40 – [14]
Stem – – 30 [6]
Fruit 6.0 70 – [15]
Stem – 50–60 – [16]
Table 10.2 Reported amino No. of residues

acid composition of ananain,
comosain and bromelain [10] Amino acid Bromelain Ananain Comosain
Asx 18 19 18
Thr 9 8 7
Ser 17 18 17
Glx 16 13 13
Gly 22 24 25
Ala 25 20 20
Val 14 14 13
Met 3 2 3
Ile 17 14 12
Leu 6 9 9
Tyr 14 12 12
Phe 6 5 7
His 1 2 2
Lys 15 11 10
Arg 6 10 11
Cys 7 7 7
stem bromelain differed from the other enzymes in the number of polar and charged
amino acids, particularly lysine and arginine, and in the number of several aliphatic
amino acids, such as alanine and isoleucine (Table 10.2). Bromelain has been fur-
ther differentiated from other pineapple proteases as reported by Maurer [15], and
the details are presented in Table 10.3.
146 A. Amid et al.
Table 10.3 Cysteine proteases derived from pineapples

Name (EC Molecular mass Isoelec- Sequences Glycosylation References
number) (Dalton) tric point
Stem bromelain 23,800 > 10 212 amino glycosylated [17]
(EC 3.4.22.32) (sequence + sugar) acids
Ananain (EC 23,800 > 10 216 amino Not glycosylated [9]
3.4.22.31) [10] acids
Comosain 24,400 > 10 Glycosylated
Fruit bromelain 23,000 4.6 Not glycosylated [2]
Fig. 10.1 Cysteine protease mechanism of action [18]
The amino acid sequences of these enzymes were initially discovered by Lee
[9]. The amino acid sequence alignment indicates that the enzymes share the same
active site Cys-25. Similarly, the position of the active site His-157 is identical
among ananain, stem bromelain, papain, chymopapain and actinidin. Consequently,
these proteases belong to the cysteine protease group [10]. The latest partial cod-
ing sequence for stem bromelain can be accessed at http://www.ncbi.nlm.nih.gov
with the reference number JF332148 [11]. Our most recent search for a bromelain
crystal structure on the protein database was not successful, only the papain crystal
structure has been identified. Therefore, studies focused on obtaining the bromelain
crystal structure are required.
10.1.3 Bromelain Mechanism of Action
The mechanism of action of bromelain is referred to as general CP activity. The

mechanism of action for CPs involves the hydrolysis of carboxylic acid derivatives
through a double-displacement pathway composed of a general acid-base forma-
tion and hydrolysis of an acyl-thiol intermediate [18]. The initial catalysis step (as
described in Fig. 10.1, where Im and +Him refer to the imidazole and protonated
imidazole, respectively) involves the noncovalent binding of the free enzyme to the
substrate to form a complex. This is followed by the acylation of the enzyme and the
formation and release of the first product, the amine R’-NH2. Deacylation follows,
where the acyl-enzyme reacts with a water molecule to release the second product
while regenerating the free enzyme, as exhibited in Fig. 10.1.
10.2 Bromelain in Food Industries
10.2.1 Bread Industry
Bromelain is used in baking such as in bread making because it can strengthen

gluten. Gluten is a protein that is present in foods processed from wheat. Bromelain
proteolysis hydrolyzes both gliadins and glutenins in gluten, thus improving the
dough structure. It encourages dough relaxation, preventing dough shrinkage, and
promotes better bread volume. This enzyme is suitable for industrial applications
due to its rapid reaction and broad optimal pH and temperature ranges [19]. Brome-
lain is active in the dough and during unbaked bread leavening, but is inactivated
by high temperatures during baking. Some of the residues remain in the inactivated
enzyme form, which can be metabolized similar to other proteins [20]. Furthermore,
bromelain has been used to generate hypoallergenic wheat flour due to its ability to
degrade the wheat glutenin IgE epitope Gln-Gln-Gln-Pro-Pro [21].
10.2.2 Brewing Industry
Crude bromelain and other proteases [22] are used in the brewing industry to obtain
good colloidal properties at low temperatures, which eliminate cloud formation
[23]. The bromelain protease activity prevents aggregation of insoluble complexes
by hydrolyzing the proteinous substances that normally precipitate polyphenols and
oligosaccharides during cold storage [24]. Additionally, bromelain can solubilize
protein from barley adjuncts and release peptides and amino acids that can then be
utilized during fermentation as a nitrogen source [25].
10.2.3 Meat Industry
Meat softness and tenderness are the most vital determinants of consumer satisfac-
tion and taste perception [26]. Proteolytic enzymes are typically used to improve
meat tenderness. Furthermore, myofibril integrity and the contribution of connective
tissue affect meat tenderness. Bromelain is extremely powerful hydrolyzing fibrous
proteins and connective tissue [27], thus, it is used to tenderize tough meat. Ad-
ditionally, bromelain increased tenderness and degraded collagen more extensively
148 A. Amid et al.
than contractile proteins, whereas ficin exhibits the most balanced degradation of
both myofibrillar and collagen proteins [28].
10.2.4 Fish Industry
Countries with large fishing industries generate problematic waste materials at

manufacturing plants processing marine species. Endogenous visceral enzymes are
used to produce fish protein hydrolysate (FPH) to obtain higher added values for
such wastes. These processes may either generate sources of bioactive peptides or
used as nitrogenous substrates for microbiological media [29, 30]. Consequently,
plant proteases such as bromelain, papain, ficin, and alcalase (contains ananain)
have been widely used to generate FPHs [31].
10.2.5 Soy Source Industry
Soybeans are well-established, rich food sources due to their large content of quali-
tative protein. Proteases such as bromelain have been widely used to prepare soy
sauce and other soy products. The proteolytic hydrolysis of soy proteins enhances
their functional properties. Consequently, treating soy proteins with bromelain or al-
calase generates soluble hydrolysates that exhibit good yield and low bitterness [24].
10.3 Bromelain in the Nutraceutical and Pharmaceutical

Industries
Pineapples, which are a large source of bromelain, have been traditionally and
widely used by South American, Chinese and Southeast Asian populations [32].
Bromelain systemically affects several cellular and molecular targets. Some brome-
lain’s relevant therapeutic applications are summarized in this section.
10.3.1 Bromelain as an Anti-Inflammatory Agent
Bromelain has been demonstrated to be an effective anti-inflammatory agent. The

major mechanisms of action appear to be proteolytic in nature and mediated via the
following factors: increased serum fibrinolytic activity [33], reduce plasma fibrino-
gen levels [34] and decrease bradykinin levels, which result in reduced vascular per-
meability and thereby a reduction in edema and pain [35]. Bromelain can modify the
leukocyte expression of cell surface molecules. Specifically, bromelain can remove
cell surface molecules, including the CD128 chemokine receptors, by preventing the
firm adhesion of leukocytes to blood vessels at the site of inflammation [36]. Studies
conducted by Bhui and co-workers, Huang and co-workers and Gaspani and co-
workers [37–39] suggested that bromelain inhibit cyclooxygenase-2 (Cox-2) expres-
sion and thus decreases other inflammatory cascade proteins, including prostaglandin
E2 (PGE2). Cox-2 is an enzyme (EC 1.14.99.1) that participates in the formation of
important biological mediators called prostanoids, including prostaglandins, prosta-
cyclin and thromboxane. Hale and co-workers [40] also observed that bromelain re-
moved several types of cell surface molecules, thereby reducing leukocyte adhesion
and activation and thus decreasing inflammation. Moreover, Gaspani and co-workers
[38] revealed that bromelain-treated rats exhibited reduced concentrations of pros-
taglandin E2 (PGE2), which is a key mediator of the immune response. In addition,
bromelain decreases the levels of PGE2 and thromboxane A2 and modulates certain
immune cell surface adhesion molecules, which participate in the pathogenesis of
arthritis [40–44]. Bromelain can reduce cell surface receptors, such as the hyaluro-
nan receptor CD44 that is associated with leukocyte migration and an induction of
proinflammatory mediators [40, 45]. Manhart and co-workers [46] have supported
those findings when they demonstrated that bromelain significantly reduced CD4+ T
lymphocytes, which are the primary effectors in animal models of inflammation. Sev-
eral in-vivo results have revealed that bromelain acts as an anti-inflammatory agent.
In fact, numerous clinical trials with bromelain have demonstrated its efficacy in
treating various inflammation-based conditions, including sepsis in children [47], rhi-
nosinusitis [48], breast engorgement during lactation [49], urogenital inflammation
[50] and osteoarthritis of the knee and hip [51, 52]. In a mouse model of inflamma-
tory bowel disease, bromelain decreased the clinical and histological rigorousness of
spontaneous colitis and colonic inflammation [53]. Moreover, Secor and co-workers
[54] established that systemic bromelain decreased the inflammatory process in a
mouse model of allergic airway disease. Additionally, anecdotal evidence indicates
that bromelain may be effective in treating mild ulcerative colitis [55].
10.3.2 Bromelain as an Anti-Tumor Agent
Studies have demonstrated that bromelain can reduce tumor growth. Bromelain
was first reported to reduce malignant growth in 1972 by Gerard [56], followed
by Nieper in 1974 [57]. The anti-cancer effects of bromelain are largely attributed
to its protease activities [58]. Bromelain is believed to act on cancer cells via the
proteolysis of extracellular proteins such as CD44. CD44 is a glycoprotein adhesion
molecule that critically participates in tumor growth and metastasis [59, 60]. It is
a cell receptor for hyaluronic acid and participates in many stages of cancer me-
tastasis [61–63]. Multiple studies have implicated chronic inflammation, immune
suppression and deregulation of the hemostatic system in carcinogenesis [32]. This
suggests that bromelain may target pathways that directly participate in cancer ini-
tiation, growth and development. Current evidence reveals that bromelain may rep-
resent a potential target for developing oral enzyme therapies for cancer. In the past,
adjuvant therapy with external proteases has produced positive results in cancer
treatment; the therapy side effects were reduced and survival was prolonged [64]. In
150 A. Amid et al.
vivo studies have reliably demonstrated the tumor-inhibitory effects of bromelain.

In chemically induced mouse skin papillomas, bromelain decreased tumor forma-
tion and tumor volume and promoted apoptotic cell death [65]. These findings are
consistent with other studies where bromelain decreased metastasis [66] and local
tumor growth [67], thereby increasing survival rates. Moreover, in vitro bromelain
treatment in mouse tumor cell lines inhibited cell growth and Matrigel invasion
capabilities [68]. Another study revealed that bromelain inhibited cell adhesion and
migration in glioblastoma cell lines [69]. The same study also discovered that bro-
melain reduced the invasive capacity of glioblastoma cells and de novo protein
synthesis [69], suggesting that bromelain is a good cancer therapy candidate.
10.3.3 Burn Debridement and Wound Healing
Bromelain has been extensively used in burn debridement [70–72]. Therefore, the
enzyme could be a tenable option for surgical escharotomy in deep burn patients.
An in vitro study demonstrated that bromelain preparations can effectively debride
full-thickness burns in pig skin within 1 day. The enzyme affected only burned
skin and resulted in minimal blood loss [73]. In a porcine model of burn-induced
compartment syndrome, circumferential limb burns treated with bromelain ex-
hibited a significant reduction in intra-compartmental pressures [71]. In addition,
Rosenberg an co-workers [70] reported complete scar debridement (burned and
traumatized tissue) after one to two brief bromelain applications with minimal side
effects and no blood loss. Moreover, topical bromelain cream has been reported to
achieve complete debridement of experimental burns in rats within approximately
2 days [74].
Similarly, the enzyme may exert beneficial effects on soft tissue wound healing.
More rapid reductions in edema and bruising have been reported in patients with
episiotomy wounds treated with bromelain [73]. In a study of wound healing, bro-
melain treatment at the early phase decreased the soft tissue wound healing period
[75]. In another study conducted by Hu [76], bromelain greatly simplified the man-
agement of high-velocity gunshot wounds in a pig model. Moreover, bromelain was
also demonstrated hydrolyze devitalized tissue in wound tracks without apparent
injuries to the surrounding normal tissue and enhanced firearm wound healing [77].
10.3.4 Diarrhea Treatment
The anti-diarrheal activity of bromelain has been previously established [78, 79].
These studies have suggested that the proteolytic activity of bromelain inactivates
a specific glycoprotein receptor located on the intestinal mucosa and blocks at-
tachment of enterotoxigenic bacteria. Studies conducted by Mynott [80] have
indicated that stem bromelain exhibited anti-secretory properties by prevent-
ing fluid secretion mediated by secretagogues that act through cAMP (cyclic-3,
5- adenosine monophosphate), cGMP (cyclic-3, 5- guanosine monophosphate)
and calcium-dependent signaling pathways. Because toxins that cause diarrhea

activate one of these pathways, bromelain likely exerts anti-diarrheal effects. The
enzyme can also block secretory changes caused by prostaglandin E, theophyl-
line, calcium-ionophore A23187, 8-Br-cAMP 2 (8-bromocyclic-3, 5-adenosine
monophosphate) and 8-Br-cGMP (8- bromocyclic-3, 5-guanosine monophosphate),
which are well-characterized intracellular mediators of ion secretion. Experiments
performed by Roselli [81], which assessed the effects of different plant extracts and
natural substances (PENS) on membrane damage in pig intestinal cells, demon-
strated the protective effects of bromelain.
10.3.5 Bromelain as Anti-Thrombotic Agent
Bromelain can prevent the aggregation of human blood platelets and prevent or
minimize the severity of angina pectoris and transient ischemic attacks (TIA). The
enzyme is also essential for preventing and treating thrombosis and thrombophle-
bitis as well as for degrading cholesterol plaques and exerting fibrinolytic activity
[82]. Hale and co-workers [40] revealed that in vitro bromelain treatment of leuko-
cytes in whole blood modified 25 % of the leukocyte markers studied. The brome-
lain induced loss of CD41 and CD42a can be expected to reduce platelet function
and, hence, inhibit thrombus formation [74]. Similarly, Felton [83] suggested that a
bromelain plasminogen activator may produce plasmin in rat experiments. Plasmin
cleaves the Hageman factor and leads to a strong release of kallikrein but a weak
release of thrombin; moreover, a combination of fibrinolytic and antithrombic prop-
erties appear to be effective, as two large-scale tests in heart patients demonstrated
an almost complete elimination of thrombosis. In addition, bromelain was observed
to increase the permeability of vessel walls to oxygen and nutrients while increas-
ingly concentrating blood, both of which assist bromelain as antithrombotic agent
[84] Furthermore, Metzig and co-workers [84] revealed that pre-incubating human
platelets with bromelain completely prevented thrombin-induced platelet aggrega-
tion in vitro. Correspondingly, the authors reported that bromelain inhibited in vivo
thrombus formation in a model of laser-induced thrombosis in rats.
10.3.6 Enhancing the Immune System
Bromelain bolsters the immune system by increasing cytokine production, which

are hormones produced by white blood cells to improve immunity. Several studies
have established the ability of bromelain to remove T-cell CD44 molecules from
lymphocytes [40, 85, 86]. In a study conducted by Munzig and co-workers [87],
highly purified bromelain protease F9 decreased the expression of CD44 ten times
more than crude bromelain, resulting in an approximately 97 % inhibition of CD44
expression. Similarly, Roep and co-workers [86] discovered that protease treatment
reduced the expression of cell surface receptors on T-cells and antigen-presenting
cells. Moreover, protease therapy has been reported to reduce CD44 expression
152 A. Amid et al.
Table 10.4 Bromelain-based personal care products

Brand Country Remarks Price (USD) Function
NOW Food USA 2500 GDU 29.99 Digestive aid
SOLARAY UK Mix with quercetin and 28.09 Dietary supplement
vitamin C
Doctor’s BEST USA Mix with quercetin 35.99 Food supplement
Natrol USA Maximum strength 21.99 Digestive aid
Natural Radiance USA Mix with glucosamine 58.98 Pain relieving cream
PERFECT USA Mix with papaya extract 34.95 Facial peel s
IMAGE LLC and alpha hydroxy acids
Kate Somerville USA – 75.00 Dark circle eye
cream
on lymphocytes from patients with multiple sclerosis [86, 87]). Furthermore, Hale
[44] discovered that bromelain exhibits a strong immunogenicity subsequent to oral
dosing. Additionally, Hale and co-workers [88] revealed that repeated exposure was
necessary for the maturity of anti-bromelain antibodies with a dose-dependent ex-
posure period of 3–6 weeks.
10.4 Bromelain in the Personal Care Industry
Bromelain has relatively recently become available for commercial purchase at

numerous personal care outlets. It helps to degrade dead, dry surface skin cells,
leading to softer and smoother skin. This effect is helpful for dry and/or blemished
skin. In fact, bromelain degrades the connecting structure that holds surface skin
cells together, which is exfoliating but can be irritating. However, more studies are
needed to demonstrate how bromelain acts on the skin. Bromelain is also consumed
as a food supplement and formulated as a topical cream to relieve pain. Table 10.4
displays several examples of bromelain-based personal care products including the
brand, activity or strength offered and price.
10.5 Bromelain in the Animal Feed Industry
Feed enzymes are typically added to animal feed to increase nutrient bioavail-
ability by acting on feed components prior to or after consumption. Theoretically,
bromelain would digest proteins in animal feeds into smaller units and promote
their absorption by the digestive track. Bromelain is used in animal feeds, es-
pecially for ruminant animals, as a digestive aid in the lumen and as a mastitis
preventative. Additionally, bromelain supplements have been demonstrated to
increase milk protein and milk fat in dairy goats [89]. An independent study by
Table 10.5 Bromelain sold as a food additive in animal feeds

Product Company Country
Bromelain powder Rolling Dies Manufacturer Thailand
Bromelain powder Wisapple China
Bromelain powder K&G Global Business Corp Taiwan
Bromelain powder BIO-CAT Inc Spain
Tománková and Kopečný [90] indicated that bromelain had the highest protein
degradation efficiency compared with pronase E and papain [90]. Table 10.5 pro-
vides examples of companies that sell bromelain suitable for use in animal feeds.
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73. Orsini RA (2006) Bromelain. Plast reconstr surg 118:1640–4
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81. Roselli M, Britti MS, Le Huerou-LuronI, Marfaing H, Zhu WY, Mengheri E (2007) Effect
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82. Kelly GS (1996) Bromelain: a literature review and discussion of its therapeutic applications.
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bromelain. Anim Feed Sci Technol 53:71–80
Chapter 11
Case Study: Recombinant Bromelain Cloning,
Characterization and Upstream Processes
Abstract This chapter presents the recombinant stem bromelain cloning procedure
followed by its characterization and finally upstream processing. Most of the pro-
cedures had patents filed in Malaysia, Europe and the United State of America with
application numbers PI 20095434, 10015711.4, and 12968766, respectively. Por-
tions of the experimental procedures were also presented in our publication (Amid
et al., Process Biochem 46:2232–2239, 2011). The entire cloning process, charac-
terization and the upstream processing of the recombinant bromelain production are
briefly explained.
11.1 Cloning of Recombinant Bromelain [1]
Detail procedures have been discussed by Amid and co-workers [1]. In most cases,
experiments aimed at identifying suitable genetic material for use in the cloning
procedure. In our case, active recombinant bromelain must be able to be expressed
in simple organisms such as E. coli because E. coli requires simple fermentation
media and can be harvested after a short period. Therefore, suitable genetic mate-
rial should be full length mRNA encoding stem bromelain because mRNA contains
only coded nucleic acids versus genomic DNA which contains coded and non-trans-
lated regions. In fact, each gene requires unique cloning procedures dependent upon
the amount of available target gene information and the final aim of the cloning pro-
cedures. For stem bromelain, the detailed mRNA sequence was obtained from the
National Centre for Biotechnology (www.ncbi.nlm.nih.gov) and our cloning aim to
express stem bromelain protein in a prokaryote host. Therefore, the suitable cloning
A. Amid () · M. J. A. Jamaluddin
M. J. A. Jamaluddin
N. A. Ismail
Campus Puncak Alam, University Technology Mara, Shah Alam, Selangor, Malaysia
DOI 10.1007/978-3-319-12397-4_11
160 A. Amid et al.
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Fig. 11.1 Flow chart summarizing the entire cloning procedure
methods were PCR for target gene amplification, direct ligation into the cloning
vector, transformation into a suitable host ( E. coli) and finally, protein expression.
Figure 11.1 summarizes the cloning procedures.
11 Case Study: Recombinant Bromelain Cloning, Characterization … 161
Next, we needed to identify source material for total RNA isolation. Because
stem bromelain was the target protein, pineapple stem was the most suitable plant
material RNA source. In this study, pineapple stems were collected from the Ma-
laysia Agricultural Research and Development Institute (MARDI), Jalan Kebun,
Klang, Selangor, Malaysia.
After source material collection, total RNA must be extracted. There are multiple
total RNA extraction techniques and many commercial extraction kits are available.
RNA extraction kit selection is normally based on individual experience and other
factors such as simplicity, availability and reproducibility. In this experiment, total
RNA extraction was performed using the RNeasy Plant Mini Kit (QIAgen, Ger-
many). However, all equipment must be RNAse free treated to ensure a sufficient
RNA amount can be isolated.
11.1.1 Complementary DNA (cDNA) Synthesis
When using mRNA as a genetic source material, the cDNA synthesis step is re-
quired because mRNA does not have the stability for genetic manipulation. In this
section, mRNA (total RNA containing mRNA, tRNA and rRNA) is used to synthe-
size cDNA using the SuperScript II Reverse Transcriptase. Again, the choice of kit
is dependent on a scientist’s experience and preference.
11.1.2 Bromelain Gene Amplification
There are multiple procedures for obtaining the correct DNA fragment for cloning
purposes such as restriction enzyme digestion, mechanical restriction and polymerase
chain reaction (PCR). However, each method has its advantages and disadvantages.
Because the complete stem bromelain mRNA sequence was available in public data-
bases, gene amplification by PCR techniques was simplified. Initially, proper primer
pair design needs to be performed. Approximately 30 years ago, scientists designed
primer pairs manually to ensure the stringency of primer binding because primer
binding can affect the number of PCR products during amplification. General con-
siderations are primer length, melting temperature, a unique template DNA sequence
and avoidance of repetitive and single-base sequences [2]. Currently, there are many
software programs that are useful for designing primers such as Prime3 version 4
(http://frodo.wi.mit.edu/), Autoprime (http://www.autoprime.de/AutoPrimeWeb),
and Primer Premier (http://www.premierbiosoft.com/primerdesign/index.html). Af-
ter primers design, primer synthesis is performed. Many scientific supply companies
provide this service. Normally, primers can be received within three working days
along with the necessary details. After the primers arrive, primer optimization can
be performed. Our laboratory has a standard reagent mixture for PCR components
and only annealing temperature optimization was performed. The following proto-
col is used in our laboratory to determine the optimal annealing temperature. The
aim of these experiments is to obtain a single PCR product band. The PCR product
162 A. Amid et al.
Fig. 11.2 A single band

representing a desirable
PCR product after annealing
temperature optimization. M
denotes the molecular weight
marker
M PCR
was analyzed using gel electrophoresis, Ethidium Bromide (EtBr) staining and was
documented using a gel documentation system (Fig. 11.2).
11.1.3 Verifying the Correct DNA Fragment for the Target Gene
The next step after DNA fragment amplification is verifying that the amplified se-
quence is correct. Usually, Basic Alignment Search Tool (BLAST) is used for this
purpose. This resource is provided by the NCBI public database. However, before
proceeding to BLAST analyses, amplified DNA fragments are first purified fol-
lowed by sequencing. Many scientific companies can provide sequencing services.
Researchers provide the company with the primer details used during the PCR
process. When the sequencing results arrive, BLAST analyses can be performed.
BLAST results determine if the amplified DNA sequences are suitable for liga-
tion into the desired cloning vector. The sequencing results were aligned with the
complete stem bromelain mRNA sequence using the “nucleotide BLAST” program
(http://blast.ncbi.nlm.nih.gov/).
11.1.4 Bromelain Gene Ligation into the Entry Vector
A fragment of DNA material cannot propagate itself to produce enough genetic ma-
terial for subsequent generations. The DNA fragment requires a host for propaga-
tion. However, the DNA fragment cannot be inserted into a suitable host without the
assistance of a vector. A vector is usually double stranded DNA equipped with an
origin of replication (ORI), the initial site for DNA replication, together with other
necessary tools such as unique restriction sites for ligation, selectable markers for
the selection process and must be small enough for genetic manipulation. Vectors
intended for different applications i.e., gene structural studies or protein expres-
sion. Some vectors have a broad range of hosts, referred to as shuttle vectors. This
study used pENT/TEV/D-TOPO vector. This vector is equipped with a kanamycin
resistance gene as a selectable marker, the pUC ori gene as the origin of replica-
tion as well as other specific features (from pENTR™ Directional TOPO® Cloning
Kit User Guide 2012 http://tools.invitrogen.com/content/sfs/manuals/pentr_dtopo_
man.pdf) (Fig. 11.3).
Fig. 11.3 Vector map for

vector used to clone brome-
lain gene
11.1.5 Introduction into a Host Cell for Amplification
11.1.5.1 Transformation into E. coli Cells
After the candidate gene is cloned into a suitable vector, the new recombinant vec-
tor is transferred into a suitable host, in this case, E. coli. The simplest procedure
is the heat shock method. During this procedure, the surface of the E. coli host,
which has undergone competent cell preparation, is surrounded by the recombinant
vector at ice cold temperatures. The lipid bilayers that structure the cell membrane
are compressed at 4 °C. When the cell is rapidly transferred to a high temperature
(42 °C), the lipid bilayers expand and form pores in the cellular membrane. This
mechanism allows the recombinant plasmid to enter the cell. When the cell is again
cooled to 4 °C the pores closed. The cell is now referred to as recombinant E. coli
and harbors a recombinant vector. Once inside the host cells, the recombinant vec-
tor replicates and transfers to host daughter cells during cellular propagation.
164 A. Amid et al.
11.1.6 Clone Selection
11.1.6.1 Colony PCR to Identify Positive Transformants
During transformation not all competent cells receive the recombinant vector.
Colony PCR is a rapid method to identify positive transformants without having
to extract the recombinant plasmid. The PCR procedure is similar to the previous
reaction except the DNA template is a 1 µL sample from colonies grown on LB
agar plates supplemented with 50 µg/mL kanamycin from the previous experiment.
During DNA sampling, half of the colony is subcultured onto new LB containing
kanamycin plates and the other half is mixed with 10 µl sterile distilled water. The
results were obtained by analyzing the PCR product using agarose gel electropho-
resis. Single bands similar in size to the bromelain gene need to be recovered to
confirm the positive transformant.
11.1.6.2 Verification of Correct Insert Orientation in the Recombinant

Plasmid by Restriction Enzyme Digestion
Identifying the correct orientation is very important to ensure the gene was tran-
scribed and translated correctly. Without correct transcription and translation, no
active protein will be expressed. Therefore, ensuring correct insert orientation is a
useful method for avoiding repeated cloning procedures.
11.1.7 Subclone into Destination Vector by LR Recombinase

Reaction
The recombinant plasmid must be subcloned into a destination vector after confir-
mation of the insert orientation. Destination vector selection is based on host type
and final product. In this case, the pDEST 17 was chosen as the destination vector.
11.1.8 Pilot Expression Study
It is crucial to determine if the entire cloning process was successful. In this step,
samples are divided into two groups: one group is induced with L-arabinose; one
group is not (control). Supernatant is extracted from both samples and used in an
enzyme activity assay. Figure 11.4 shows the results from the expression study us-
ing L-arabinose as an inducer.
Next, recombinant bromelain expressed by E. coli must be purified to be suit-
able for characterization experiments. Cell pellets were collected from an induced
Fig. 11.4 SDS page analysis

to determine protein expres-
sion after L-arabinose induc-
tion. Lanes 1 to 4 show the
protein collected at hourly
time points after L-arabinose
induction. Arrows indicate
the presence of recombinant
bromelain in the cell lysate
Fig. 11.5 SDS-PAGE and

western blot results after
protein purification under
native conditions. M is the
protein marker (10–150 kDa)
followed by the protein
sample by SDS-PAGE ( 1)
and western blot ( 2)
culture and are then lysed to isolate the intracellular protein. The cells were lysed,
washed and eluted using sodium dihydrogen phosphate (NaH2PO4), sodium chlo-
ride (NaCl) and imazadole at pH 8.0. Purification under native conditions indicates
the target protein is soluble in the cytoplasm. Conversely, when the purified protein
(native) does not appear as a band in the SDS-PAGE, the recombinant enzyme is
within inclusion bodies, which is undesirable because the protein it is not in active
form. The appearance of purified recombinant bromelain was confirmed using So-
dium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis.
Figure 11.5 shows the SDS-PAGE analysis results, clearly identifying recombinant
bromelain as a single band after purification using a Ni-NTA column.
11.2 Recombinant Bromelain Characterization
11.2.1 Recombinant Bromelain Concentration
To determine the recombinant bromelain concentration in the purified samples, an

enzyme-linked immunosorbent assay (ELISA) was performed. The ELISA method
was used to accurately quantify the purified recombinant bromelain in the samples
without the addition of other protein mixtures. Initially, a commercial bromelain
166 A. Amid et al.
Fig. 11.6 Recombinant bromelain concentrations in unpurified and purified samples. Standard
error is based on duplicate values
standard curve was constructed using ELISA. Figure 11.6 shows the recombinant
bromelain concentration for two types of samples; unpurified and purified. Com-
paring both conditions, before and after purification, recombinant bromelain con-
centration in the unpurified sample is higher than the purified sample. After the
purification process, the concentration was reduced because the majority E. coli
proteins are removed during the purification process. Consequently, purification
reduces the amount of recombinant protein in the sample [3]. Therefore, specific
recombinant bromelain quantification can be accurately obtained using ELISA.
11.2.2 Recombinant Bromelain Activity
Recombinant bromelain activity was determined using titrametric and spectropho-

tometric methods. The optimal recombinant bromelain activity temperature and pH
were measured using a continuous spectrophotometric procedure at 340 nm. In the
titrimetric assay of recombinant bromelain activity, gelatin was used as a substrate
and the proteolytic activity of recombinant bromelain was measured using the Gela-
tin Digestion Unit (GDU). GDU is used to determine the rate at which bromelain
catalyzes the degradation of the protein gelatin (substrate) at specific temperatures
and pH. Gelatin is a purified structural protein derived from animal tissues that is
high in collagen such as tendons and cartilage. The rate at which gelatin (substrate)
is degraded or the rate that the products of this digestion (amino acids) are produced
can be related to bromelain activity. One unit of bromelain will hydrolyze 1.0 mg
of amino nitrogen (Alanine, Lysine, Glycine, Tyrosine) from gelatin in 20 min at
pH 4.5 and 45 °C. Figure 11.7 shows the enzyme activity of recombinant bromelain
compared with commercial bromelain.
Fig. 11.7 Gelatin digestion

unit of recombinant and com-
mercial bromelain. Standard
error is based on duplicate
values
Table 11.1 Comparison of activity between recombinant and commercial bromelain

Samples Recombinant bromelain Commercial bromelain
Enzyme activity (U/mL) 48 33
Amount of total protein (mg/mL) 39 39
Specific activity (U/mg) 1.231 0.846
The digestion activity of recombinant bromelain against gelatin was 1750 GDU/g

at pH 4.5 and 45 °C, whereas commercial bromelain produced 1680 GDU/g. The
activity of recombinant bromelain was higher and closely related to the commercial
bromelain (Fig. 11.7). This may be due to the higher purity of recombinant brome-
lain compared with commercial bromelain.
11.2.3 Recombinant Bromelain Specific Activity
The specific activity of bromelain was calculated based on the activity of the en-
zyme divided by amount of protein in the samples. Specific activity is normally
expressed as units/mg and is an important measurement of enzyme purity and qual-
ity. Specific activity is a constant value across different batches of pure enzyme pro-
duction. The specific activity of recombinant bromelain increases as the protein is
purified (Table 11.1). The higher activity displayed by purified recombinant brome-
lain indicates an efficient purification strategy with 41-fold purification. The higher
purity of the enzyme and higher specific activity are amenable to pharmaceutical
and therapeutic applications.
11.2.4 Effect of Temperature on Recombinant Bromelain Activity
Bromelain activity at different temperatures ranging from 15 to 65 °C is shown

in Fig. 11.8. The activity of recombinant bromelain is increases from 15 °C until
it reaches maximal activity at 45 °C where the activity is 3.794 U/mg. Bromelain
168 A. Amid et al.
Fig. 11.8 Enzyme activity of

purified recombinant brome-
lain at different temperatures
activity decreases at 55 °C and continues decreasing until the activity is abolished
to 0 U/mg at 65 °C. Using the LNPE substrate, the purified recombinant bromelain
demonstrated the highest hydrolytic activity at 45 °C under routine assay condi-
tions. The activity of recombinant bromelain was moderate between 15 to 35 °C and
the enzyme was devoid of detectable activity at 65 °C.
Based on the optimum temperature for recombinant bromelain activity, recombi-
nant bromelain can be used for most industrial applications, particularly the baking
and dairy industries [4]. Because the recombinant bromelain retains its activity at
higher temperatures, it also may be used as a meat tenderizer in the food processing
industry. Additionally, recombinant bromelain could be used as a biological deter-
gent together with papain [5] and lipase in industrial purposes [6].
11.2.5 Effect of pH on Recombinant Bromelain Activity
Figure 11.9 shows recombinant bromelain activity versus pH ranging from pH 1 to

10. The purified recombinant bromelain demonstrated optimal activity at pH 4.6
for LNPE hydrolysis. The recombinant enzyme retains partial activity below pH
4.6 but fails to demonstrate significant activity beyond pH 6. Pillai and co-workers
and Voegeli and co-workers demonstrated that pH 4.6 was the optimal condition
for bromelain activity [7, 8] The maximum bromelain activity occurred at pH 4.6
following the theory mentioned in the bromelain assay description [9]. The maxi-
mal activity was achieved at 2.254 U/mg followed by the lowest point at pH 5,
0.135 U/mg. After reaching maximal activity, recombinant bromelain activity de-
creased to 0 U/mg activity. Recombinant bromelain activity ceases at pH 6–10.
These results indicate that recombinant bromelain is active in acidic conditions
rather than alkaline conditions. Recombinant bromelain is also inactive at neutral
pH. This coincides with the majority of research results demonstrating that most
proteases are active in acidic conditions excepting alkaline proteases such as kera-
tinase [10]. The higher activity achieved in acidic conditions may be beneficial for
Fig. 11.9 Enzyme activity of

purified recombinant brome-
lain at different pH levels
the use of recombinant bromelain in certain therapeutic applications. Recombinant

bromelain could be used as a digestive enzyme in the acidic conditions of the stom-
ach. Recombinant bromelain would also be active throughout the gastrointestinal
tract [5, 11]. Moreover, recombinant bromelain could be used in cosmetic applica-
tions because would remain active in acidic environments, which is appropriate for
the majority of the human body and skin [7, 8].
11.3 Recombinant Bromelain Upstream Processes
To ensure high recombinant bromelain expression and productivity, the most suit-
able media had to be identified. During cloning and bench scale experiments, Luria
Bertani agar and broth were utilized; however, the amount of recombinant brome-
lain produced was not sufficient for the commercial scale. Therefore, the upstream
processing team investigated the most suitable media formulation for recombinant
bromelain production. The team then optimized the fermentation process using the
selected media. The entire experimental design is described below in Fig. 11.10.
11.3.1 Identifying a Suitable Media Formulation
Nine media formulations were tested to observe their effect on the growth of recom-
binant E. coli harboring the bromelain gene and recombinant bromelain expression.
The chosen media were 2X YT, 5XLB, Studier Autoinduction, TY, LB (Miller), GE,
Teriffic Broth (TB), Super Broth (SB) and M9 Minimal Media. Specific activity and
cell dry weight were measured for analysis.
Figure 11.11 shows that the media formulation named Studier Autoinduction
performed the best formulation by producing highest recombinant E. coli biomass
and specific recombinant bromelain activity. Because Studier Autoinduction media
170 A. Amid et al.
Fig. 11.10 Process flow diagram for the experimental design to determine the optimal media for-
mulation and fermentation conditions for large scale recombinant bromelain production
Fig. 11.11 Preliminary media screening. Comparison of recBromelain specific activities (right bar)
and cell dry weights (left bar) as a function of culture media
Fig. 11.12 The standardized effect of all parameters from Plackett-Burman screening. The col-
umns shaded in dark grey represents highly significant parameters where p<0.0001; light grey
represent significant parameters where p<0.05; and medium-dark grey represents insignificant
parameters where p>0.05
has 22 components, the most significant media component had to be identified to

minimize tedious experimental design. A Plackett-Burman experimental design was
chosen to identify the most significant media component for examination in the
subsequent media optimization experiment.
All tested media components are listed in Fig. 11.12. A positive effect is repre-
sented by a positive value and a negative effect is represented by a negative value.
Figure 11.12 shows that several media components had significant effects, but three
were chosen for subsequent experiments: tryptone, NH4Cl and CoCl2. To charac-
terize the suitable range of each parameter, an OFAT experimental design was em-
ployed. The results indicated that 4–14 % (w/v) tryptone, 100–260 mM NH4Cl and
0.4–1.2 µM CoCl2 were suitable ranges for use in media optimization experiments.
Table 11.2 shows the results from the media formulation optimization which
indicated that the highest specific activity, 0.672 ± 0.31 U/mg, was obtained when
14 % tryptone, 100 mM NH4Cl and 0.4 µM CoCl2 were added and the remaining
components were fixed. Optimal media formulation alone does not ensure high
recombinant bromelain production. Therefore, the team proceeded to optimize the
fermentation process in a small scale bioreactor.
11.3.2 Optimizing the Fermentation Process Condition
The parameters obtained in the small scale fermentation process were used to scale
up the fermentation process to obtain high recombinant bromelain production. Four
parameters were tested during the fermentation process optimization: dissolved
172 A. Amid et al.
Table 11.2 Results and the experimental design to determine the optimal media formulation for
high recombinant bromelain specific activity
oxygen (15–35 % saturation), aeration (1–4 vvm), pH (6.6–7.4) and temperature

reduction (20–30 °C).
The contour graph in Fig. 11.13 indicates that the optimal process conditions for
2 L bioreactor (1 L working volume) fermentations are: dissolved oxygen, 35 %;
pH, 7.4; temperature reduction to 30 °C; aeration, 1 vvm. The optimized media for-
mulation and fermentation process were used in the scale up trial.
11.3.3 Scaling up Fermentation Volume for Recombinant

Bromelain Production
Two scale up methods were tested for recombinant bromelain production in 30 L
bioreactors (18 L working volume) with constant tip speeds and kLa ranges.
Table 11.3 summarizes our observations while scaling up the fermentation vol-
ume for recombinant bromelain production. Based on the results obtained, the con-
stant kLa range method is suitable for scale up because the biomass only reduced
Fig. 11.13 The generated 3-D plot with respect to dissolved oxygen and pH, derived from statisti-
cal model equation
Table 11.3 Methods for scaling up fermentation volume for recombinant bromelain production
/SURGXFWLRQ /SURGXFWLRQ
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&DVFDGHVHWXSUSP FDOFXODWHG FDOFXODWHG
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)L[HGVHWSRLQWV $HUDWLRQ YYP
RSWLPL]HG 7HPSHUDWXUH &KRXUVDQG&KRXUV
'LVVROYHGR[\JHQ
S+
:HWFHOOZHLJKWJ/ 33.3 (↓11.4%) 35.0 (↓6.9%)
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8PJSURWHLQ
6.9 % compared with the constant tip speed method (11.4 % reduction). These re-
sults are also suitable for the specific activity of recombinant bromelain, where
the constant kLa range method produced a 6.5 % reduction compared with 38.7 %
using the constant tip speed method. Therefore, the constant kLa range method
was chosen for scaling up the fermentation volume for recombinant bromelain
production.
174 A. Amid et al.
References
tion of a recombinant stem bromelain from Ananas comosus. Process Biochem 46:2232–
2239
2. Nicholl DST (2009) An introduction to genetic engineering, 3rd edn. Cambridge University
Press, Cambridge
3. Koller CA, Kohli V (1993) Purification of genomic DNA using heparin to remove nuclear
proteins. Nucleic Acids Res 21:2952
4. Vanhanen M, Tuomi T, Hokkanen H, Tupasela O, Tuomainen A, Holmberg PC et al (1996)
Enzyme exposure and enzyme sensitisation in the baking industry. Occup Environ Med
53:670–676.
5. Liu ZB, Weis R, Glieder A (2004) Enzymes from higher eukaryotes for industrial biocataly-
sis. Food Technol Biotech 42:237–249
6. Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT (1999) The realm of
microbial lipases in biotechnology. Biotechnol Appl Bioc 29:119–131
7. Pillai S, Oresajo C, Hayward J (2005) Ultraviolet radiation and skin aging: roles of reac-
tive oxygen species, inflammation and protease activation, and strategies for prevention of
inflammation-induced matrix degradation—a review. Int J Cosmet Sci 27:17–34
8. Voegeli R, Rawlings AV, Doppler S, Heiland J, Schreier T (2007) Profiling of serine protease
activities in human stratum corneum and detection of a stratum corneum tryptase-like en-
zyme. Int J Cosmet Sci 29:191–200
9. Heinrikson RL, Kezdy FJ (1976) Acidic cysteine protease inhibitors from pineapple stem.
Methods Enzymol 45:740–751
10. Rawlings ND, Barrett AJ (1994) Families of cysteine peptidases. Methods Enzymol 244:461–
486
11. Walsh G, Headon DR (1994) Protein biotechnology. Wiley, New York
Chapter 12
Case Study: Recombinant Bromelain
Downstream Processing
Azura Amid, Zatul Iffaf Mohd Arshad and Muhd Ezza Faiez Othman
Abstract This chapter presents details on the purification, formulation and drying
of recombinant bromelain. The wide range of applications of recombinant brome-
lain has increased interest in finding viable purification techniques for large-scale
production. An affinity chromatography technique was developed by Amid and
co-workers (Expression, purification, and characterization of a recombinant stem
bromelain from Ananas comosus, Process Biochem 46:2232–2239, 2011) to purify
recombinant bromelain. However, this technique presented low recovery and small
sample loading capacity and thus is not suitable as a purification tool in the large-
scale production of recombinant bromelain. An aqueous two-phase system is one
alternative method that we use to purify recombinant bromelain, as it reliable and
easy to scale up and has a low cost. As part of avoiding cysteine degradation, spray
drying the purified recombinant protein with maltodextrin as an excipient provides
the possibility of preserving its activity and creating fine particles that are suit-
able for end-product application. The processes of the purification, formulation and
spray drying of recombinant bromelain are explained briefly.
Keywords Back extraction · Continuous ultrasonication · Gibbs free energy · Inlet

air temperature · Maltodextrin · PEG-rich phase · Salting-out · Immiscible
A. Amid () · Z. I. M. Arshad
M. E. F. Othman
DOI 10.1007/978-3-319-12397-4_12
176 A. Amid et al.
Fig. 12.1 Contour plot on the effect of cycles, A (passes), and flow rate, B (ml/min), on the spe-
cific activity, Y (U/mg-protein), of continuous cell disruption by ultrasonication
12.1 Cell Disruption by Continuous Ultrasonication
A normal cell disruption technique applied for large-scale processing is homogeni-

zation. However, the produced recombinant bromelain is heat sensitive, and heat
generated during the homogenization process deactivates the enzyme, and thus, a
continuous ultrasonication technique was used in our case. Figure 12.1 shows the
effect of the flow rate and bursting cycles during the ultrasonication process on the
specific activity of our recombinant bromelain product. These data suggest that a
medium bursting cycle and medium flow rate provide the highest specific activity
of recombinant bromelain. A high flow rate might not be able to disrupt most of the
cells in the medium, whereas too high of a bursting cycle might reduce the specific
activity of the enzyme.
12.2 Partial Purification of Recombinant Bromelain by

Ammonium Sulfate Precipitation
Partial purification by ammonium sulfate precipitation was performed using am-

monium salt powder as reported in Doonan [2] with a slight modification. At
50 % (w/v) ammonium sulfate saturation and above, darker visible bands could be
12 Case Study: Recombinant Bromelain Downstream Processing 177
Fig. 12.2 Precipitate protein

fraction under SDS-Page.
Lanes: ( 1) a PageRuler®
marker and ( 2) 25 %, ( 3)
50 %, ( 4) 75 % and ( 5) 100 % .'D
ammonium sulfate saturation
observed. Based on the previous report by Amid and co-workers [1], the band for
recombinant bromelain is positioned at approximately 50 kDa (Fig. 12.2).
12.3 Purification of Recombinant Bromelain
The ATPS system involves the formation of two immiscible phases after the mixing
of polyethylene glycol (PEG)-salt in an aqueous solution. In this work, we evaluate
the performance of PEG8000/K2HPO4 in purifying recombinant bromelain using
a two-step ATPS method: forward and back extraction. The factors affecting the
partition behavior of recombinant bromelain, such as the pH, type of salt and con-
centration of PEG8000 and K2HPO4, were also investigated to elucidate the opti-
mum conditions for purification of the enzyme. Figure 12.3 summarizes the ATPS
extraction method.
12.3.1 Selection of the Salt Types
To identify a suitable salt for the purification of recombinant bromelain, several

ATPSs were conducted using different salt types and amounts of K2HPO4, Na2SO4
and (NH4)2SO4. A phase system containing 17 % w/v PEG8000 and 20 % w/v
K2HPO4 provided the highest specific activity, 4.194084 unit/mg, as shown in
Fig. 12.4, and this system was selected for further study. The amount of lysate r-
bromelain used in this study was 20 % (w/v). The ability of K2HPO4 to produce
a higher salting-out effect led to an effective partition of recombinant bromelain
in the PEG-rich top phase. This is because anions with higher valence, such as
178 A. Amid et al.
Fig. 12.3 Flow chart showing the ATPS extraction method
Fig. 12.4 Specific activity of

recombinant bromelain with
different salt types
K2HPO4, are better as salting out agents and decrease the amount of water avail-
able to hydrate polymers [3]. This findings follows the order of Gibbs free energy
of hydration: ΔGhyd(HPO42−) = − 1789 kJ/mol > ΔGhyd(SO42−) = − 1080 kJ/mol. A
superior salting-out ability is related to a higher negative value of Gibbs free energy
of hydration [4, 5].
12.3.2 pH Screening
Figure 12.5 shows the recombinant bromelain specific activities versus pH, ranging
from pH 6 to 9, with 17 % w/v of PEG8000 and 20 % w/v K2HPO4. The amount
of lysate r-bromelain used in this study was 20 % (w/v). The maximum recom-
binant bromelain activity occurred at pH 5 [1]; however, under ATPS conditions,
it presented an apparent optimum specific activity at pH 8 of 8.433884 Unit/mg.
Under alkaline conditions, PEG8000 appears to stabilize the biological activity of
Fig. 12.5 Specific activity
6SHFLILFDFWLYLW\XQLWPJ
of recombinant bromelain at 7RS
different pH values

S+
Fig. 12.6 Specific activity of

6SHFLILFDFWLYLW\XQLWPJ
recombinant bromelain at dif-

ferent K2HPO4 concentration

7RS

.+32 FRQFHQWUDWLRQZY
recombinant bromelain [6]. At higher pH values, the recombinant bromelain is neg-

atively charged and will partition more into the PEG-rich phase [7].
12.3.3 Salt Concentration Screening
The effect of the K2HPO4 concentration on the recombinant bromelain partition is

shown in Fig. 12.6. All of these experiments were performed at pH 8.0 with 17 %
w/v PEG8000 and different concentrations of K2HPO4, from 14 to 20 % w/v. The
amount of lysate r-bromelain used in this study was 20 % (w/v). The maximum
value of specific activity in the top phase is achieved at a concentration of 16 % w/v,
and thus, it was selected for further experiments. When the K2HPO4 concentration
was higher than 16 % w/v, the specific activity decreased due to the accumulation
of recombinant bromelain in the interface of the bottom phase lower partition in the
PEG-rich phase [7].
12.3.4 Screening of PEG8000 Concentration
The effect of the PEG8000 concentration, ranging from 12 to 20 % w/v, was used to
study the partitioning of recombinant bromelain, as shown in Fig. 12.7. During the
180 A. Amid et al.
Fig. 12.7 Specific activ- 2

ity of recombinant brome- 1.8
Specific activity, Unit/mg

lain at different PEG8000 1.6
concentration 1.4
1.2
1
0.8 Top
0.6
0.4
0.2
0
12% 14% 18% 20%
PEG8000 concentration ( % w/v)
experiment, the K2HPO4 concentration was maintained at 16 % w/v, pH 8.0, with
20 % (w/v) lysate r-bromelain. It was found that the highest specific activity was
achieved at 18 % w/v of PEG8000. It can be observed that the specific activity of
recombinant bromelain is marginally increased from 12 to 16 % w/v and then starts
to decrease at 20 % w/v. An increase in the polymer concentration causes the space
available for recombinant bromelain to partition into the top phase to be reduced,
and consequently, the protein tends to partition into the bottom phase [8].
12.3.5 Optimizing the ATPS Process Conditions
All of the values obtained from OFAT screening were used in the optimization of
the ATPS process conditions. Three parameters were tested during the purifica-
tion process optimizations: pH (6.5–8.5), K2HPO4 concentration (14–18 % w/v),
and PEG8000 concentration (16–20 % w/v). The response surface plot graph in
Fig. 12.8 indicates that the optimal process conditions for ATPS were 20 % (w/v)
PEG8000 and 18 % (w/v) K2HPO4 solutions at pH 6.5. Next, these optimized pro-
cess conditions were applied to the back extraction purification method.
12.3.6 Back Extraction ATPS Method
In the forward extraction, the chosen ATPS conditions included 20 % (w/v) PEG8000
and 18 % (w/v) K2HPO4 solutions (pH 6.5). Most of the recombinant bromelain par-
titioned to the top phase, and the bottom phase was discarded. A purification fold of
16.92 and yield of 20.29 % were obtained in the forward extraction. In the second
ATPS, fresh 18 % (w/v) K2HPO4 and 8 % (w/v) (NH4)2SO4 solutions were added,
and most of the recombinant bromelain was shifted to the top phase. After two-
step extractions, the purification fold of the top phase was 3748.48, and the yield
was 13.85 %. The purification fold was higher compared with the single extraction
method reported by Ketnawa and co-workers of (3.44-fold) [9]; and Ketnawa and
Fig. 12.8 Response surface plot of two variables pH and K2HPO4 concentration
Table 12.1 Purification table of recombinant bromelain in PEG8000/K2HPO4 ATPS by two-step

extraction at pH 6.5 and 25 °C
Total protein Enzyme activ- Specific enzyme Purification Yield
(mg/ml) ity (unit/ml) activity (unit/mg) fold (%)
Crude 1.05 0.42 0.39 1 100
Forward Top 0.074 0.50 6.71 17.21 20.29
extraction phase
Back Top 0.015 22.70 1486.63 3811.87 13.85
extraction phase
co-workers (2.23-fold) [10]; Therefore, the two-step extraction method can facili-
tate the separation of recombinant bromelain from the PEG-rich phase in addition
to helping achieve a high purification factor. Table 12.1 shows a summary of ATPS
purification involving both the forward and back extraction method.
12.4 F
ormulation and Spray Drying of Recombinant
Bromelain
Spray drying is commonly used in the production of pharmaceutical powders. In the

present study, the top phase sample consisting of purified recombinant bromelain
in a PEG-rich phase was collected before undergoing the spray drying process. The
procedure was initiated by conducting the excipient screening. Five distinguished
and commonly used excipients in the food and pharmaceutical industry were cho-
sen. The main aim was to select a suitable excipient that could protect the recombi-
nant enzyme solution from rapid dehydration during spray drying, thus retaining as
much specific activity of the final spray dried recombinant bromelain as possible.
182 A. Amid et al.
Fig. 12.9 Excipients screening with different types of excipients
The five selected excipients used for screening were Arabic gum, lactose, maltodex-
trin (dextrose equivalent, DE 10), mannitol and sucrose, which have been reported
as binder agents, filler agents and microencapsulation agents for protecting bioac-
tive compounds or active pharmaceutical compounds in many types of food and
pharmaceutical products.
Figure 12.9 shows the clear graphical results of the excipients types used
in this screening. The initial activity before the addition of excipients was
0.327 ± 0.004 U/mg-protein. The lowest activity obtained was with Arabic gum with
0.137 ± 0.024 U/mg-protein and 51.35 % of activity lost. Thus, Arabic gum appears
to have an inhibitory potential toward enzymatic activity [11]. Next, the suitable ex-
cipient was used in the formulation to produce dry recombinant bromelain through
the spray drying technique.
The parameters chosen were the inlet air temperature, gas flow height and feed
flow rate. The output response of the recombinant bromelain specific activity (U/mg-
protein) varied from 0.020 ± 0.002 to 0.112 ± 0.001 U/mg-protein. Figure 12.10
shows the effect of the feed flow rate and inlet air temperature by a 3D response
surface plot, and the highest point in the red region clearly shows the optimal range
of response. It clearly shows that the increase of inlet temperature to a maximum
with a decrease of gas flow height to a minimum reduces the specific activity of the
spray dried recombinant bromelain. In the inlet temperature region of 110 to 130 °C,
with a gas flow height from 40 to 50 mm, the specific activity is optimal. Devakate
and co-workers [12] reported that desirable high activity was achieved using spray
drying under low temperature conditions. Denaturation of protein might occur at
high inlet temperature, which reduces the activity before the product is dried [12].
Fig. 12.10 3D surface plot of the effect of inlet air temperature, A (°C), and gas flow height, B
(mm), toward specific activity (U/mg-protein) of spray dried recombinant bromelain
Fig. 12.11 SEM images of excipient-free spray dried recombinant bromelain. Three stages of
magnification at ( A) 1000X, ( B) 3000X and ( C) 5000X
The enzyme inactivation for bromelain reported by Devakate and co-workers [12]
occurred at 65–70 °C, but with the addition of maltodextrin, the recombinant bro-
melain activity in this study was maintained at a high temperature of more than
120 °C with 50 % activity recovery
Dried recombinant bromelain was further subjected to a scanning electron
micrograph (SEM) for particle morphology observation. Figure 12.11 shows the
images captured at different magnifications, 1000X, 3000X and 5000X. The results
in the figure indicate that without excipient-free recombinant bromelain powder, the
particle shape is crust-like. In excipient-free conditions, the recombinant bromelain
enzyme was affected by the rapid dehydration process and thermal change during
spray drying. This condition occurs due to the liquid evaporation from the enzyme
184 A. Amid et al.
Fig. 12.12 SEM images of excipient-free spray dried recombinant bromelain. There stages of
magnification at ( A) 1000X, ( B) 3000X and ( C) 5000X
mixture and shrinkage of the droplets due to the water escaping during the transition
of heat in the spray drying [13, 14]. The moisture content was relatively low due to
the rapid water removal during the drying process, which reduced the surface area
[14, 15].
It can be observed (negative control) with the naked eye that the excipient-free
(2.23-fold) powder has a darker color and rough surface characteristics. Because of
the low moisture content, the absorption of moisture from the ambient surroundings
promotes the hygroscopicity of the dried powder.
In contrast with the recombinant bromelain mediated with maltodextrin as the
excipient shown in Fig. 12.12, the powder had a smooth spherical shape, with a
large particle size, and was non-porous and slightly damped. The function of ex-
cipients is not just preventing the recombinant enzyme solution from rapid dehydra-
tion but creating a cohesive film that maintains the heat associated with the rapidly
hydrating sample [16]. By adding maltodextrin to the solution, the moisture content
is relatively preserved, and thus, the slow activity loss of spray dried recombinant
bromelain was prevented [17]. For other conditions, Phisut [17] mentioned that
maltodextrin causes an increase in bulk density and inhibits dried powder hygro-
scopicity. In this study, the dextrose equivalent used was DE10. In addition, higher
residual bromelain activity was achieved by applying moderate pump settings at
12 % and maintaining the outlet temperature.
References
tion of a recombinant stem bromelain from Ananas comosus. Process Biochem 46:2232–
2239
2. Doonan S (2004) Bulk purification by fractional precipitation. Methods Mol Biol 244:117–
124
3. Omidinia E, Shahbaz Mohamadi H, Dinarvand R, Taherkhani H-A (2010) Investigation of
chromatography and polymer/salt aqueous two-phase processes for downstream processing
development of recombinant phenylalanine dehydrogenase. Bioprocess Biosyst Eng 33:317–
329
4. Xie X, Han J, Wang Y, Yan Y, Yin G, Guan W (2010) Measurement and correlation of the
phase diagram data for PPG400 + (K3PO4, K2CO3, and K2HPO4) + H2O aqueous two-phase
systems at T = 298.15 K. J Chem Eng Data 55:4741–4745
5. Zhang W, Hu Y, Wang Y, Han J, Ni L, Wu Y (2013) Liquid-liquid equilibrium of aqueous
two-phase systems containing poly(ethylene glycol) of different molecular weights and sev-
eral ammonium salts at 298.15 K. Thermochim Acta 560:47–54
6. Ketnawa S, Chaiwut P, Rawdkuen S (2011) Extraction of bromelain from pineapple peels.
Food Sci Technol Int 17:395–402
7. Banik RM, Santhiagu A, Kanari B, Sabarinath C, Upadhyay SN (2003) Technological as-
pects of extractive fermentation using aqueous two-phase systems. World J Microbiol Bio-
technol 19:337–348
8. Pakhale SV, Vetal MD, Rathod VK (2013) Separation of bromelain by aqueous two phase
flotation. Sep Sci Technol 48:984–989
9. Ketnawa S, Chaiwut P, Rawdkuen S (2011) Aqueous two-phase extraction of bromelain
from pineapple peels (‘Phu Lae’ cultv.) and its biochemical properties. Food Sci Biotechnol
20:1219–1226
10. Ketnawa S, Rawdkuen S, Chaiwut P (2010) Two phase partitioning and collagen hydrolysis
of bromelain from pineapple peel Nang Lae cultivar. Biochem Eng J 52:205–211
11. Rowe RC, Sheskey PJ, Weller PJ (2003) Handbook of pharmaceutical excipients. Pharma-
ceutical Press, London
12. Devakate RV, Patil VV, Waje SS, Thorat BN (2009) Purification and drying of bromelain.
Sep Purif Technol 64:259–264
13. Maa YF, Nguyen PA, Andya JD, Dasovich N, Sweeney TD, Shire SJ et al (1998) Effect of
spray drying and subsequent processing conditions on residual moisture content and physi-
cal/biochemical stability of protein inhalation powders. Pharm Res 15:768–775
14. Saluja V, Amorij JP, Kapteyn JC, de Boer AH, Frijlink HW, Hinrichs WLJ (2010) A compari-
son between spray drying and spray freeze drying to produce an influenza subunit vaccine
powder for inhalation. J Control Release 144:127–133
15. Phisut N (2012) Spray drying technique of fruit juice powder: some factors influencing the
properties of product. Int Food Res J 19:1297–1306
16. Elversson J, Millqvist-Fureby A (2006) In situ coating-an approach for particle modification
and encapsulation of proteins during spray-drying. Int J Pharm 323:52–63
17. Phisut N (2012) Spray drying technique of fruit juice powder: some factors influencing the
properties of product. Int Food Res J 19:1297–1306
Index
A Chromatographic, 51, 52, 64, 66, 67, 68, 69,

Affinity tags, 69 77, 117, 118, 120, 123
Airlift bioreactor, 102 Clarification, 66, 67, 76
Ammonium sulfate precipitation, 123 Clone selection, 164
Aqueous Two-Phase Separation, 67 Cloning, 11, 12, 13, 14, 15, 17, 159, 161, 162,
164, 169
B Colorimetric protein assay, 21
Back extraction, 180 Continuous ultrasonication, 176
Basic Local Alignment Search Tool, 47 Crystallization, 45, 117, 120, 121
Bread industry, 147 Cysteine protease, 144, 146
Brewing industry, 147
Bromelain, 25, 93, 131, 133, 135, 136, 137, D
138, 143, 144, 145, 146, 147, 148, 149, Depyrogenation, 118, 121, 122
150, 151, 152, 161, 164, 165, 167, 168, Desalting, 65, 66, 67
171, 176, 177, 179, 181, 182, 183, 184 Desiccation, 117, 121, 122
amino acid composition of, 144 Diarrhea treatment, 150
as anti-inflammatory agent, 148 Dissolved oxygen, 89, 90, 91, 103, 104, 105,
as anti-thrombotic agent, 151 106, 111, 131, 172
as anti-tumor agents, 149 Downstream processing, 63, 65, 77, 115, 116,
crude, 147 117, 118, 122, 124, 126
effect of temperature on, 167
gene amplication, 161 E
in animal feed industry, 152 Enzyme active site, 43, 53, 54
in food industry, 147 Enzyme-linked immunosorbent assay, 165
in personal care industry, 152 Expression of recombinant protein, 63, 65, 90
mechanism of action, 146 Extracellular, 1, 65, 124, 125, 149
recombinant activity, 166, 168, 169, 172,
177, 178, 180 F
specific activity of, 167 Fermentation, 1, 2, 3, 16, 17, 19, 20, 21, 22,
Bubble column, 102 24, 81, 89, 90, 91, 93, 95, 96, 99, 100,
101, 102, 103, 104, 106, 108, 109, 110,
C 111, 116, 117, 126, 131, 138, 141, 147,
Carbon flux, 95 159, 169, 171, 172
Casein, 24, 31 File patent, 7
Catalytic activity, 42, 43, 45 Filtration, 65, 66, 67, 68, 76, 77, 117, 118,
cDNA, 11, 13, 14, 161 119, 122
Centrifugation, 66, 118 Fish industry, 148

DOI 10.1007/978-3-319-12397-4
188 Index
Flocculation, 117, 118, 119 P

Fluidized bed bioreactor, 102 PEG-rich phase, 74, 179, 181
Food industry, 2, 42 pH, 2, 3, 42, 43, 45, 52, 53, 69, 70, 71, 73, 74,
Formulation, 1, 42, 50, 81, 82, 85, 87, 131, 91, 122, 123, 131, 136, 144, 147, 165,
135, 169, 171, 181, 182 166, 168
Pilot expression study, 164
G Plackett-Burman design, 82
Genomic, 11, 12, 159 Plasmid stability, 16, 93, 94
Gibbs free energy, 178 Polishing, 51, 69, 117, 118, 121
Glutathione S-transferase, 64, 70 Polymerase chain reaction, 161
Post-translational modification, 17, 46
Power input per liquid volume, 106, 109
H
Precipitation, 67, 74, 76, 77, 117, 118, 119,
Halal, 3
120, 121, 123, 176
Host, 1, 3, 9, 11, 12, 15, 16, 17, 63, 65, 69, 81,
Pre-commercialization grant, 4
87, 89, 93, 94, 101, 116, 117, 122, 124,
Process simulation, 131
125, 126, 159, 162, 163, 164
Product isolation, 117
Hygroscopic, 122
Protein assay, 21, 64
I
Q
Immiscible, 71, 118, 177
Quality control, 32, 131
Immunoassays, 64
Impeller tip speed, 108, 109, 110
Initial velocity, 54, 55, 56 R
Inlet air temperature, 182 Reaction velocity, 43
Intracellular, 65, 122, 125, 140, 151, 165 Recombinant proteases, 2
Investor, 8
S
J Salting-out, 73, 74, 177
Joint-venture, 6 SDS polyacrylamide gel electrophoresis, 64
Sedimentation, 117, 118, 119
Settling, 117, 118, 119
K
Soy source industry, 148
Key performance index, 4
Specific activity, 78, 85, 89, 90, 91, 92, 167,
168, 169, 171, 176, 177, 178, 179, 180,
L 181, 182
Licensing, 6 Stirred tank bioreactor, 102
Luminescence, 50 Substrate specificity, 42, 53, 136
Lyophilization, 5, 117, 121
T
M Tagged recombinant protein, 62, 63, 68
Maltodextrin, 182, 183, 184 Transformation, 11, 160, 163, 164
Maltose binding protein, 70 Turbidity, 20
Meat industry, 147
Media formulation, 81, 87, 105, 169, 171, 172
U
Metabolic loads, 93, 94
Ultraviolet, 49, 50
Michaelis constant, 54, 55, 56
Mixing time, 108, 110, 111
V
Vector, 1, 11, 13, 14, 15, 48, 63, 160, 162,
O
163, 164
OFAT design, 85
Optimizing, 9, 64, 65, 82, 91, 95, 123, 171,
180 X
Oral toxicity, 136 X-ray structures, 46
Oxidative stress, 89 Xylanase, 3, 24, 70

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Recombinant Enzymes—From Basic Science

ISBN 978-3-319-12396-7 ISBN 978-3-319-12397-4 (eBook)

Library of Congress Control Number: 2014955170

© Springer International Publishing Switzerland 2015

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Recombinant enzyme is a substance that has many applications in industry. Studies

1 Introduction to Recombinant Enzyme Pre-commercialization������������� 1

2 Recombinant Enzyme: Cloning and Expression������������������������������������� 11

3 Common Laboratory Procedure�������������������������������������������������������������� 19

4 Characterization of Recombinant Enzymes�������������������������������������������� 41

5 Purification of Recombinant Protein for Industrial Use������������������������ 61

6 Recombinant-Enzyme Fermentation������������������������������������������������������� 81

7 Scaling-Up Recombinant Enzyme Fermentation������������������������������������ 99

8 Downstream Processing of Recombinant Enzymes

9 Economic and Environmental Evaluation of Recombinant

10 Case Study: Recombinant Bromelain Selection�������������������������������������� 143

11 Case Study: Recombinant Bromelain Cloning,

12 Case Study: Recombinant Bromelain Downstream Processing������������ 175

Abstract This chapter discusses a number of matters involving the commercializa-

Keywords File patent · Investor · Joint-venture · Key performance index · Pre-

A recombinant enzyme is an enzyme that is produced through recombinant DNA

The purification process has been facilitated by the availability of protein-tagging

1.1.2 The Differences Between Natural and Recombinant

1.2 Why Choose Recombinant Enzyme

1.2.1 Shorter Production Time Using Fermentation

1.2.2 Production of Rare and Difficult Enzymes For Industry

Recombinant DNA technology and protein engineering offer a superior method of

1.3 Reason for Commercialization

Commercialization is generally an unpopular activity for academics because it

1.3.2 Researcher Key Performance Index

Commercialization activities do not directly contribute to the key performance in-

Governments allocate money to universities to conduct fundamental research. If

The effectiveness of a product cannot be validated on the small scale of a laboratory

It is undeniable that when an academician successfully commercializes a product

When a product is successfully commercialized there are certainly financial re-

1.5 Strategies for Commercialization

1.5.1 Identifying Suitable Product

Prior to commercializing a biotechnology product, the researcher must choose

1.5.2 File Patent For Product Protection?

1.5.3 Promoting and Marketing

The most challenging part of commercialization for researchers is marketing. The

session may help promote researcher innovation output to investors. In Malaysia,

1.5.4 Links with Industries

1.5.5 Identifying Possible Investor

In an effort to make Malaysia a high-income nation, the Agensi Inovasi Malaysia

1.5.6 Identifying and Applying For Commercialization Grant

In addition to AIM, the Malaysian Technology Development Corporation

1.6 Obstacle During the Pre-commercialization Stage

1.6.1 Each Product Is Unique

1.6.2 Bench Scale Procedures Are Not Suitable

1.6.3 Fine-Tuning Each Unit Processes

1.6.4 Unethical Business Competitors

Azura Amid and Norhidayah Hassan

Keywords Commercialization grant · cDNA · Cloning · Genomic · Host ·

For commercialization purposes, the enzymes produced must be in an active

Fig. 2.1 Simplified cDNA

recombinant vector insertion

Fig. 2.2 Strategies of PCR cloning; blunt-end and TA cloning (www.invitrogen.com)

Bacteria system e.g. E. coli

Tag protein gene Yeast

Promoterr Insect cell

Fig. 2.3 Outline of the recombinant protein cloning

1 Introduction to Recombinant Enzyme Pre-commercialization�� 1

2 Recombinant Enzyme: Cloning and Expression�� 11

3 Common Laboratory Procedure�� 19

4 Characterization of Recombinant Enzymes�� 41

5 Purification of Recombinant Protein for Industrial Use�� 61

6 Recombinant-Enzyme Fermentation�� 81

7 Scaling-Up Recombinant Enzyme Fermentation�� 99

8 Downstream Processing of Recombinant Enzymes

9 Economic and Environmental Evaluation of Recombinant

10 Case Study: Recombinant Bromelain Selection�� 143

11 Case Study: Recombinant Bromelain Cloning,

12 Case Study: Recombinant Bromelain Downstream Processing�� 175

1.1.2 The Differences Between Natural and Recombinant

1.2 Why Choose Recombinant Enzyme

1.2.1 Shorter Production Time Using Fermentation

1.2.2 Production of Rare and Difficult Enzymes For Industry

1.3 Reason for Commercialization

1.3.2 Researcher Key Performance Index

1.5 Strategies for Commercialization

1.5.1 Identifying Suitable Product

1.5.2 File Patent For Product Protection?

1.5.3 Promoting and Marketing

1.5.4 Links with Industries

1.5.5 Identifying Possible Investor

1.5.6 Identifying and Applying For Commercialization Grant

1.6 Obstacle During the Pre-commercialization Stage

1.6.1 Each Product Is Unique

1.6.2 Bench Scale Procedures Are Not Suitable

1.6.3 Fine-Tuning Each Unit Processes

1.6.4 Unethical Business Competitors

2.2 Vector for Cloning

2.3 Host for the Recombinant Enzyme

3.2 Estimating Bacterial Growth by Turbidity Measurement

3.2.3 Materials and Methods

3.2.4 Results and Discussion

3.3 Total Protein Assay

3.3.3 Materials and Method

3.3.4 Results (Table 3.2)

3.4 Enzyme Activity Assays

3.4.1 Enzyme Activity Assay Using LNPE as Substrate

3.4.1.3 Materials and methods

3.4.1.4 Results and Discussions

3.4.2 Enzyme Activity Assay Using Casein as Substrate

3.4.2.3 Materials and methods

3.4.2.5 Results and Discussions (Table 3.7)

3.5 Sodium Dodecyl Sulfate Polyacrylamide Gel

3.5.3 Materials and Methods

4.2.1 Enzyme Active Site Residues and Catalysis

4.2.2 Effects of Temperature on Enzyme Activity and Stability

4.2.3 Effects of pH on Enzyme Activity and Stability

4.3 Biophysical and Structure Characterization

4.4 Characterizing Post-Translational Modifications

4.5 Computational Methods and Bioinformatics for

4.6.1.1 Ultraviolet Absorption Spectroscopy

4.6.2 Chromatographic Methods