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Future prospects of enzyme engineering

Future prospects of enzyme engineering

E nzyme engineering is the recent technology growing rapidly due to its higher application in a
lot of fields and due to having bright and clear future vision. A most exciting development over the last few
years is the application of genetic engineering techniques to enzyme technology. There are a number of
properties which may be improved or altered by genetic engineering including the yield and kinetics of the
enzyme, the ease of downstream processing and various safety aspects. Enzymes from dangerous or
unapproved microorganisms and from slow-growing or limited plant or animal tissue may be cloned into
safe high-production microorganisms. The amount of enzyme produced by a microorganism may be
increased by increasing the number of gene copies that code for it. For example; The engineered cells,
aided by the plasmid amplification at around 50 copies per cell, produce penicillin – G – Amidase
constitutively and in considerably higher quantities than does the fully induced parental strain. Such
increased yields are economically relevant not just for the increased volumetric productivity but also
because of reduced downstream processing costs, the resulting crude enzyme being that much purer. New
enzyme structures may be designed and produced in order to improve on existing enzymes or create new
activities. Much protein engineering has been directed at Subtilisin (from Bacillus amyloliquefaciens), the
principal enzyme in the detergent enzyme preparation, Alcalase. This has been aimed at the improvement
of its activity in detergents by stabilizing it at even higher temperatures, pH and oxidant strength. A number
of possibilities now exist for the construction of artificial enzymes. These are generally synthetic polymers
or oligomers with enzyme-like activities, often called synzymes. Enzymes can be immobilized i.e., an
enzyme can be linked to an inert support material without loss of activity which facilitates reuse and
recycling of the enzyme.Use of engineered enzyme to form biosensor for the analytical use is also recent
activity among the developed countries. Some enzymes make use in diseases diagnosis so they can be
genetically engineered to make the task easier. Thus it is obvious that there is huge scope of the enzyme
technology in the future as well as in present.
Introduction
Enzymes are Organic compounds, produced in the living cells to speed up chemical reaction in the
biological systems so that they can take place at relatively lower temperature, but themselves remain
apparently unchanged during the process. Therefore enzymes are termed as biocatalysts. Biocatalysts are
either proteins (enzymes) or, in a few cases, they may be nucleic acids (ribozymes; some RNA molecules
can catalyze the hydrolysis of RNA. Today, we know that enzymes are necessary in all living systems, to
catalyze all chemical reactions required for their survival and reproduction – rapidly, selectively and
efficiently. Isolated enzymes can also catalyze these reactions. In the case of enzymes however, the
question whether they can also act as catalysts outside living systems had been a point of controversy
among biochemists in the beginning of the twentieth century. It was shown at an early stage however that
enzymes could indeed be used as catalysts outside living cells, and several processes in which they were
applied as biocatalysts have been patented These excellent properties of enzymes are utilized in enzyme
technology. For example, they can be used as biocatalysts to catalyze chemical reactions on an industrial
scale in a sustainable manner. Their application covers the production of desired products for all human
material needs (e.g., food, animal feed, pharmaceuticals, fine and bulk chemicals, fibers, hygiene, and
environmental technology), as well as in a wide range of analytical purposes, especially in diagnostics. In
fact, during the past 50 years the rapid increase in our knowledge of enzymes – as well as their biosynthesis
and molecular biology – now allows their rational use as biocatalysts in many processes, and in addition
their modification and optimization for new synthetic schemes and the solution of analytical problems
Enzymes have become big business. They are used in many industrial processes to catalyze
biological reactions. Enzymes are exploited in a variety of manufacturing processes such as food
processing and for the synthesis of medicines such as antibiotics like artificial penicillin. They are also used
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Future prospects of enzyme engineering

to clean up factory effluents and pollution in water and soil. Many processes can be made faster and
cheaper by using the right enzyme and conditions.
Optimum conditions are maintained during factory production by use of bioreactors. These are vessels
which are designed to provide the ideal environment for reactions involving enzymes or living organisms.
Source of enzymes used commercial production is plant, animal and microbial cells. Animal enzymes used
currently are lipases, tripsin, rennets etc. Most prevalent plant enzymes are papain, proteases, amylases and
soybean lipoxygenase. These enzymes are used in food industries, for example, papain extracted from
papaya fruit is used as meat tenderizer and pancreatic protease in leather softening and manufacture of
detergents. In addition microbial enzymes have gained much popularity. Production of primary and
secondary metabolites by microorganism is possible only due to involvement of various enzymes. They are
of two types: the extracellular and the intracellular enzymes. There is a wide range of extracellular enzymes
produced by pathogenic and saprophytic microorganisms such as cellulose, polymethylegalactouronase,
pectinmethylesterase etc. These enzyme helps in establishment in host tissues or decomposition of organic
substrates. The intracellular enzyme like invertase, uricoxidase, asparaginase are of high economic value
and difficult to extract as they produced inside the cell. They can be extracted by breaking the cells by
means of a homogenizer or a ball mill and extracted them through the biochemical process.
Biotechnology offers an increasing potential for the production of goods to meet various human
needs. In enzyme technology – a sub-field of biotechnology – new processes have been and are being
developed to manufacture both bulk and high added- value products utilizing enzymes as biocatalysts, in
order to meet needs such as food (e.g., bread, cheese, beer, vinegar), fine chemicals (e.g., amino acids,
vitamins), and pharmaceuticals. Enzymes are also used to provide services, as in washing and
environmental processes, or for analytical and diagnostic purposes. The driving force in the development of
enzyme technology, both in academia and industry, has been and will continue to be:
• The development of new and better products, processes and services to meet these needs; and/or
• The improvement of processes to produce existing products from new raw materials as biomass.
The goal of these approaches is to design innovative products and processes that are not only competitive
but also meet criteria of sustainability. A positive effect in all these three fields is required for a sustainable
process. Criteria for the quantitative evaluation of the economic and environmental impact are in contrast
with the criteria for the social impact, easy to formulate. In order to be economically and environmentally
more sustainable than an existing processes, a new process must be designed to reduce not only the
consumption of resources (e.g., raw materials, energy, air, water), waste production and environmental
impact, but also to increase the recycling of waste per kilogram of product.

Sources of enzymes: Biologically active enzymes may be extracted from any living organism. A very wide range
of sources are used for commercial enzyme production from Actinoplanes to Zymomonas, from spinach to snake
venom. Of the hundred or so enzymes being used industrially, over a half are from fungi and yeast and over a third
are from bacteria with the remainder divided between animal (8%) and plant (4%) sources. A very much larger
number of enzymes find use in chemical analysis and clinical diagnosis. Non-microbial sources provide a larger
proportion of these, at the present time. Microbes are preferred to plants and animals as sources of enzymes because:
1. they are generally cheaper to produce.
2. their enzyme contents are more predictable and controllable,
3. reliable supplies of raw material of constant composition are more easily arranged, and
4. plant and animal tissues contain more potentially harmful materials than microbes, including
phenolic compounds (from plants), endogenous enzyme inhibitors and proteases.

Table 1 . Some important industrial enzymes and their sources.

Enzyme EC number Source Intra/extra Scale of Industrial use

-cellular production
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Future prospects of enzyme engineering

Animal enzymes

Catalase 1.11.1.6 Liver I - Food

Chymotrypsin 3.4.21.1 Pancreas E - Leather

Lipase 3.1.1.3 Pancreas E - Food

Rennet 3.4.23.4 Abomasum E + Cheese

Trypsin 3.4.21.4 Pancreas E - Leather

Plant enzymes

Actinidin 3.4.22.14 Kiwi fruit E - Food

a-Amylase 3.2.1.1 Malted barley E +++ Brewing

b-Amylase 3.2.1.2 Malted barley E +++ Brewing

Bromelain 3.4.22.4 Pineapple latex E - Brewing

b-Glucanase 3.2.1.6 Malted barley E ++ Brewing

Ficin 3.4.22.3 Fig latex E - Food

Lipoxygenase 1.13.11.12 Soybeans I - Food

Papain 3.4.22.2 Pawpaw latex E ++ Meat

Bacterial enzymes

a-Amylase 3.2.1.1 Bacillus E +++ Starch

b-Amylase 3.2.1.2 Bacillus E + Starch

Asparaginase 3.5.1.1 Escherichia coli I - Health

Glucose isomerase 5.3.1.5 Bacillus I ++ Fructose syrup

Penicillin amidase 3.5.1.11 Bacillus I - Pharmaceutical

Protease 3.4.21.14 Bacillus E +++ Detergent

Pullulanase 3.2.1.41 Klebsiella E - Starch

Fungal enzymes

a-Amylase 3.2.1.1 Aspergillus E ++ Baking

Aminoacylase 3.5.1.14 Aspergillus I - Pharmaceutical

Glucoamylase 3.2.1.3 Aspergillus E +++ Starch

Catalase 1.11.1.6 Aspergillus I - Food

Cellulase 3.2.1.4 Trichoderma E - Waste

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Future prospects of enzyme engineering

Dextranase 3.2.1.11 Penicillium E - Food

Glucose oxidase 1.1.3.4 Aspergillus I - Food

Lactase 3.2.1.23 Aspergillus E - Dairy

Lipase 3.1.1.3 Rhizopus E - Food

Rennet 3.4.23.6 Mucor miehei E ++ Cheese

Pectinase 3.2.1.15 Aspergillus E ++ Drinks

Pectin lyase 4.2.2.10 Aspergillus E - Drinks

Protease 3.4.23.6 Aspergillus E + Baking

Raffinase 3.2.1.22 Mortierella I - Food

Yeast enzymes

Invertase 3.2.1.26 Saccharomyces I/E - Confectionery

Lactase 3.2.1.23 Kluyveromyces I/E - Dairy

Lipase 3.1.1.3 Candida E - Food

Raffinase 3.2.1.22 Saccharomyces I - Food

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Future prospects of enzyme engineering

Once the enzyme has been purified to the desired extent and concentrated, the manufacturer's main
objective is to retain the activity. Enzymes for industrial use are sold on the basis of overall activity. To
achieve stability, the manufacturer should follow the recent advanced technology even genetic engineering
thechniques.Most industrial enzymes contain relatively little active enzyme (< 10% w/w, including
isoenzymes and associated enzyme activities), the rest being due to inactive protein, stabilisers,
preservatives, salts and the diluent which allows standardisation between production batches of different
specific activities.The key to maintaining enzyme activity is maintenance of conformation, so preventing
unfolding, aggregation and changes in the covalent structure. Three approaches are possible: use of
additives, the controlled use of covalent modification, and enzyme immobilization. So if the genetic
engineering along with the advanced technique for enzyme engineering are employed there might be the
great possibility of increasing the half life of active protein and their stability as well as specificity which
will certainly reduce conventional methods for
stabilizing the enzymes.
Screening for novel enzymes: One of the major skills of
enzyme companies and suitably funded academic
laboratories is the rapid and cost-effective screening of
microbial cultures for enzyme activities. Natural
samples, usually soil or compost material found near
high concentrations of likely substrates, are used as
sources of cultures.
Preparation of enzymes: After the screening of the
novel enzyme having great commercial as well as
industrial use, enzyme is prepared by optimizing the
condition of higher production with available resources.
Purification of enzyme after preparation depends upon its
future use. Often the enzyme may be purified several
hundred-fold but the yield of the enzyme may be very
poor, frequently below 10% of the activity of the
original material. In contrast, industrial enzymes will be
purified as little as possible, only other enzymes and
material likely to interfere with the process which the
enzyme is to catalyze, will be removed.
Fig.1 Flow diagram for the preparation of enzymes.

Genetic Protein Engineering of Enzymes

A most exciting development over the last few years is the application of genetic engineering techniques to
enzyme technology. Recombinant DNA technology has allowed the transfer of useful enzyme genes from
one organism to another. Thus, when an enzyme has been identified as a good candidate enzyme for
industrial use, the relevant gene can be cloned into a more suitable production host microorganism and an
industrial fermentation carried out. In this way, it becomes possible to produce industrial enzymes of very
high quality and purity. A recent example of this technology is the detergent enzyme Lipolase produced by

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Future prospects of enzyme engineering

Novo Nordisk A/S, which has improved removal of fat stains in fabrics. The enzyme was first identified in
the fungus Humicola languinosa at levels inappropriate for commercial production. The gene DNA
fragment for the enzyme was cloned into the fungus Aspergillus oryzae and commercial levels of enzyme
achieved. The enzyme has proved to be efficient under many wash conditions. The enzyme is also very
stable at a variety of temperature and pH conditions relevant to washing.
There are a number of properties which may be improved or altered by genetic engineering including the
yield and kinetics of the enzyme, the ease of downstream processing and various safety aspects. Enzymes
from dangerous or unapproved microorganisms and from slow-growing or limited plant or animal tissue
may be cloned into safe high-production microorganisms.
All proteins, including enzymes, are based on the same 20 different amino acid building blocks
arranged in different sequences. Enzyme proteins typically comprise sequences of several
hundred amino acids folded in a unique three-dimensional structure. Only the sequence of these 20 building
blocks determines the three-dimensional structure, which in turn determines all properties such as catalytic
activity, specificity and stability. Nature has been performing ‘protein engineering’ for billions of years
since the very start of evolution. Natural spontaneous mutations in the DNA coding for a given protein
result in changes of the protein structure and hence its properties. This natural variation is part of the
adaptive evolutionary process continuously taking place in all living organisms, allowing them to survive in
continuously changing environments. Natural variants of enzyme proteins are adapted to perform
efficiently in different environments and conditions. This explains why in nature enzymes belonging to the
same enzyme family but isolated from different organisms and environments often show a variation in
amino acid sequence of more than 50%. The properties of enzymes used for industrial purposes sometimes
also require some adaptations in order to function more effectively in applications for which they were not
designed by nature. Traditionally, such enzyme optimization is performed by screening naturally occurring
microorganisms, followed by classical mutation and selection. The disadvantage of this method is,
however, that it may take a very long time until the enzyme with the desired properties is found. This is
why protein engineering was developed.

Assumptions for Protein Engineering

While attempting protein engineering, one should recognize the following properties of enzymes:

(i) many amino acid substitutions, deletions or additions lead to no change in enzyme activity, so that they
are silent mutations;

(ii) proteins have a limited number of basic structures and only minor changes are superimposed on them
leading to variation;

(iii) similar patterns of chain folding and domain structure can arise from different amino acid sequences,
which show little or no homology (although same amino acid sequence never gives different folding and
domain structures).
The above properties suggest that while many major changes sometimes may lead to no alteration in
function, some of the minor changes at specific positions may lead to the desired favourable change.

For example, a single amino acid replacement (glycine to aspartic acid) in E. coli asparate transcarbamylase
leads to

(i) loss of activity and to

(ii) an alteration in the binding of catalytic and regulatory subunits. Another example involved the
engineering of a single chain biosynthetic antibody binding site (BARS), which is though only one sixth of
the size of the complete antibody, but retains its antigen binding specificity.

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Future prospects of enzyme engineering

This synthetic fragment has heavy and light chain variable regions (V H and V J connected by a 15 - amino
acid linker. A synthetic gene has also been prepared for the fragment, which expressed in E. coli. This
fragment binds to digoxin, a cradiac glycoside. Single amino acid replacements in BABS fragment have
sometimes led to major changes in its binding affinity.

In view of the above, it is necessary to examine not only the crystal structure but also the active sites
therein, so that the gene may be modified or artificially synthesized for protein engineering to meet the
desired needs.
Methods for Protein Engineering –
A variety of methods have been used and proposed for future use in protein engineering. In this connection
mutagenesis, selection, and recombinant DNA are being used and will be increasingly utilized in future.
1. Mutagenesis and Selection for Protein Engineering - Mutagenesis and selection can be effectively
utilized for improving a specific property of an enzyme. Following are some of the examples of selection of
mutant enzymes:

(i) E. coli anthranilate synthetase enzyme is normally sensitive to tryptophan inhibition due to feedback
inhibition. An MTR 2 mutation of E. coli was found to possess an altered form of enzyme anthranilate
synthetase that is insensitive to tryptophan inhibition. They may help in continuous synthesis of tryptophan
without any inhibition by tryptophan accumulated as a product.

(ii) Xanthine dehydrogenase enzyme oxidizes 2 hydroxy-purine at position 8, but a mutant has been
inolated which oxidizes 2 hydroxy-purine at position 6.

(iii) Lactate dehydrogenase (LDU) from a bacterial system was modified to malate dehydrogenase able a
natural mutation leading to a single amino acid substitution (Gln 02... Arg; see later m thIS chapter).

In the above and other cases of naturally occurring mutant enzymes, single amino acid modification or
addition/deletion has been observed.

However, if improvement requires changes in several amino acids, such a mutant will be rare or
nonexistent and modifications of this type will be possible only through gene modification techniques
discussed in the following section.
2. Production of Artificial Semi Synthetic Oxido Reductases - Flavo Enzymes - Artificial oxido
reductases can be prepared by covalently attaching redoxactive prosthetic groups to existing sites. Linking
of 10-methyilsoalloxazine derivatives (as redox-active groups) to specific sites of several proteins has been
achieved. The efficiency of these semisynthetic enzymes (e.g. flavopapain) compares favourably with that
of naturally occurring flavoenzymes.
3. Modification of Proteases into Peptide Ligases -Peptide ligation to native enzymes may lead to high
specificity and stereoselecitivity, and may suppress side reactions. Therefore, synthesis of any enzyme that
may catalyze peptide ligation will be most welcome.

Protease 'subtilisin' has been modified (by converting a serine into cysteine or seleno-cysteine) into thiol-
and selenolsubtilisin, the two semi synthetic enzymes (they are damaged proteases), which can catalyse
peptide ligation. Both these damaged proteases are efficient peptide ligases. Similarly histidine residue can
also be modified to yield peptide ligases.
4. Enzyme PEG Conjugates - An enzyme L- asparaginase (isolated from microbes) has antitumour
properties, but is toxic with a life time of less then 18hr thus reducing its utility. It has been shown that E.
coli L-asparaginase can be modified by polyethylene glycol derivatives to produce PEG-asparaginase
conjugates , which differ from the native enzyme in following features:
(i) it retains only 52% of the catalytic activity of native enzyme;
(ii) it becomes resistant to proteolytic degradation; (Hi) it does not cause allergy. In view of this, PEG-
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Future prospects of enzyme engineering

asparaginase has been used to treat malignant murine (mouse), canine (cats, etc.) and human tumours. PEG
conjugates of a large number of enzymes (adenosine deaminase, uricase, catalase, etc.) have been prepared
and will be utilized in industry also.
5. Production of Site Specific Nucleases - Restriction Enzymes - The DNA recognition and binding
properties of proteins can be combined using chemical cleavage agents. Cys178 of E. coli CAP protein; has
been modified using 'S-iodoacetamide -1, 10- phenanthroline' yielding a DNA cleaving agent that
recognized and cleaved DNA at the centre of the recognition site (22 bp) for CAP.

This may give restriction enzymes recognizing upto 20 bases instead of 6 or 8 bases and may, therefore, be
useful for isolating long DNA fragments needed for sequencing and mapping. Nucleases may also be
produced by fusion of non-specific phosphodiesterases to oligonucleotides of defined sequence.

For a nuclease from Staphylococcus modified by this approach, it was shown that oligonucleotide
component of fused product pairs with its complementary sequence and the hybrid enzyme hydrolyses
single stranded DNA or RNA adjacent to the oligonucleotide binding site. This approach thus can also be
used for developing artificial restriction enzymes.
Protein engineering and how it is applied to enzymes
A most exciting development over the last few years is the application genetic engineering techniques to
enzyme technology. Protein engineering of enzymes is a faster, more controlled, more targeted and more
accurate method to optimize the properties of enzymes for a specific industrial application than the
traditional method described above. It makes it possible to sidestep the high number of natural isolate
screenings that would otherwise be necessary to find the enzyme with the desired properties, and increases
the likelihood that a suitable enzyme will be found. The protein engineering technique involves genetic
modification by means of recombinant DNA technology of the enzyme producing microorganism, in
particular the enzyme encoding gene, resulting in substitution of one or more amino acids in the amino acid
sequence of the enzyme protein. Strategies for making such amino acid substitutions and developing
protein engineered enzymes are based on the knowledge of the structure/function relationships of enzymes,
computer modeling and techniques for creating and testing enzyme variants.
Enzyme technology is the application of modifying an enzyme's structure (and thus its function) or
modifying the catalytic activity of isolated enzymes to produce new metabolites, to allow new (catalyzed)
pathways for reactions to occur, or to convert from some certain compounds into others
(biotransformation). These products will be useful as chemicals, pharmaceuticals, fuel, food or agricultural
additives. An enzyme reactor consists of a vessel containing a reactional medium that is used to perform a
desired conversion by enzymatic means. Enzymes used in this process are free in the solution or
immobilized in particulate, membranous or fibrous support. There are many directions in which enzyme
technologists are currently applying their art and which are at the forefront of biotechnological research and
development. Some of these have already been examined in some detail earlier. At present, relatively few
enzymes are available on a large scale (i.e. > kg) and are suitable for industrial applications. These
shortcomings are being addressed in a number of ways:
1. New enzymes are being sought in the natural environment and by strain selection
2. Novel enzymes are being designed and produce by genetic engineering;
3. New organic catalysts are being designed and synthesized using the 'knowhow' established from
enzymology; and
4. More complex enzyme systems are being utilized.
Each of these areas has a extensive and rapidly expanding literature. Some advances possibly belong more
properly to other areas of science. Thus, the development of genetically improved enzymes is generally
undertaken by molecular biologists and the design and synthesis of novel enzyme-like catalysts is in the

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provenance of the organic chemists. Both groups of workers will, however, base their science on data
provided by the enzyme technologist.
There are a number of properties which may be improved or altered by genetic engineering
including the yield and kinetics of the enzyme, the ease of downstream processing and various safety
aspects. Enzymes from dangerous or unapproved microorganisms and from slow growing or limited plant
or animal tissue may be cloned into safe high-production microorganisms. In the future, enzymes may be
redesigned to fit more appropriately into industrial processes; for example, making glucose isomerase less
susceptible to inhibition by the Ca2+ present in the starch saccharification processing stream.
The amount of enzyme produced by a microorganism may be increased by increasing the number of gene
copies that code for it. This principle has been used to increase the activity of penicillin-G-amidase in
Escherichia coli. The cellular DNA from a producing strain is selectively cleaved by the restriction
endonuclease HindIII. This hydrolyses the DNA at relatively rare sites containing the 5'-AAGCTT-3' base
sequence to give identical 'staggered' ends.

[Fig2]
intact DNA cleaved DNA
The total DNA is cleaved into about 10000 fragments, only one of which contains the required genetic
information. These fragments are individual cloned into a cosmid vector and thereby returned to E. coli.
These colonies containing the active gene are identified by their inhibition of a 6-amino-penicillanic acid-
sensitive organism. Such colonies are isolated and the penicillin-G-amidase gene transferred on to pBR322
plasmids and recloned back into E. coli. The engineered cells, aided by the plasmid amplification at around
50 copies per cell, produce penicillin-G-amidase constitutively and in considerably higher quantities than
does the fully induced parental strain. Such increased yields are economically relevant not just for the
increased volumetric productivity but also because of reduced downstream processing costs, the resulting
crude enzyme being that much purer.
The process starts with the isolation and characterisation of the required enzyme. This information is
analysed together with the database of known and putative structural effects of amino acid substitutions to
produce a possible improved structure. This factitious enzyme is constructed by site-directed mutagenesis,
isolated and characterised. The results, successful or unsuccessful, are added to the database, and the
process repeated until the required result is obtained.

Another extremely promising area of genetic engineering is protein engineering. New enzyme structures
may be designed and produced in order to
improve on existing enzymes or create
new activities. An outline of the process of
protein engineering is shown in Figure 2.
Such factitious enzymes are produced by
site-directed mutagenesis (Figure 3).
Unfortunately from a practical point of
view, much of the research effort in

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protein engineering has gone into studies concerning the structure and activity of enzymes chosen for their
theoretical importance or ease of preparation rather than industrial relevance. This emphasis is likely to
change in the future. Figure 2. The protein engineering cycle.
As indicated by the method used for site-directed mutagenesis (Figure 3), the preferred pathway for
creating new enzymes is by the stepwise substitution of only one or two amino acid residues out of the total
protein structure. Although a large database of sequence-structure correlations is available, and growing
rapidly together with the necessary software, it is presently insufficient accurately to predict three-
dimensional changes as a result of such substitutions. The main problem is assessing the long-range effects,
including solvent interactions, on the new structure. As the many reported results would attest, the science
is at a stage where it can explain the structural consequences of amino acid substitutions after they have
been determined but cannot accurately predict them. Protein engineering, therefore, is presently rather a hit
or miss process which may be used with only little realistic likelihood of immediate success. Apparently
quite small sequence changes may give rise to large conformational alterations and even affect the rate-
determining step in the enzymic catalysis. However it is reasonable to suppose that, given a sufficiently
detailed database plus suitable software, the relative probability of success will increase over the coming
years and the products of protein engineering will make a major impact on enzyme technology.
Much protein engineering has been directed at subtilisin (from Bacillus amyloliquefaciens), the principal
enzyme in the detergent enzyme preparation, Alcalase. This has been aimed at the improvement of its
activity in detergents by stabilising it at even higher temperatures, pH and oxidant strength. Most of the
attempted improvements have concerned alterations to:
1. the P1 cleft, which holds the amino acid on the carbonyl side of the targeted peptide bond;
2. the oxyanion hole (principally Asn155), which stabilises the tetrahedral intermediate;
3. the neighbourhood of the catalytic histidyl residue (His64), which has a general base role; and
4. the methionine residue (Met222) which causes subtilisin's lability to oxidation.
It has been found that the effect of a substitution in the P1 cleft on the relative specific activity between
substrates may be fairly accurately predicted even though predictions of the absolute effects of such
changes are less successful. Many substitutions, particularly for the glycine residue at the bottom of the P1
cleft (Gly166), have been found to increase the specificity of the enzyme for particular peptide links whilst
reducing it for others. These effects are achieved mainly by corresponding changes in the K m rather than the
Vmax. Increases in relative specificity may be useful for some applications. They should not be thought of as
the usual result of engineering enzymes, however, as native subtilisin is unusual in being fairly non-specific
in its actions, possessing a large hydrophobic binding site which may be made more specific relatively
easily (e.g. by reducing its size). The inactivation of subtilisin in bleaching solutions coincides with the
conversion of Met222 to its sulfoxide, the consequential increase in volume occluding the oxyanion hole.
Substitution of this methionine by serine or alanine produces mutants that are relatively stable, although
possessing somewhat reduced activity.

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Figure 3. An outline of the process of site-directed mutagenesis, using a hypothetical example. (a) The
primary structure of the enzyme is derived from the DNA sequence. A putative enzyme primary structure is
proposed with an asparagine residue replacing the serine present in the native enzyme. A short piece of
DNA (the primer), complementary to a section of the gene apart from the base mismatch, is synthesised.
(b) The oligonucleotide primer is annealed to a single-stranded copy of the gene and is extended with
enzymes and nucleotide triphosphates to give a double-stranded gene. On reproduction, the gene gives rise
to both mutant and wild-type clones. The mutant DNA may be identified by hybridisation with
radioactively labelled oligonucleotides of complementary structure.
An example of the unpredictable nature of protein engineering is given by trypsin, which has an active site
closely related to that of subtilisin. Substitution of the negatively charged aspartic acid residue at the
bottom of its P1 cleft (Asp189), which is used for binding the basic side-chains of lysine or arginine, by
positively charged lysine gives the predictable result of abolishing the activity against its normal substrates
but unpredictably also gives no activity against substrates where these basic residues are replaced by
aspartic acid or glutamic acid.
Considerable effort has been spent on engineering more thermophilic enzymes. It has been found that
thermophilic enzymes are generally only 20-30 kJ more stable than their mesophilic counterparts. This may
be achieved by the addition of just a few extra hydrogen bonds, an internal salt link or extra internal
hydrophobic residues, giving a slightly more hydrophobic core. All of these changes are small enough to be
achieved by protein engineering. To ensure a more predictable outcome, the secondary structure of the
enzyme must be conserved and this generally restricts changes in the exterior surface of the enzyme.
Suitable for exterior substitutions for increasing thermostability have been found to be aspartate ,
glutamate, lysine , glutamine, valine , threonine, serine , asparagine, isoleucine , threonine, asparagine ,
aspartate and lysine , arginine. Such substitutions have a fair probability of success. Where allowable, small
increases in the interior hydrophobicity for example by substituting interior glycine or serine residues by
alanine may also increase the thermostability. It should be recognised that making an enzyme more
thermostable reduces its overall flexibility and, hence, it is probable that the factitious enzyme produced
will have reduced catalytic efficiency.
Artificial enzymes:
A number of possibilities now exist for the construction of artificial enzymes. These are generally synthetic
polymers or oligomers with enzyme-like activities, often called synzymes. They must possess two
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structural entities, a substrate-binding site and a catalytically effective site. It has been found that producing
the facility for substrate binding is relatively straightforward but catalytic sites are somewhat more difficult.
Both sites may be designed separately but it appears that, if the synzyme has a binding site for the reaction
transition state, this often achieves both functions. Synzymes generally obey the saturation Michaelis-
Menten kinetics . For a one-substrate reaction the reaction sequence is given by

synzyme + S (synzyme-S complex) synzyme + P

Some synzymes are simply derivatised proteins, although covalently immobilised enzymes are not
considered here. An example is the derivatisation of myoglobin, the oxygen carrier in muscle, by attaching
(Ru(NH3)5)3+ to three surface histidine residues. This converts it from an oxygen carrier to an oxidase,
oxidising ascorbic acid whilst reducing molecular oxygen. The synzyme is almost as effective as natural
ascorbate oxidases.
It is impossible to design protein synzymes from scratch with any probability of success, as their
conformations are not presently predictable from their primary structure. Such proteins will also show the
drawbacks of natural enzymes, being sensitive to denaturation, oxidation and hydrolysis. For example,
polylysine binds anionic dyes but only 10% as strongly as the natural binding protein, serum albumin, in
spite of the many charges and apolar side-chains. Polyglutamic acid, however, shows synzymic properties.
It acts as an esterase in much the same fashion as the acid proteases, showing a bell-shaped pH-activity
relationship, with optimum activity at about pH 5.3, and Michaelis-Menten kinetics with a Km of 2 mm and
Vmax of 10-4 to 10-5 s-1 for the hydrolysis of 4-nitrophenyl acetate. Cyclodextrins (Schardinger dextrins) are
naturally occurring toroidal molecules consisting of six, seven, eight, nine or ten a-1, 4-linked D-glucose
units joined head-to-tail in a ring (a-, b-, g-, d- and e-cyclodextrins, respectively: they may be synthesised
from starch by the cyclomaltodextrin glucanotransferase (EC 2.4.1.19) from Bacillus macerans). They
differ in the diameter of their cavities (about 0.5-1 nm) but all are about 0.7 nm deep. These form
hydrophobic pockets due to the glycosidic oxygen atoms and inwards-facing C-H groups. All the C-6
hydroxyl groups project to one end and all the C-2 and C-3 hydroxyl groups to the other. Their overall
characteristic is hydrophilic, being water soluble, but the presence of their hydrophobic pocket enables
them to bind hydrophobic molecules of the appropriate size. Synzymic cyclodextrins are usually
derivatised in order to introduce catalytically relevant groups. Many such derivatives have been examined.
For example, a C-6 hydroxyl group of b-cyclodextrin was covalently derivatised by an activated pyridoxal
coenzyme. The resulting synzyme not only acted a transaminase but also showed stereoselectivity for the
L-amino acids. It was not as active as natural transaminases, however. Polyethyleneimine is formed by
polymerising ethyleneimine to give a highly branched hydrophilic three-dimensional matrix. About 25% of
the resultant amines are primary, 50% secondary and 25% tertiary:

[8.6]
Ethyleneimine polyethyleneimine
The primary amines may be alkylated to form a number of derivatives. If 40% of them are alkylated with 1-
iodododecane to give hydrophobic binding sites and the remainder alkylated with 4(5)-
chloromethylimidazole to give general acid-base catalytic sites, the resultant synzyme has 27% of the
activity of a-chymotrypsin against 4-nitrophenyl esters. As might be expected from its apparently random
structure, it has very low esterase specificity. Other synzymes may be created in a similar manner.

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Antibodies to transition state analogues of the required reaction may act as synzymes. For example,
phosphonate esters of general formula (R-PO2-OR')- are stable analogues of the transition state occurring in
carboxylic ester hydrolysis. Monoclonal antibodies raised to immunising protein conjugates covalently
attached to these phosphonate esters act as esterases. The specificities of these catalytic antibodies (also
called abzymes) depends on the structure of the side-chains (i.e. R and R' in (R-PO2-OR')-) of the antigens.
The Km values may be quite low, often in the micromolar region, whereas the V max values are low (below 1
s-1), although still 1000-fold higher than hydrolysis by background hydroxyl ions. A similar strategy may be
used to produce synzymes by molecular 'imprinting' of polymers, using the presence of transition state
analogues to shape polymerising resins or inactive non-enzymic protein during heat denaturation.

Coenzyme-regenerating systems
Many oxidoreductases and all ligases utilise coenzymes (e.g. NAD+, NADP+, NADH, NADPH, ATP),
which must be regenerated as each product molecule is formed. Although these represent many of the most
useful biological catalysts, their application is presently severely limited by the high cost of the coenzymes
and difficulties with their regeneration. These two problems may both be overcome at the same time if the
coenzyme is immobilised, together with the enzyme, and regenerated in situ.
A simple way of immobilising/regenerating coenzymes would be to use whole-cell systems and these are,
of course, in widespread use. However as outlined earlier, these are of generally lower efficiency and
flexibility than immobilised-enzyme systems. Membrane reactors (may be used to immobilise the
coenzymes but the pore size must be smaller than the coenzyme diameter, which is extremely restrictive.
Coenzymes usually must be derivatised for adequate immobilisation and regeneration. When successfully
applied, this process activates the coenzymes for attachment to the immobilisation support but does not
interfere with its biological function. The most widely applied synthetic routes involve the alkylation of the
exocyclic N6-amino nitrogen of the adenine moiety present in the coenzymes NAD+, NADP+, NADH,
NADPH, ATP and coenzyme A.
In some applications, such as those using membrane reactors it is only necessary that the coenzyme has
sufficient size to be retained within the system. High molecular weight water-soluble derivatives are most
useful as they cause less diffusional resistance than insoluble coenzyme matrices. Dextrans,
polyethyleneimine and polyethylene glycols are widely used. Relatively low levels of coenzyme attachment
are generally sought in order to allow greater freedom of movement and avoid possible inhibitory effects.
The kinetic properties of the derived coenzymes vary, depending upon the system, but generally the
Michaelis constants are higher and the maximum velocities are lower than with the native coenzymes.
Coenzymes immobilised to insoluble supports presently have somewhat less favourable kinetics even when
co-immobilised close to the active site of their utilising enzymes. This situation is expected to improve as
more information on the protein conformation surrounding the enzymes' active sites becomes available and
immobilisation methods become more sophisticated. However, the cost of such derivatives is always likely
to remain high and they will only be economically viable for the production of very high value products.
There are several systems available for the regeneration of the derivatised coenzymes by chemical,
electrochemical or enzymic means. Enzymic regeneration is advantageous because of its high specificity
but electrochemical procedures for regenerating the oxidoreductase dinucleotides are proving competitive.
To be useful in regenerating coenzymes, enzymic processes must utilise cheap substrates and readily
available enzymes and give non-interfering and easily separated products. Formate dehydrogenase and
acetate kinase present useful examples of their use, although the presently available commercial enzyme
preparations are of low activity:

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Future prospects of enzyme engineering

Genetically Engineered Enzymes

Enzymes are naturally occurring proteins that speed up biochemical processes. They're used to
produce everything from wine and cheese to corn syrup and baked goods. Enzymes allow the manufacturer
to produce more of a particular product in a shorter amount of time, thus increasing profit.
Generally, the use of enzymes is beneficial. In some cases, they can replace harmful chemicals and reduce
water and energy consumption in food production. However, enzymes produced by genetically engineered
organisms are cause for concern. Not enough is known about the long-term effects of these enzymes on
humans and the ecosystem for them to be used across the board.
FDA regulations on enzyme use is a gray area. Enzymes used in the processing of foods do not have to be
listed on product labels because they are not considered foods. Also, when enzymes are genetically
engineered, the manufacturer is not required to notify the FDA that the enzymes have been modified. The
lists of GE enzymes known by the FDA is, by their own admission, "probably incomplete."
Worldwide, the enzyme market is a $1.3 billion industry. One of the largest enzyme manufacturers are
Novo Nordisk, which manufactures GE and non-GE enzymes. The FDA provided us with this partial list of
genetically engineered enzymes:
• Chymosin—used in the production of cheese
• Novamyl(TM)—used in baked goods to help preserve freshness
• Alpha amylase—used in the production of white sugar, maltodextrins and nutritive carbohydrate
sweeteners (corn syrup)
• Aspartic (proteinase enzyme from R. miehei)—used in the production of cheese
• Pullulanase—used in the production of high fructose corn syrup
If you want to absolutely avoid genetically engineered enzymes you will have two choices: avoid foods in
the following categories, or call the food manufacturers directly and ask them if their enzymes are
genetically engineered. They will probably have no idea. Ask them to check and call them back again. Let
us know if you get written confirmation.
• Beers, wines and fruit juices—(Enzymes used: Cereflo, Ceremix, Neutrase, Ultraflo, Termamyl, Fungamyl,
AMG, Promozyme, Viscozyme, Finizym, Maturex, Pectinex, Pectinex Ultra SP-L, Pectinex BE-3L, Pectinex
AR, Ultrazym, Vinozym, Citrozym, Novoclairzym, Movoferm 12, Glucanex, Bio-Cip Membrane, Peelzym,
Olivex/Zietex)
• Sugar—Enzymes used: Termamyl, Dextranase, Invertase, Alpha Amylase

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• Oils—Enzymes used: Lipozyme IM, Novozym 435, Lecitase, Lipozyme, Novozym 398, Olivex, Zeitex
• Dairy products—Enzymes used: Lactozym, Palatase, Alcalase, Pancreatic Trypsin Novo (PTN),
Flavourzyme, Catazyme, Chymosin
• Baked goods—Enzymes used: Fungamyl, AMG, Pentopan, Novomyl, Glutenase, Gluzyme
In many cases the enzymes named above are brand names. They may appear under other names as well.
Enzymes are usually found in minuscule quantities in the final food product. The toxin found in genetically
engineered tryptophan was less than 0.1 percent of the total weight of the product, yet it was enough to kill
people. The use of enzymes is pervasive in the food industry. Nothing is known about the long-term effects
of genetically engineered enzymes. We include this information so you can make an informed choice about
whether you want to eat them or not.

Enzymes produced by genetically modified microorganisms

Novozymes’ enzymes produced by genetically modified microorganisms
Novozymes A/S markets a range of enzymes for various industrial purposes. Many of these enzymes are produced by
fermentation of genetically modified microorganisms (GMMs).

There are several advantages of using GMMs for the production of enzymes, including:

• It is possible to produce enzymes with a higher specificity and purity

• It is possible to obtain enzymes which would otherwise not be available for economical, occupational health
or environmental reasons
• Due to higher production efficiency there is an additional environmental benefit through reducing energy
consumption and waste from the production plants
• For enzymes used in the food industry particular benefits are for example a better use of raw materials (juice
industry), better keeping quality of a final food and thereby less wastage of food (baking industry) and a
reduced use of chemicals in the production process (starch industry)
• For enzymes used in the feed industry particular benefits include a significant reduction in the amount of
phosphorus released to the environment from farming
Due to an efficient separation process the final enzyme product does not contain any GMMs.
The enzymes are produced by fermentation of the genetically modified micro organisms (the production
strain) which then produces the desired enzyme. The process takes place under well-controlled conditions
in closed fermentation tank installations.
After fermentation the enzyme is separated from the production strain, purified and mixed with inert
diluents for stabilisation.
The following is a list of Novozymes' enzymes produced by genetically modified organisms.

Food Applications:

Brand name Type of enzymes Main Application

Amylase® AG XXL Glucoamylase Juice Industry

Dextrozyme® Pullulanase / Amyloglucosidase Starch industry

Finizym® W Phospholipase Starch industry

Gluzyme® Mono Glucose oxidase Baking industry

Lecitase® Novo Lipase Oils and fats industry

Maltogenase® Maltogenic amylase Starch industry

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Maturex® Alpha-acetodecarboxylase Brewing industry

NovoCarne® Tender Protease Meat industry

Novoshape® Pectinesterase Fruit processing

Novozym® 27080 Carbohydrase / Lipase Baking industry

NOVOZYM® 27122 Xylanase Protein Hydrolysis

Novozym® 33081 Polygalacturonase Juice Industry

Novozym® 46016 Phospholipase Dairy industry

Novozym® 46019 Cellobiose oxidase Dairy Industry

Pectinex® XXL Pectin lyase / Polygalacturonase Juice Industry

Promozyme® D2 Pullulanase Starch industry

Saczyme® Glucoamylase Alcohol Industry

Toruzyme® Transferase Starch industry

Feed Applications:

Brand name Type of enzymes Main Application

Bio-Feed® Wheat Xylanase Animal feed industry

Bio-feed® Phytase Phytase Animal feed industry

Other Applications:

Brand name Type of enzymes Main Application

Alcalase® Subtillisin Detergent industry

Aquazym® LT-L Alpha-amylase Textile industry

BioPrep® Pectate lyase Textile industry

Carezyme® Cellulase Detergent industry

Clear-Lens® LIPO Lipase Personal care industry

DeniLite® Laccase Textile industry

DeniMax® 601 Cellulase Textile Industry

Duramyl® Alpha-amylase Detergent industry

Everlase® Subtillisin Detergent industry

Extruzyme® Pro Alpha-amylase Pet food industry

Greasex® Lipase Leather industry

Kannase® Subtillisin Detergent industry

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Future prospects of enzyme engineering

Lipex® Lipase Detergent industry

Lipolase® Lipase Detergent industry

Liquanase® Subtilisin Detergent industry

Liquozyme® Alpha-amylase Starch and Ethanol industry

Mannaway® Mannanase Detergent industry

NovoBate® 100 Trypsin Leather Industry

Chemical Modification of Enzymes –

We know that the proteins synthesized under the control of gene sequences in a cell undergo post
translational modification. This leads to stability, structural integrity, altered solubility and viscosity of
individual proteins. This may also alter the chemical reactivity.

These alterations can be achieved in vitro and may .sometimes even create entirely new enzyme, by
creating new active sites or modifying the old ones. Some of the examples will be described in this section.

Protein Modelling

Utilizing the data generated through X-ray diffraction and NMR studies, models can be constructed with
the help of computer graphics. There are computer programmes available (interactive colour graphics
programmes) with the help of which a protein structure can be fitted to the electron density map (obtained
from X-ray diffraction) by simultaneous display on the screen of computer monitor. Similarly, Van der
Waals surfaces for the protein can be displayed and interaction between several molecules simulated.

There are also other interactive molecular graphics which can be used (with the help of programmes) to
find out the perturbations (disturbances) in protein structure that will result from specific modifications of

amino acid sequences. We know that to some extent the three dimensional structure of a protein can be
predicted from the amino acid sequence, but we still have to depend partly on X-ray diffraction patterns for
determining the three dimensional structure.

In future when the three dimensional structure can be accurately predicted from amino acid sequence data,
this will lead to long term success in protein engineering. The models of proteins, made on the basis of
amino acid alterations, can then be tested for the predictions about structure function relationships.

Multienzyme Systems by Gene Fusion ( Bi and Polyfunctional Enzymes) –

Multienzyme systems have been artificially synthesized, which can catalyze sequential reactions in many
biotechnological production processes. Although, proximity of more than one enzymes can also be
achieved by co-immobilization and chemical cross linking, gene fusion appears to have the highest
potential in enzyme technology. The gene fusion technology, for preparation of bi-and polyfunctional
enzymes, involves joining of structural genes of two or more enzymes. The translational stop singal at the
3' end of the first gene is removed and ligated in frame to the A TG start codon of the second gene.
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Future prospects of enzyme engineering

Alternatively, short linkers (2-10 amino acids) are used. The novel chimaeric gene gives a single
polypeptide chain carrying active sites of both genes. This fusion may involve
(i) two monomeric enzymes
(ii) a monomeric and a dimeric enzyme or

(iii)two dimeric enzymes.

Rationale of Protein Enzyme Engineering - Although thousands of proteins have been
characterized in prokaryotes and eukaryotes, only few became commercially important. This is due to the
high cost of isolating and purifying enzymes in sufficient quantities.

Although the cost aspect has been overcome by producing an enzyme in large quantities in bacteria, for its
industrial application, an enzyme (outside the cell) should also have some characteristics in addition to
those of enzymes in the cells. These characteristics may include the following:
(i) enzyme should be robust with a long life;
(ii) enzyme should be able to use the substrate supplied in the industry even if it differs slightly from that in
the cell;
(iii) enzyme should be able to work under conditions (e.g. extremes of pH, temperature and concentration)
of the industry even if they differ from those in the cell.

In view of the above, enzyme should be engineered to meet the altered needs. Therefore, efforts have been
made to alter the properties of the enzymes. Following is the list of properties that one needs to alter in a
predictable manner for protein or enzyme engineering.

(1)Kinetic properties of enzyme turnover and Michaelis Constant, Km.

(2) Theremostability and the optimum temperature for the enzyme.
(3) Stability and activity of enzyme in nonaqueous solvents.
(4) Substrate and reaction specificity.
(5) Cofactor requirements.
(6) Optimun pH.
(7) Protease resistance.
(8) Allosteric regulation.
(9) Molecular weight and subunit structure.

For a particular class of enzymes, variation in nature may occur for each of the above properties, so that
one may like to combine the optimum properties to get the most efficient form of the enzyme.

This aspect of protein engineering will be illustrated using the example of glucose isomerases, which
convert glucose into other isomers like fructose and are used to make high fructose corn syrup vital for
soft drink industry. It exhibits wide variation in its properties.
Sometimes, it may not be possible to get a combination of optimum properties. For instance, an enzyme
with highest activity may not be the most stable. Therefore, a compromise in properties may have to be
made, if we have to select an enzyme from the available variability or even if we create variability by
mutagenesis.
However, if structure and function relationship of an enzyme is known, the structural features for
desirable function may be combined and protein engineering techniques may then be used to create a
novel enzyme exhibiting a combination of all desirable functional properties.

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Future prospects of enzyme engineering

Glucose isomerase belongs to a TIM barrel family of enzymes which resemble each other in having a
highly characteristic domain called TIM barrel, with active site for catalytic action at one end. This TIM
barrel may be found in enzymes that may differ in sequences and may catalyze different reactions.
As earlier discussed, since similarities of structure of protein meant similarities in function, TIM barrel
presents a challenge to this concept. However, it is curious tbat some enzymes in this family appear in
pairs in their metabolic pathways so that they catalyse two consecutive steps thus showing coupling of
their functions.
As an example of two enzymes of TIM barrel family, while 'triose phosphate isomerase' is one of the
most efficient catalysts, 'glucose isomerase' is relatively very inefficient.
Therefore, if 'glucose isomerase' enzyme is redesigned to use the highly efficient domain of TIM barrel
family, it will be a remarkable achievement for soft drink industry. Efforts in this direction are being
made (see later for methods of protein engineering).

Acheivements of Protein Engineering

A number of proteins are known, now, where efforts have been made to know the effects of site specific
mutagenesis involving substitution of one or more amino acids. Efforts have also been made to study in
detail the function of different regions of a protein. Following are some results of such efforts.

β-lactamase. This enzyme functions in the periplasmic space of bacterial cells. The enzyme hydrolyses and
inactivates the beta- lactam ring of penicillin derivatives and helps in transport across the inner membrane.
During transport a polypeptide (signal sequence peptide of 23 amino acids) is cleaved off.

Detailed analysis suggested that, transport and processing does not depend on this polypeptide of 23 amino
acids alone. An active site involving amino acid serine has also been identified, since its replacement by
cysteine leads to reduction in the activity of this enzyme.

Dihydrofolate reductase. In this enzyme, replacement of a single amino acid, aspartic add (ASP) by
asparagine (ASN), led to a decrease in specific activity by a thousand fold, suggesting that aspartic acid is
very important.(or the active site. Other similar modifications were also examined.

Insulin. It consists of A and B chains linked by C-peptide of 35 amino acids. It was shown that a sequence
of 6 amino acids for C-peptide was adequate for the, linking function.

Lactose permease (product of, gene of 'lac' operon). This enzyme is involved in transport of lactose and
a cysteine to glycine substitution showed that this amino acid was not essential for transport. Further, out of
four histidine residues, two at positious 35 and '39 do' not play any essential role in transport, while the
mutation in any of the other two histidines at positions 208 and 322, lead to loss of transport function.

T4 lysozyme. A mutation of isoleucine to cystine in this enzyme leading to formation of a disulphide

bridge led to thermal stability and a 200 fold increase in enzyme activity even at 6T'C.

Human beta interferon. Removal of one of the three cysteine residues' I led to an improvement in stability
of the enzyme.

λ repressor. This protein could be engineered to develop a specific site for cro protein, since the alteration
led to development of a cro recognition site.I

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Future prospects of enzyme engineering

Acetylcholine receptor. This protein is involved in transport, of acetylcholine through. the membrane.
Specific regions of this protein involved in acetylcholine binding and channel formation have been,
identified.

Cytochrome C. A phenylalanine residue has been identified to be non-essential for electron transfer but is
involved in determining the reduction potential of the protein.

Trypsin. It could be redesigned to have altered substrate specificity.

Subtilisin. Another successful alteration of substrate specificity involved the enzyme subtilisin reported in
1986-87.

Lactate dehydrogenase. A lactate dehydrogenase (LDH) from Bacillus stearothermophilus was modified
separately by each of the three substitutiens of amino acids (resulting from mutations; Asp197... Asn;
Thr246"'Gly; Gln102...Arg). The substitution, Gln102"'Arg, led to change in specificity from lactate to
malate, with high efficiency, comparable to that which the native LDH had for lactate.

Lactic protease. Substrate specificity of lactic protease (in E. coli), has been shown to be dramatically
modified by replacing active site methionine by alanine (Met192-->Ala; Mer13-->Ala).

Protein Structures for Future Enzyme Engineering – Following are some of the proteins, whose
structures are known and for which. cloned genes are available, so that they can be used for protein
engineering:
(i) immunoglobulins,

(ii) glucose isomerases,

(iii) amylases,

(iv) p-hydroxybenzoate hydroxylase,

(v) ribulose-1, 5, bisphosphate carboxylase (ruBP), (vi) repressor proteins, and (g) restriction edonucleases.

Drug Designing by Blocking Enzyme Activity –

Since most drugs act by modifying or blocking the activity of an enzyme, a deeper understanding of
suitably chosen target enzymes should lead to major advances in rational drug designing. Following two
examples will illustrate the utility of drug designing as an important area of biotechnology research.

Trimethoprim (TMP). This clinically important antimicrobial drug provides an important example to
demonstrate drug designing. This drug inhibits the enzyme dihydrofolate reductase (dHFR) in bacteria and
is frequently used to treat urinary tract infections, but in high concentrations it attacks even human dHFR,
thus becoming harmful (toxic), if used by the patient.

In view of this, the structures of different dHFRs have been compared, so that TMP with greater specificity
for bacterial enzyme may be designed. Since TMP shows flexibility in its three dimensional structure in
association with enzyme dHFR, efforts have been made to synthesize TMP, which will have a rigid three

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Future prospects of enzyme engineering

dimensional structure in association with bacterial enzyme dHFR, so that it may not be able to attack
human dHFR.

Inhibitor of renin. Inhibitors of the enzyme 'renin' are also being actively modelled. The enzyme catalyses
the first in a series of reactions that lead to elevated blood pressure. Models of renin have been prepared on
the basis of known structures of similar other proteins like aspartic proteinase and pepsin.
This led to the designing of non peptide inhibitors that mimic intermediate product in the reaction of renin
with its substate, so that native renin will not function. These inhibitors may be useful for treating
hypertensions.
Novel Applications and Future Uses

• The exploitation of enzymes as electrocatalysts (specific biosensors);

• Enzymes as analytical tools to measure specific compounds, for the regeneration of specific
metabolites;
• Enzyme utilization in the synthesis of bulk organic materials and the production of fragrances and
cosmetics;
• Enzyme utilization in the formation of food flavours and aroma compounds;
• The use of enzymes as tools for the detoxification of pesticide residues;
• Enzymes as monitors of toxic chemical levels in food and water.
Biomedical Applications of Enzyme Technology
The synthesis of new anti-microbial compounds
Enzyme replacement therapy
Enzymes in the treatment of cancer
Enzyme graft and dermatological applications
Enzymes as activators of precursor biomolecules
Enzyme technology in the prevention of dental cavities.

The use of enzymes in analysis

Enzymes make excellent analytical reagents due to their specificity, selectivity and efficiency. They are
often used to determine the concentration of their substrates (as analytes) by means of the resultant initial
reaction rates. This is being achieved by the production of biosensors which exploit biological systems in
association with advances in micro-electronic technology.
Principle of Biosensors
A biosensor is a sensor that is based on the use of biological material for its sensing function. The
biocomponent specially reacts or interacts with the analyte of interest resulting in a detectable chemical or
physical change. A biosensor is an analytical device which converts a biological response into an electrical
signal. The term ‘biosensor’ is often used to cover sensor devices used in order to determine the
concentration of substance and other parameters of biological interest even where they do not utilize a
biological system directly. A successful biosensor must possess at least some of the following beneficial
features:

Conclusions

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Future prospects of enzyme engineering

Enzyme technology is presently going through a phase of maturation and evolution. The maturation is
shown by the development of the theory concerning how enzymes function and how this is related to their
primary structure through the formation and configuration of their three-dimension structure. The evolution
is shown by the ever-broadening range of enzymic applications.
There still remains much room for the development of useful processes a materials based on this hard-won
understanding. Enzymes will clearly be more widely used in the future and this will be reflected in the
number enzymes available on an industrial (and research) scale, the variety of reactions catalysed and the
range of environmental conditions under which they will operate. Established enzymes will be put to new
uses and novel enzymes, discovered within their biological niches or produced by design using enzyme
engineering, will be used to catalyse hitherto unexploited reactions. This is just the start of the enzyme
technology era.
References:
Dubey R.C. “Text book of biotechnology” S.chnad publications
http://www.molecular-plant-biotechnology.info
http://www.biotechweblog.com/50226711/synthetic_heparin_from_engineered_enzyme
s.php
http://www.codexis.com/pdf/LuetzPerspective.pdf
http://www.faqs.org/abstracts/Science-and-technology/Green-chemistry-awards-
Engineered-enzymes-catalyze-a-new-field.html
http://dspace.mit.edu/handle/1721.1/38917

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