1.

INTRODUCTION

Proteases produced by enzymatic method were more

environment friendly when compared with the chemical process and it has
tremendous potential in the leather industry and in other several industries.
However optimization of protease could involve several variables such as
temperature, pH and incubation period. In this regard the Bacillus species
were exploited for their ability to produce these enzymes.

In the animal kingdom, fishes are a large group consisting of

24000 species showing wide morphological and habitat variations. They
occupy marine and fresh water environments, while a few are able to survive
in both. Besides this, some undertake anadromous and catadromous
migrations for spawning. These diverse conditions of habitat and feeding
preferences have influenced the biochemical composition of fish species.
Even within in the species variations can occur depending on physiological
condition, season etc.

Unlike the seafood processing sector, fresh water fish or the

inland fisheries sector is un-organized and hence poses a different level of
waste disposal problems. These by-products are rich in protein and fat which
make them more perishable. As per one estimate, visceral waste alone
contributes to the total of 3, 00, 000 tones (Mahendrakar, 2000). Further,
viscera have been reported to be a good source of proteins including enzymes
and fats. Also, the visceral waste harbors a microbial population that can

1
produce proteolytic/lipolytic enzymes which, if identified, can be used for
further applications like lipid hydrolysis and for producing protein
hydrolysates sector.

Since fish is harvested from natural water bodies including

farms, it harbors a number of microorganisms found in the environment from
where it is caught. These native microorganisms may include fish spoilage
bacteria as well as pathogens. In addition to these inherent microorganisms,
the fish can be contaminated with other microorganisms during handling,
transportation and processing, right from the point of catch to the end product.
These microorganisms can be hazardous to the health of the consumer. The
most important pathogens which gain entry into fish during handling,
transportation, and processing are Salmonella, Vibrio cholera, Staphyloccous
aureus, and Listeria monocytogenes. In the addition, Entropathogenic
Escherichia coli (EREC), Clostridium perfringes and Bacillus cereus may
also gain entry into fish.

Bacillus species are aerobic, sporulating, rod-shaped bacteria

that are ubiquitous in nature. Bacillus species are used in many medical,
pharmaceutical, agricultural, and industrial processes that take advantage of
their wide range of physiologic characteristics and their ability to produce a
host of enzymes, antibiotics, and other metabolites. Early in 1977,Priest et al.,
it was, reported that the gram-positive, spore forming bacterium Bacillus
subtilis produces and secretes proteases, esterases, and other kinds of
exoenzymes at the end of the exponential phase of growth. Bacitracin and
polymyxin are two well-known antibiotics obtained from Bacillus species.
Several species are used as standards in medical and pharmaceutical assays.
Certain Bacillus species are important in the natural or artificial degradation

2
of waste products. Some Bacillus insect pathogens are used as the active
ingredients of insecticides. On the other hand many Bacillus species are being
resistant to heat, radiation, disinfectants, and desiccation, they are difficult to
eliminate from medical and pharmaceutical materials and can be a cause of
contamination. In addition, Bacillus species are well known in the food
industries as troublesome spoilage organisms. Hence, techniques learnt and
used in this study can also be applied in quality assurance and quality control
departments of medical and pharmaceutical industries as well as in food
processing

The family Bacillaceae, consisting of rod-shaped bacteria that

form endospores, has two principal subdivisions: the anaerobic spore-forming
bacteria of the genus Clostridium, and the aerobic or facultatively anaerobic
spore-forming bacteria of the genus Bacillus frequently known as ASB
(aerobic spore-bearers). Bacterial cells of Bacillus cultures are Gram positive
when young, but in some species become Gram negative as they age and
hence, it is to be ensured that enzyme production be done when the cultures
are in exponential phase.

Proteolytic enzymes are ubiquitous in occurrence, being found in

all living organisms, and are essential for cell growth and differentiation. The
extracellular proteases are commercial value and find multiple applications in
various industrial sectors. Although there are many microbial sources
available for producing proteases, only a few are recognized as commercial
producers (Gupta, et al., 2002b). Of these, strains of Bacillus sp. dominate the
industrial sector (Gupta et al., 2002a). In addition to that, several workers
investigated the production of protease and alkaline protease from Bacillus

3
subtilis (Uchida et al., 1972; Daguerre et al., 1975; Remeikaite, 1979;
Massucco, 1980; Gomaa et al., 1987) and explaining that only small amounts
are produced by them and hence the comprehensive method to purify and
clone isolates for production and enzyme purification (Andrade et al., 2002).

Proteases represent one of the most important groups of industrial

enzymes and account for at least a quarter of the total global enzyme
production (Layman, 1986). Different species of bacteria produce acidic,
neutral and alkaline proteases. The production of extra cellular proteases is
governed, at least in part, of available individual nutrients (North, 1982).Since
microorganisms can be made to propagate rapidly and profusely, they are an
ideal source for enzymes. (Rehm, 1986)

Proteases are active at mild conditions, with pH optima in the

range of 6 to 8; they are robust and stable, do not require stoichiometric
cofactors and are also highly stereo and regioselective (Bordusa, 2002). These
properties are quite relevant to use them as catalysts in organic synthesis. This
is possible because proteases can not only catalyze the cleavage of peptide
bonds but also their formation (Capellas et al., 1997; Björup et al., 1999; So et
al., 2000), as well as other reactions of relevance for organic synthesis, for
instance: the regiospecific hydrolysis of esters and the kinetic resolution of
racemic mixture, (Khmelnitsky et al., 1997; Carrea and Riva, 2000; Bordusa,
2002 Extracelluar ) . Subtilisin, chymotrypsin, trypsin and papain have been
widely used proteases in the chemical synthesis of peptides.

4
 Protease
families

Serine Threonine Cysteine Aspartic Metallic

groups

There are five families of proteases in which serine, threonine, cysteine,

aspartic or metallic groups play a primary catalytic role. Serine, cysteine and
threonine proteases are quite different from aspartic and metalloproteases. In
the first three groups, the nucleophile in the catalytic center is part of an
amino acid residue, while in the second two groups the nucleophile is an
activated water molecule. In cysteine proteases the nucleophile is a sulfhydryl
group and the catalytic mechanism is similar to the serine proteases in which
the proton donor is a histidine residue.

PROTEASES OR
PEPTIDASES

ENDOPEPTIDASES EXOPEPTIDASES
Peptidases

Peptidases hydrolyze peptide bonds within the protein chain,

previously called endopeptidase while proteases hydrolyse large polypeptides
into smaller molecules. (Adinarayana et al., 2004).

5
Endopeptidases

These cleave peptide bonds at points within the protein and

remove amino acids sequentially from either N or C-terminus respectively.
The term proteinase is also used as a synonym word for endopeptidase and
four mechanistic classes of proteinases are recognized by the IUBMB
(International Union of Biochemistry and Molecular Biology 1984).

Exopeptidases

The exopeptidase act only near the ends of polypeptide chains at

the N or C terminus. Those acting at a free N terminus liberate a single amino
acid residue (amino peptidases), a peptide (dipeptidyl-peptidases) or a
tripeptide ( tripeptidyl peptidases). The exopeptidases acting at a free carbon
terminus liberate a single amino acid (carboxyl peptidases) or a dipeptide
(peptidyl-dipeptidases). Some exopeptidases are specific for dipeptidases or
remove terminal residues that are substituted, cyclised or linked by isopeptide
bonds. Isopeptide bonds are peptide linkages other than those of a carboxyl to
an amino acid groups and this group of enzymes is redenominated omega.

Physiological functions of proteases

Proteases execute a large variety of complex physiological functions. Their
importance in conducting the essential metabolic and regulatory functions is
evident from their occurrence in all forms of living organisms. Proteases play
a critical role in many physiological and pathological processes such as
protein catabolism, blood coagulation, cell growth and migration, tissue

6
arrangement, morphogenesis and development, inflammation, tumor growth
and metastasis, activation of zymogens, release of hormones and
pharmacologically active peptides from precursor proteins, and transport of
secretory proteins across membranes. In general, extracellular proteases
catalyze the hydrolysis of large proteins to smaller molecules for subsequent
absorption by the cell whereas intracellular proteases play a critical role in the
regulation of metabolism. In contrast to the multitude of the roles
contemplated for proteases, our knowledge about the mechanisms by which
they perform these functions is very limited.

Protein turn over

All living cells maintain a particular rate of protein turnover by continuous,
albeit balanced, degradation and synthesis of proteins. Catabolism of proteins
provides a ready pool of amino acids as precursors of the synthesis of
proteins. Intracellular proteases are known to participate in executing the
proper protein turnover for the cell. In E. coli, ATP-dependent protease La, the
lon gene product, is responsible for hydrolysis of abnormal proteins (Chung et
al., 1981). The turnover of intracellular proteins in eukaryotes is also affected
by a pathway involving ATP-dependent proteases ( Hershko et al., 1984).
Evidence for the participation of proteolytic activity in controlling the protein
turnover was demonstrated by the lack of proper turnover in protease-deficient
mutants.

1.1 MODE OF ACTION

Proteolytic enzymes are involved in a great variety of physiological processes

and this action can be divided in to two different categories.

7
1) Limited proteolysis

2) Unlimited proteolysis.

Limited proteolysis
In this type, the protease cleaves only one or a limited number of peptide
bonds of a target protein leading to the action or maturation of the formerly
inactive protein e.g. conversion of prohormones to hormones.

Unlimited proteolysis
In this case, proteins are degraded into their amino acid constituents. The
proteins to be degraded are usually first conjugated to multiple molecule of
the polypeptide ubiquitin. This modification makes them for rapid hydrolysis
by the proteasome in the presence of ATP. Another pathway consists in the
compartmentation of proteases e.g. lysomes. Proteins transferred into this
compartment undergo a rapid degradation.

1.2 SOURCES OF PROTEASE

Proteases are found in all forms of microbes, plants and animals.

 Proteases from microbes

Proteases are found in several microorganisms such as viruses,

protozoa, bacteria, yeast and fungi. The inability of the plant and animal
proteases to meet current world demands has led to an increased interest in

8
microbial proteases. Microorganisms represent an excellent source of
enzymes owing to their broad biochemical diversity and their susceptibility to
genetic manipulation. Proteins are degraded by microorganisms, and they
utilize the initiated proteinases (endopeptidases) secreted by microorganisms
followed by further hydrolysis by peptidases (exopeptidases) at the extra or
intracellular site. Numerous proteinases are produced by microorganisms
depending on the species of the produces the strains even belonging to the
same species. Several proteinases are also produced by the same strain under
various cultural conditions.

Candida albicans and C.tropicalis are the medically more

important opportunistic pathogens causing infections in immunocompromised
patients. Their extracellular enzyme, an aspartic proteinase is considered to be
a major virulence factor. Most commercial serine proteases, mainly neutral
and alkaline, are produced by organisms belonging to the genus Bacillus.
Some of the gram-negative bacteria producing proteases were identified as
Pseudomonas aeruginosa (Morigara, 1964), Vibrio chlorae (Deane et al.,
1987), Xathomonas maltophila (Debette, 1991). Some rare microorganisms
produce alkaline proteases. Kurthia spiroforme was reported to produce
protease (Green et al. 1989).

Halophiles were described to produce alkaline proteases includes

Halobacterium species (Ahan et al., 1990). Similar enzymes are also produced
by other bacteria such as Thermus caldophilus and Desulfurococus mucosus,
Streptomyces, Sermons and Escherichia genera. Fungi produce several serine
proteinases. Cysteine proteinases are not so widely distributed as seen with
serine and aspartic proteinases.

9
 Trichomonas vaginalis, a flagellated protozoan responsible for
trichomonosis, one of the most common sexually transmitted diseases, has
numerous cysteine and some metallo proteinases. The cysteine enzymes are
involved in the damage to the host by the parasite.

Microbial proteases account to approximately 40% of the worldwide

enzymes sales. In addition, proteases from microbial sources are preferred to
the enzymes from plant and animal sources since they posses almost all
characteristics desired for their biotechnological applications.

 Proteases from plants

Papain is obtained from the leaves and unripe fruit of the

Carica papaya. Papain has the property to transform albuminoids into
peptones in either acid or alkaline or neutral medium it superior to pepsin.
Another plant based proteolytic enzyme bromalain comes from the stems of
pineapple.

 Proteases from animals

The most familiar proteases of animal origin are pancreatic

trypsin, chymotrypsin, pepsin and rennin (boyer, P.D.1971) these are prepared
in pure form in bulk quantities. However, their production depends on the
availability of livestock for slaughter, which in turn in governed by political
and agricultural policies (Hoffman 1974). Rennin (mainly chymosin) obtained
from the stomach (abomasums) of unweaned calves has been used in the

10
production of cheese. Digestive enzymes such as trypsin, chymotrypsin etc.
from the animals are proteases.

1.3 APPLICATIONS OF PROTEASE

Proteolytic enzymes account for nearly 60% of the industrial

market in the world. They find application in a number of biotechnological
processes, viz. in food processing, and Pharmaceuticals, leather industry, silk,
bakery, soy processing, meat tendering and brewery industries. However, its
application in the production of peptide synthesis in organic media is limited
by the presence of organic solvents. (Rahman et al., 2005).

 Protease in detergent industry

Proteases are one of the standard ingredients of all kinds of detergents

ranging from those used for household laundering to reagents used for
cleaning contact lenses or dentures. The use of proteases in laundry detergents
accounts for approximately 25% of the total worldwide sales of enzymes. The
preparation of the first enzymatic detergent, “Burnus,” dates back to 1913; it
consisted of sodium carbonate and a crude pancreatic extract. The first
detergent containing the bacterial enzyme was introduced in 1956 under the
trade name BIO-40. In 1960, Novo Industry A/S introduced alcalase,
produced by Bacillus licheniformis; its commercial name was BIOTEX.
Maxatase, a detergent made by Gist-Brocades, followed this. The ideal

11
detergent protease should possess broad substrate specificity to facilitate the
removal of a large variety of stains due to food, blood, and other body
secretions. Activity and stability at high pH and temperature and compatibility
with other chelating and oxidizing agents added to the detergents are among
the major prerequisites for the use of proteases in detergents. The use of
enzyme is mainly due to shorter period of agitation, lower wash temperature
often after a preliminary period of soaking(Nielsen Jensen and Outlrup 1981)
the interest in using alkaline enzymes in automatic dishwashing detergents has
also increased recently.
A combination of lipase, amylase and cellulase is expected to enhance
the performance of protease in laundry detergents. All detergent proteases
currently used in the market are serine proteases produced by Bacillus strains.
An alkaline protease from Conidiobolus coronatus was found to be
compatible with commercial detergents used in India and retained 43% of its
activity at 50°C for 50 min in the presence of Ca2+ (25 mM) and glycine (1M).
(Bhosale et al, 1995).

 Removal of blood stain

Alkaline proteases showed high capability for removing proteins and
stains from cloth so it is used in detergent powder or solutions. Protease from
Spilosoma oblique was used for removal of blood (Anwar and Saleemuddin,
1997). Properties of the microbial protease such as alkaline pH, thermo
stability and can digest collagen, which helps in dehairing.

12
  Protease in wool industries

The uses of application of protease primarily were found in think

proof wool industry. Wool fibers are covered in overlapping scales pointing
towards the fiber tip. A successful method involved the partial hydrolysis of
scale tips with the protease, papain. This method was abandoned few years
ago, primarily for economic reasons.

 Protease in Silver Recovery

Alkaline proteases find potential application in the bioprocessing

of used x-ray films for sliver recovery. The enzymatic hydrolysis of the
gelatin layers on the x-ray film enables not only the silver but also the
polyester films base, to be recycled. The alkaline protease from Bacillus sp.
B21-2 (Fujiwar and Yamamotto, 1987) decomposed the gelatinous coating on
the used x-ray films from which the silver was recovered.

 Protease used in Food Industry

Certain proteases have been used in food processing for centuries

and any record of the discovery of their activity have been lost amid sty
time. Papain from the Kaves and unripe fruit of carica papaya has been used

13
to tenderize meat. Proteases play a prominent role in meat tenderization,
especially of beef. An alkaline elastase (Takage et al., 1992) and
thermophillic alkaline protease (Wilson et al., 1992) have proved to be
successful and promising meat tenderizing enzymes, as they possess the
ability to hydrolyze connective tissue proteins as well as muscle fiber
proteins. A patented method used a specific combination of neutral and
alkaline proteases for hydrolyzing raw meat. The reason for this may be that
the preferential specificity was favorable when metalloprotease and serine
protease were used simultaneously (Pender son et al., 1994).Current trend in
similar research shows yet another alkaline protease from
B.amyloliquefaciens resulted in the production of a methionine rich protein
hydrolysate form chickpea protein. (George et al., 1997).

 Medical applications

It also regulates various metabolic processes such as blood

coagulation, fibrinolysis, complement activation, phagocytosis and blood
pressure control (Adinarayana et al., 2004). Collagenases with alkaline
protease activity are increasingly used for therapeutic applications in the
preparation of slow-release dosages forms. Elastosterase, a preparation with
high electrolytic activity from B.subtilis 316M was immobilized on a bandage
for the therapeutic application in the treatment of burns and purulent wounds,
carbuncles, furanches and deep abscess (kudrya and Simonanko, 1994) and

14
alkaline proteases having fibrinolytic activity have been used as a
thrombolytic agent. (Kim et al., 1996).

1.4Advantages of enzymatic dehairing

(i) Significant reduction or even complete elimination of the use of sodium

sulphide.
(ii) Recovery of hair of good quality and strength with a good saleable value.
(iii) Creation of an ecologically conducive atmosphere for the workers.
(iv) Enzymatically dehaired leathers have shown better strength properties and
greater surface area.
(v) Simplification of pre-tanning processes by cutting down one step, viz.
bating.
(vi) A significant nature of the enzymatic dehairing process is the time factor
involved. The lime-sulphide process takes about 16 h, whereas the enzymatic
dehairing would be also completed within 12 hrs.
Proteolytic enzymes are of great commercial importance, contributing
to more than 40% of the world commercially produced enzymes.
Approximately 50% of the enzymes used as industrial process aids are
proteolytic enzymes. Proteolytic enzymes are more efficient in enzymatic
dehairing than amylolytic enzymes.
Proteolytic enzymes derived from a large number of Bacillus sp. and
Streptomyces sp. have been used in dehairing of hides and skins. A lime and
sulphide-free process of dehairing has been developed for the manufacture of
suede from sheep skins using protease from B. subtilis. Schlosser et al. have
reported a method of depilation in an acid medium containing Lactobacillus
culture.

15
1.5 Waste treatment and digestion of natural proteins

Alkaline proteases provide potential application for the management

of wastes from various food processing industries and household activities.
These proteases can solubilize proteins in wastes through a multistep process
to recover liquid concentrates or dry solids of nutritional value for fish or
livestock (Shoemaker, 1986 and Shih, 1993). Dalev reported an enzymatic
process using a B. subtilis alkaline protease in the processing of waste
feathers from poultry slaughter houses (Dalev, 1994). Feathers constitute
approximately 5% of the body weight of poultry and can be considered a high
protein source for food and feed, provided their rigid keratin structure is
completely destroyed. Pretreatment with NaOH, mechanical disintegration,
and enzymatic hydrolysis resulted in total solubilization of the feathers. The
end product was a heavy, grayish powder with a very high protein content,
which could be used as a feed additive.

Similarly, many other keratinolytic alkaline proteases were used in

feed technology (Dhar et al., 1984., Chandrasekaran et al., 1986 and Bockle et
al,, 1997) for the production of amino acids or peptides (Lin et al., 1996, Kida
et al., 1995) , for degrading waste keratinous material in household refuse
(Mukhopadhyay, 1992), and as a depilatory agent to remove hair in bath tub
drains, which caused bad odors in houses and in public places (Takami et al.,
1992) . Microbial proteases have the capability to digest different natural
substrates with base of fibrin, albumin and collagen suggesting usefulness for
different applications.

16
FUTURE SCOPE
Proteases are a unique class of enzymes, since they are of immense
physiological as well as commercial importance. They possess both
degradative and synthetic properties. Since proteases are physiologically
necessary, they occur ubiquitously in animals, plants, and microbes. However,
microbes are a goldmine of proteases and represent the preferred source of
enzymes in view of their rapid growth, limited space required for cultivation,
and ready accessibility to genetic manipulation. Microbial proteases have been
extensively used in the food, dairy and detergent industries since ancient
times. There is a renewed interest in proteases as targets for developing
therapeutic agents against relentlessly spreading fatal diseases such as cancer,
malaria, and AIDS. Advances in genetic manipulation of microorganisms by
SDM of the cloned gene opens new possibilities for the introduction of
predesigned changes, resulting in the production of tailor-made proteases with
novel and desirable properties. The advent of techniques for rapid sequencing
of cloned DNA has yielded an explosive increase in protease sequence
information. Analysis of sequences for acidic, alkaline, and neutral proteases
has provided new insights into the evolutionary relationships of proteases.
Despite the systematic application of recombinant technology and protein
engineering to alter the properties of proteases, it has not been possible to
Obtain microbial proteases that are ideal for their biotechnological
applications. Industrial applications of proteases have posed several problems
and challenges for their further improvements. The biodiversity represents an
invaluable resource for biotechnological innovations and plays an important
role in the search for improved strains of microorganisms used in the industry.
A recent trend has involved conducting industrial reactions with enzymes
reaped from exotic microorganisms that inhabit hot waters, freezing Arctic

17
waters, saline waters, or extremely acidic or alkaline habitats. The proteases
isolated from extremophilic organisms are likely to mimic some of the
unnatural properties of the enzymes that are desirable for their commercial
applications. Exploitation of biodiversity to provide microorganisms that
produce proteases well suited for their diverse applications is considered to be
one of the most promising future alternatives. Introduction of extremophilic
proteases into industrial processes is hampered by the difficulties encountered
in growing the extremophiles as laboratory cultures. Revolutionary robotic
approaches such as DNA shuffling are being developed to rationalize the use
of enzymes from extremophiles. The existing knowledge about the structure-
function relationship of proteases, coupled with gene-shuffling techniques,
promises a fair chance of success, in the near future, in evolving proteases that
were never made in nature and that would meet the requirements of the
multitude of protease applications.

A century after the pioneering work of Louis Pasteur, the science of

microbiology has reached its pinnacle. In a relatively short time, modern
biotechnology has grown dramatically from a laboratory curiosity to a
commercial activity. Advances in microbiology and biotechnology have
created a favorable niche for the development of proteases and will continue
to facilitate their applications to provide a sustainable environment for
mankind and to improve the quality of human life.

18
 2. REVIEW OF LITERATURE

2.1. LITERATURE REVIEW ON PROTEASE ENZYME

• Today, proteases account for approximately 40% of the total

enzyme sales in various industrial market sectors, such as detergent,
food, pharmaceutical, leather, diagnostics, waste management and
silver recovery. This dominance of proteases in the industrial market
is expected to increase further by the year 2005 (Godfrey and West
1996).

• However, until today, the largest share of the enzyme market has
been held by detergent alkaline proteases active and stable in the
alkaline pH range. Microbial proteases have been reviewed several
times, with emphasis on different aspects of proteases.

• Aunstrup (1980) focused on microbial selection and fermentation

of proteases, whereas Ward (1985) mainly dealt with the sources of
microbial proteases and their possible functional role in nature.

• Kalisz (1988) updated the available information with a detailed

description of the types of proteases and their commercial
applications, whereas Outtrup and Boyce (1990) focused on the

19
 industrially important proteases, their applications and the role of
molecular biology in protease research.

• Protease synthesis is also affected by rapidly metabolizable nitrogen

sources, such as amino acids in the medium. Besides these, several
other physical factors, such as aeration, inoculum density, pH,
temperature and incubation, also affect the amount of protease
produced. ( Hameed et al., (1999); Puri et al., (2002))

• In order to scale up protease production from microorganisms at the

industrial level, biochemical and process engineers use several
strategies to obtain high yields of protease in a fermentor. Controlled
batch and fed-batch fermentations using simultaneous control of
glucose, ammonium ion concentration, oxygen tension, pH and salt
availability and chemostat cultures (Frankena et al. 1985, 1986)
have been successfully used for improving protease production for
long-term incubations, using a number of microorganisms. (Hameed
et al., (1999); Hubner et al., (1993); Mao et al., (1992); Van
Putten et al., (1996))

• In a recent study by our group, the overall alkaline protease yield

from B. mojavensis was improved up to 4-fold under semi-batch and
fed-batch operations by separating biomass and protease production
phases, using intermittent de-repression and induction during the
growth of the organism. (Beg et al., (2002))

• In recent years, there has been a great amount of research and

development effort focusing on the use of statistical approach

20
 methods, using different statistical software packages during process
optimization studies, with the aim of obtaining high yields of
alkaline protease in the fermentation medium. De Coninck et al.,
(2000); Puri et al., (2002); Varela et al., (1996).

• The vast diversity of proteases, in contrast to the specificity of their

action, has attracted worldwide attention in attempts to exploit their
physiological and biotechnological applications (Fox et al., 1991,
Rao et al., 1998.).

2.2. LITERATURE REGARDING ISOLATION OF PROTEASE

• The quantitative and qualitative distribution of bacteria in freshly

caught fish fairly depicts the natural bacterial population (Varma et
al, 1982., Surendran et al and Gopakumar, 1982.,
Nirmalathamparun et al, 1983).

• Fresh water fishes are documented to harbor higher percentage of

Gram positives belonging to the family Micrococcaciae and
Bacillaceae, which together comprised 50% of the total bacterial
load. Gram negatives Pseudomonas and Enterobacteriaceae have
also been reported (Surendran et al, 1985).

• In the case of freshly caught fish from marine or fresh water areas
the total plate count are reported to be in the range of 10²-106/g (Lee,
1969., Cann et al, 1971., Lee and Pfeifer, 1977). Very high counts
of the order 107-108/g have been reported in the intestines of fish and
shrimp (animal sources) (Vanderzant et al, 1970).

21
• Microbial proteases account for approximately 40% of the total
worldwide enzyme sales (Godfrey et al, 1996)

• Daisuke Tsuru et al.,(1965) described the physiochemical

properties of Bacillus subtilis neutral protease and they compared
these properties with those of other bacterial alkaline proteases.
They found that the neutral protease was quite distinct in amino acid
composition from other proteases isolated from various strains of
Bacillus subtilis.

• Aronson et al., (1971) used casein agar for the maximum

production of extracellular protease enzyme. He selected 29 mutants
of Bacillus circus and they all formed spores out of which 27 were
auxotrophs for purines and pyrimidines. Upon reversion to the
prototrophy a large fraction regained the capacity to reproduce
extracellular protease.

• Kerry Yasunobu and James McConn (1965) extracted neutral

protease from Bacillus subtilis and assayed the enzyme activity
using the casein digestion method. They studied the physio-chemical
Properties of the protease enzyme. Similar enzyme was isolated from
Bacillus amyloliquefaciens.

• Yeshodha et al., (1976) ruled the optimum conditions for the

extraction of protease from jawasse (edible sources). The enzyme
was optimally active at pH 6.0 with 2.5% egg albumin as substrate.

22
 They also revealed that the enzyme was inhibited by para-chloro-
metacresol, sodium pentachlorophenate, phenyl mercuric nitrate and
sodium trichlorophenate.

• Pinar calik et al., (1998) investigated the effects of oxygen transfer

on serine protease (SAP) production by Bacillus licheniformis on the
defined medium having citric acid as the sole carbon source. In
addition to SAP activity they also studied about the concentration of
the product (SAP) and by products, i.e., neutral protease, amylase,
amino acid and organic acids.

• Bayoudh et al., (2000) extracted alkaline protease produced by

Pseudomonas aeroginosa MN 1, isolated from an alkaline tannery
wastewater, was purified and characterized.

• Alkaline protease production from alkalophilic Bacillus sp. Isolated

from natural habitats. Enzyme and microbial technology in press.
(Genckal, H., and Tari, C., (2006))

• Hansen, G. H., Strom, E., and Olafsen, J. A., (1992), Effect of

different holding regimes on the intestinal microflora of herring
(Clupea harengus) larvae. Applied and environmental microbiology,
vol. 58, p. 461-470.

23
 • Singh et al., (2001) extracted serine alkaline protease from Bacillus
species SSR1, which was collected from sugar factory, milk plant
and clay soil etc., the above enzyme can be used in laundry industry.

• Berla Thangam and Suseela Rajkumar (2002), extracted

extracellular protease from alkalophilic bacterium Alkaligens
faecalis, purified it by combination of ion exchange and size-
exclusion chromatographic methods and their properties were also
examined.

• Brady et al., (2002) isolated the extracellular protease produced by

Proteus vulgaris and partially purified it. The maximal proteolytic
activity was at 8.0 to 9.0 pH unit range and it had a molecular mass
of 44000 daltons.

2.3. LITERATURE REGARDING ENZYMATIC DEHAIRING

• The application of the protease in the leather industry for dehairing

and bating of hides in order to substitute currently used toxic
chemicals is a relatively new development and has conferred added
biotechnological importance (Rao et al, 1998.).

• Rohm, (1910) revealed that the prepared and standardized

pancreatic glands and a declaiming and added directly to the skins
at the time of bating. Unhairing enzymes are obtained from animal
sources, plant sources or microbial sources. The uses of pancreatic

24
 enzymes for depilation the treating of skins with caustic alkali for
swelling.

• Marriott, et al., (1921) reported that enzymatic unhairing may be

made possible in two ways, acidic pH and alkaline pH. Acetic acid
treatment causes unhairing of skin.

• Kaverzneva and Oleinikova, (1934) explained that the protease

are obtained from extracts of sprouting soya beans (plant sources).
Hide powder is energetically dissolved by soya bean extracts in and
alkaline medium. The proteases easily dissolve albumen of the
hides.

• Das et al., (1953) studied those proteolytic enzymes of the latex of

madar plants (plant sources) (Calotropis gigantia) to be rich sources
of proteases and they are used in the process of unhairing.

• Madhavakrishna, et al., (1953) pointed that the proteolytic

enzymes may be obtained from various sources such as animal,
plants, commercial enzyme preparation may be constructed by
enzymes from single sources or combinations of enzymes from
more than one sources. In the process of enzymatic
dehairing/dewooling, washed and soaked skins are either painted on
the flesh aide with in a period of 24 to 48 hours.

• Rohm, and Haas, (1956) explained that the soak liquor contains
proteolytic enzyme in the presence of ammonium salts and reducing

25
 agents (eg.NaHSo3) at pH<7, to which bacterial carbohydrates have
been added. A good soaking effect is thereby obtained without
damage to the skin (or) hair. Ionic (or) non-ionic surface active
agents, as well as disinfectants may be added.

• Bose, et al., (1956) extracted the enzyme from germinated ragi

(plant sources) and used an unhairing agent.

• Madhavakrishna, et al., (1958) compared the chemical, physical

and microscopical assessments of quality of the leathers produced
by the traditional liming process and by the two enzymatic
unhairing process such as protease and amylase.

• Zhang and Jiandong, (1960) carried out enzymatic hide depilation

without sulfur pollution. The process does not use sodium sulfide,
involves lime water-soaked hide, deliming, softening, and tumbling
with basic protease to remove hair.

• Toyoda, (1960) pointed out that there was no significant change in

chrome fixation by enzymatic unhairing but formaldehyde fixation
was found decline by protease.

• Forminosano and simoncini (1964) proposed that Bacillus had an

effective unhairing power.

26
• Pfleider (1968) extracted protease from Aspergillus oryzae and
better result was observed at pH values below 5 in unhairing and
soaking process.

• Sivaparvathi and Dhar (1974) reported the proteolytic action

autolytic enzymatic unhairing action on the skin.

• Rejean Beaudet et al., (1974) reported the structural and

biochemical properties of the extra cellular protease purified from
four different strains of Staphylococcus aureus.

• Fekete et al., (1982) improved hide unhairing and liming method

resulting in reduced decomposition of products and sulfide ion
pollution by treating it with proteolytic enzyme solution.

• Cranston et al., (1986) used conventional reagent in a novel way,

the sirolime process, which allows rapid removal of hair from cattle
hide in an essentially figrous from with consequent major
environmental benefits compared with convention hair destroying
systems.

• Taylor et al., (1987) conducted research works on the uses of

enzyme in the tannery and studied the unhairing process of the
enzymes.

• Wei et al., (1991) compared china 537 proteinase with fercon M301
proteinase and vinkol A proteinase activity on shaved wet blue goat

27
 skin. They found that the skin treated with 527 acid proteinase had
good thickness, air permeability, shrink temperature and porosity.

• Wolf (1991) performed successful unhairing of sheepskins and

cattle hides with a neutral amylase containing metallic proteinase
derived out of Strptomyces hygroscopicus to reduce the sulfide
loads in the waste water of conventional unhairing process.

• Thangam et al., (2001) extracted alkaline protease from Alkaligens

faecalis for enzymatic unhairing in tannery industries.

• Paul et al., (2001) investigated the use of neutral protease enzyme,

lipase that possesses substrate specificity. The enzyme caused
loosening of the hair and associated hair loss, without damaging the
fibrous of the dermis.

28
 3. MATERIALS AND METHODS

MATERIALS

Fish samples were obtained from the stores of CLRI .The samples
were homogenized and were allowed to pass through Whatman No.40 filter
paper on a buchner flask. About 10gm of various samples were mixed in 90
ml of saline. From the various dilutions about 1ml of the samples were
transferred to the sterile plates and standard caseinate agar was poured and
kept for incubation for 48 hrs and thus zone of hydrolysis is formed.

Subculturing
The organism which cleared the zone was subcultured in the
nutrient broth and about 1ml of samples were transferred to the sterile Petri
plates and standard caseinate agar was poured and incubated at 37 c for 96
hrs. Isolates that showed a zone of hydrolysis were selected for further
examinations .This shows the presence of proteolytic activity.

MEDIA COMPOSITION
NUTRIENT BROTH Composition (g/l)
Peptone 5gm
Sodium chloride 5gm
Yeast extracts 1.5gm
Beef extract 1.5gm
Distilled water 1000ml
pH 7.5

29
3.1 IDENTIFICATION OF CULTURES

MICROSCOPIC EXAMINATION

Gram staining for the Bacteria

A drop of sterilized distilled water was taken on the middle

of the clear slide. Then a loopful of bacterial suspension
(young culture) was transferred to the sterilized drop of water
and a very thin film was prepared on the slide by spreading
uniformly. The film was fixed by passing it over the gentle
flame for two or three times. The slide was flooded with
crystal violet solution and allowed to stand for 30 sec and
then washed thoroughly with gentle stream of tap water. The
slide was then immersed in iodine solution for 1 minute and
washed thoroughly with 95% alcohol for 10 sec. Alcohol was
drained off and washed thoroughly with gentle stream of tap
water. The slide was then covered with safranin for 1 minute.
After washing with tap water and blotted dry it and examined
under microscope.

Spore staining

One drop of sterile saline water was taken on a clean

glass slide for spore staining. A loopful bacterial old slant
culture was taken in the drop and smear was made on the
slide. The film was dried over flame gentle heating. The slide

30
was then placed over a beaker and 5% malachite green was
added drop wise on the slide. Boiling of the malachite green
was avoided by adding more malachite green. The slide was
taken out of the stream and washed gently with tap water.
The preparation was needed with safranin solution for 1 min.
and washed with gentle stream of tap water, and placed
under immersion lens with immersion oil.

BIOCHEMICAL TESTS

Carbohydrate fermentation Test

Nutrient broth is used as basal medium for fermentative test. Bromo
cresol was used as an indicator. Fermentation tubes with 1.0 ml of basal
medium provided with indicator were made and pH of the medium was
adjusted at 7.5 with NaOH the medium was sterilized at 121ºC for 15 minutes
1.0 ml of filter sterilized fructose, glucose, arabinose, lactose, mannitol,
xylose and sucrose was taken in each tube. The tubes were then inoculated
with fresh culture of bacterial isolates and allow to incubate at 37 ºc for 24
hrs. The change of color of indicator to yellow indicates the production of
acid.

Catalase Test

Catalase test carried out of one drop of 30% hydrogen

peroxide was placed on a slide. One loopful of the fresh
bacterial culture was taken by a sterile needle and placed on
the drop of hydrogen peroxide. Bubble production indicated
positive result.

31
Hydrolysis of Starch

Hydrolysis of Starch was carried out with 10 gm soluble

starch in 100 ml distilled water which was heated in water
bath until dissolved. 20 ml of this solution was mixed with 100
ml of melted nutrient agar and poured in the Petridish after
sterilization. A loopful of fresh bacterial culture was picked up
by the sterile needle and stabbed on to the agar plate; After
24 hrs of incubation at 37° C, the plate was flooded with dilute
iodine solution. Hydrolysis of starch was indicated by a clear
zone around the growth and unchanged starch gave a blue
color.

Urease test
Prepare the urea broth medium then inoculate the test
organism into the urea broth. Incubate at 30-37º C for 18-24
hours.

Citrate Utilization Test

For the Citrate Utilization Test, slope culture with a 1 inch

butt of Simmon's citrate agar was inoculated by streaking
over surface with a wire needle and incubated at 37° C for up
to 3 days. The color of the medium changed from green to
bright blue due to the utilization citrate and when citrate is
not utilized, the color of the medium remain unchanged.

32
Methyl Red Test

Methyl Red (MR) Test detects acid production to a sufficient degree

(below 4.5) from glucose. One ml of fresh bacterial culture grown in glucose
phosphate medium was taken in a test tube. Five drops of methyl red reagent
was added and read immediately. Positive tests are light red and negative
yellow.

Indole Production Test

For the Indole, one loopful fresh bacterial culture (24 hrs old) was
inoculated in peptone broth and incubated at 37° C for 1-3 days, after
incubation, Kovac's solution was added and shaken vigorously for one minute.
A red colour in the reagent layer indicated positive reaction.

Nitrate Reduction Test

Nitrate reduction test was carried out in nitrate broth. The freshly prepared
cultures were inoculated in sterile nitrate broth containing tubes and incubated
at 37° C for 24 hrs. At the end of incubation 0.1 ml of solution A was added
followed by solution B in equal volume. The appearance of pink deep color
showed that bacterial isolates reduced nitrate to nitrite.

Voges Proskauer Test

33
Voges Proskauer (V.P.) Test carried out of one ml of fresh bacterial culture
was grown in phosphate peptone medium. After addition of 0.2 ml of 40%
KOH, 0.6 ml of 5% alpha napthol in absolute ethanol was added. After 10-15
minutes with vigorous shaking bright orange red color developed if acetyl
methyl carbinol was present.

METHOD

3.2 PRODUCTION OF ENZYME

250ml of nutrient broth was prepared and sterilized in autoclave
at 1 atm for 15 minutes. The culture was inoculated in nutrient broth and it
was kept in shakers for 48 hrs. Then the medium was centrifuged at 15000
rpm for 10 minutes and the supernatant was taken for the experiment. The
supernatant containing the crude enzyme was assayed for its activity and
estimated by Lowry’s method using Bovine serum Albumin as standard
(Lowry et al., 1951).

ESTIMATION OF PROTEIN

Principle
The protein content of the enzyme sample was estimated by the
Lowry’s method (Lowry et al., 1951). Protein reacts with the folin-ciocalteu
reagent (FCR) to give blue coloured complex. The colour so formed was due
to the reaction of the alkaline copper with the protein as in the biuret test and
the reduction of the phosphomolybdic-phosphotungstic components in the

34
FCR by the amino acids, tyrosine and tryptophan present in the protein. The
intensity of the blue colour is measured at 660nm in a spectrophotometer.

Reagents

Reagent- A - 2%(w/v) sodium carbonate in 0.1N sodium hydroxide.

Reagent- B - 0.5% (w/v) copper sulphate in 1% (w/v) sodium potassium

tartarate.

Reagent- C - freshly prepared solution containing 50ml of reagent A and 1ml

of reagent B.

Folin’s phenol reagent- commercially available folin’s phenol reagent was

diluted (1:2) with distilled water just before use.

Standard Bovine Serum Albumin (BSA)

100ml of BSA was made upto 100ml of dist water (µg/ml).

Procedure
Working standard solution of volume 0.2, 0.4, 0.6, 0.8 and 1ml was
pipetted into series of test tubes and the volume was made upto 1ml with
water in all the test tubes. A tube with 1ml of water serves as blank and 1ml of
the sample was taken as test. 5ml of reagent C was added and shaked
vigorously in a cyclomixer. The reaction mixture was incubated for 10
minutes. After 10 minutes 0.5 ml of folin’s phenol reagent was added and kept

35
in dark for 30 minutes. The absorbency of the solution (developed blue
colour) was measured at 660 nm.

ENZYME ASSAY
For protease assay, the method adopted by kunitz (1947) was modified
and used. The culture filtrate serves as the source of enzyme.

Principle
The enzyme protease reacts with the casein and liberates tyrosine.
The liberated tyrosine in alkaline conditions causes the reduction of phospho
molybdate and phospho tungstate in folinciocalteau reagent to give blue
colour, the colour developed is measured at 620nm. The absorbance serves as
the parameter of the estimation of tyrosine produced.

Reagents
2%- casein: 2gm in 100ml distilled water.
Citrate phosphate buffer (pH 7.0)
Solution A: 0.1M solution of citric acid (19.21gm in 1000ml).
Solution B: 0.2 M solution of dibasic sodium phosphate (53.65gm of
Na2HPO4.7H2O or 71.7 gm of Na2HPO4.12H2O in 1000 ml).
6.5 ml of solution A+43.6 ml of solution B mixed and diluted to a total of 100
ml.
10% Tri chloro acetic acid (TCA) –10 gm of TCA in 100 ml of distilled
water.
5N Sodium hydroxide– 2gm of sodium hydroxide in 100ml of distilled water.

36
Folin ciocalteau reagent
Commercially available folin‘s phenol reagent was diluted 1:2 with distilled
water just before use.
Stock Tyrosine
50mg of tyrosine was dissolved in 1N Hydrochloric acid and then made upto
100ml using distilled water in a standard flask.

Working standard
10ml of stock tyrosine was taken and made upto 100ml using distilled water
in standard flask.

Procedure

Casein (Qualigens Fine Chemicals) of concentration 2% of volume

0.5ml was taken in 2 test tubes labeled test and control (T1and C1). To this
1ml of citrate phosphate buffer was added. The tubes are incubated at 37°c for
5 minutes. Then 2ml of enzyme, whose activity was estimated, was added to
the tube labeled test. Again the tubes are incubated at 37°c for 30 minutes.
After incubation, 2ml of 10% TCA (Hi-pure) was added to both the control
and the test tubes. Then 2ml of enzyme was added to the control tube. The
tubes are centrifuged and the supernatant was taken for the assay.

Standard tyrosine of volume 0.05ml, 0.1ml, 0.15ml, 0.2ml and

0.25ml was taken in 5 test tubes labeled S1 to S5. Then 0.5ml of
supernatant was taken in 2 tubes, labeled U1 and U2. The volumes in the
tubes were made upto 2.4ml with distilled water. Sodium hydroxide of

37
concentration 0.5N of volume 2.0ml was added to all the tubes followed by
the addition of 0.6ml of folin ciocalteau reagent (Qualigens Fine Chemicals).
The tubes were incubated at room temperature for 10 to 20 minutes.
Absorbance was measured at 620 nm in spectrophotometer. Taking
concentration along the x-axis and optical density along the y-axis drew a
standard graph.

Determination of the Effect of pH on Protease Activity

Casein was dissolved in Tris HCL buffer solution and the enzyme assay was
carried out within pH range (7.0 to 9.0)

Determination of the Effect of Temperature on Protease Activity

For the determination of the effect of temperature, the reaction medium was
incubated at varied temperature and the protease activity was determined. For
this purpose the enzyme preparation was added to a mixture of 2% casein
solution, 1 ml of citrate phosphate buffer and incubated at
30º,36º,42°,48°,54°C temperatures and it is carried out by enzyme assay.
.
3.3PURIFICATION

AMMONIUM SULPHATE PRECIPITATION:

The supernatant containing the crude enzyme was purified by
precipitation with ammonium sulphate. In practice ammonium sulphate was
used because it was high soluble in water and has no deterious effect. This
process was carried out 0-10ºC to minimize denaturation. The addition of the

38
salt removes the layer of water molecule that surrounds the hydrophobic
groups of the protein surface that allows the protein to aggregate and hence
precipitation occurs.
The supernatant was fractionated by adding 30% ammonium sulphate
and incubated overnight at 4ºC ,the precipitate was removed by centrifugation
at 12000 rpm for 20 minutes at 4ºC mixed buffer and dialyzed against distilled
water.
DIALYSIS

Dialysis is a very small technique used extensively to separate

macromolecules from smaller molecules. Here the solution containing sample
and phosphate buffer was taken in a dialysis bag which allows only small
molecules and ions to pass through but larger molecules like proteins are held
back. The method was commonly used for removing salts from proteins.
The dialysis bag was boiled for 10 minutes in a beaker of
water containing sodium sulphate (2%) and EDTA (1M).
Then the bag was taken out and rinsed in distilled water. The bag
was boiled in a beaker of water containing 1mM EDTA. The bag was cooled.
One end of the bag was tied and checked for leakage. The dialysis bag was
diluted with sample and then tied at other end. The bag was then suspended in
a beaker with 500 ml distilled water and kept overnight at 4ºC.
The water was changed the next day and bag was suspended for 3 more
hours later the bag was removed and the sample was transferred to
lyophilization flask.

39
 Figure 3.3

3.4. LYOPHILIZATION

Cells were harvested while still in the exponential phase in a vessel

cooled by ice water under vigorous stirring. Nevertheless, in some cases a thin
layer of brown debris could be seen at the top of the sediment after
centrifugation (15 minutes at 4500rpm) and if present, was removed carefully.
Washing was performed 3 times with ice cold water. The cell paste obtained
was resuspended in some water, poured into petri dishes as thin layers, frozen
overnight at -20ºC and lyophilized for 24 hours in a Minilyo II apparatus.
The cells were then pestled and the powder was filled into penicillium flasks,
again lyophilized for 6 hours and then sealed under vacuum. Tablets of
samples took up considerable amounts of moisture when taken out of the
desiccators and allowed to equilibrate with the surroundings for 15 to 30 min.
To remove the residual water present, samples of cells ranging from 0.1 to 0.7
gm were weighed before and after drying at 105ºC for 24 hours. To

40
determine the ash content, these cells were burnt to constant weight in a
Bunsen flame.

3.5. DETERMINATION OF MOLECULAR WEIGHT OF THE

ENZYME

SDS-PAGE
SDS-PAGE was done according to the method proposed by
Laemmli (1970). The electrophoresis equipment consists of two parts
basically: 1) Power pack and 2) Electrophoresis unit.

Many proteins are oligomeric proteins containing two or more

subunits. By a modification of PAGE called SDS-PAGE, an oligomeric
protein may be dissociated into its subunits and the molecular weight of the
subunit can be determined.
SDS-PAGE of proteins was carried out in the presence of sodium
dodecyl sulphate – an anionic detergent that readily binds and dissociates
oligomeric proteins in the presence of reducing agent, 2-mercapto ethanol into
their subunits. The detergent binds to hydrophobic regions of the denature
protein chain in a constant ratio of about 1.4gm of SDS/gm of protein. The
bound detergent molecules carrying negative charges mask the negative
charge of the protein. In essence polypeptide chains of a constant charge/mass
ratio and uniform shape are produced. The protein SDS complex carries
negative charges, hence move towards the anode and the separation is based
on the size of the protein. There by the molecular weight of the desired protein
can be determined.

41
MATERIALS REQUIRED
i) Acrylamide 30%
ii) Ammonium per sulfate 10%
iii) Sodium dodecyl sulphate 10%
iv) Separating gel buffer
v) Stacking gel buffer
VI) Sample buffer
Vii) Staining solution
Viii) Destaining solution
ix) Storage solution
x) Running buffer

xi) Spacers
xii) Clips
xiii) Plates
xiv) Electrophoresis unit

MARKER USED
Medium range markers

REAGENTS

1. Acrylamide 30% [W/V]

30gms of acrylamide and 0.8gms of methyl bisacrylamide was
weighed and added to 5ml of deionised water .They were dissolved well and
the solution was made up to 50ml using deionised water. The solution was

42
then filtered through Whatmann no.1filterpaper and stored in brown bottles in
a refrigerator.

2. Ammonium per sulfate (APS) 10% [2/V]

APS was freshly prepared for every use 0.1 g of APS was dissolved
in 1ml of deionised water and stored at 4°C.

3. Sodium dodecyl sulphate (SDS) 10% [W/V]

1g of SDS was weighed and dissolved in 10ml of deionised water
and stored at 4°C.
4. Separating gel buffer 1.5M Tris Hcl (pH 8.8)
36.34g of 1.5M Tris was added to100ml of deionised water. The pH
of the solution was adjusted to 8.8 using concentrated Hcl and 8ml of
10%SDS was added. The solution was made upto 200ml using deionised
water. Then it was filtered through Whatmann No.1 filter paper and stored at
room temperature.

5. Stacking gel buffer 1M Tris Hcl (pH6.8)

12.1g of Tris base was added to 100ml of deionised water .The pH of
the solution was adjusted to 6.8 using concentrated Hcl and 8ml of 10%SDS
was added to this solution .Then the solution was made upto 200ml with
deionised water and it was filtered through Whatmann filter paper No.1 and
stored at room temperature.

6. Sample Buffer
Stacking buffer - 1.25ml

43
 Glycerol - 1.0ml
β-mercaptoethanol - 0.5ml
SDS - 150mg
Deionised water - 7.25ml
Bromophenol blue - 2%W/V

7. Staining solution
50% Ethanol
7% Acetic acid
2% Coomassie brilliant blue
The above ingredients were made upto 100ml using distilled water.

8. Destaining Solution
50%Ethanol
7%Acetic acid in deionised water
The above ingredients were made upto 100ml using distilled water.

9. Storage Solution
7% Acetic acid in deionised water.

10. Running Buffer

1.5g of Tris buffer and 7.2g of glycine was added to 100ml of deionised water
and to it 0.5g of SDS was added and dissolved well. The solution was made
up to 500ml using deionised water.

44
PREPARATION OF SEPARATING GEL
A beaker was taken and rinsed thoroughly with deionised water.
The following ingredients was added to the beaker
Deionised water - 5.9ml
30%Acrylamide - 5.0ml
8.8% Buffer - 3.8ml
10%SDS - 0.15ml
10%APS - 0.15ml
TEMED - 6.0µl (TEMED-Tetra ethyl methylene diamine)

PREPARATION OF STACKING GEL

Acrylamide - 0.83ml
6.8 Buffer - 0.68ml
10% APS - 0.05ml
TEMED - 0.005ml
Distilled water - 3.40ml

PREPARATION OF SAMPLE

18µl of deionised water, 12µl of sample and 10µl of the dye were
added in an empty micro centrifuge tube. The tube was placed in boiling water
bath for 2minutes with the cap open.

PREPARATION OF SDS PAGE

ASSEMBLING THE PLATES

45
 The plates were thoroughly cleaned and dried and were assembled by
inserting spacers of uniform length. The plate were then sealed using a
cellophane tape. Then the plates were clamped together with metal clips and
pressure was directly applied on the spacer.

CASTING THE SEPARATING GEL

The separating gel mixture was prepared in a small, thick walled

flask by mixing the components. The mixture was degreased for a minute, the
correct volume of the TEMED was then added and gently mixed .Then the
separating gel mixture was poured into the space between the glass plates
leaving sufficient space at the top for stacking gel to be polymerized later. The
stacking gel needs to be at least twice the height of the sample. Thus a space
of about 3.5cm needs to be left above the separating gel. Water saturated
butanol was gently layered on to the gel surface for two reasons. First, it helps
to make a straight line and second it prevent oxidation.

CASTING THE STACKING GEL

After polymerization (30-60 minutes) of separating gel, a small volume of

water was overlaid on the gel. The water was blotted and the stacking gel
solution was poured over the polymerized separating gel. The comb was
inserted immediately into the stacking gel mixture taking care to avoid
trapping of any air bubbles beneath it. The assembly was left undisturbed
during which the stacking gel polymerizes for 30 –45minutes.

ATTACHING THE GEL CASSETTE TO THE APPARATUS

46
 The comb and the spacers from the slides were removed carefully.
The slab gel was attached to the apparatus prior to sample loading. The
running buffer was first added to the upper chamber. The running buffer was
then added to the lower reservoir and bubbles that had been trapped between
the plates were removed. The sample wells were washed with a stream of
running buffer.

LOADING THE SAMPLE ON TO THE WELLS

The sample in the micro centrifuge tube was taken and loaded carefully inside
the well. The procedure was reported in a similar manner for rest of the
samples.

RUNNING THE GEL

The apparatus was then connected to the power source so that the anode (+)
was attached to the bottom reservoir. A current of 80volts was maintained
when the tracking dye moves through the stacking gel. When the tracking dye
reaches the separating gel, the current was switched off. The glass plates and
the gel were removed carefully.

STAINING THE GEL

The gel was carefully placed for staining in a petridish. This was
allowed to remain for 2 minutes.

DESTAINING THE GEL

The gel was then carefully transferred to the destaining solution and allowed
to remain for 1 hour. The protein bands appears to be visibly distinct.

STORAGE OF THE GEL

47
 The gel was then carefully removed and transferred to the storage
solution for long term storage.

Figure 3.5

3.6 APPLICATION

Removal of blood stain

A clean piece of cloth was soaked in blood and allowed to dry the
cloth. Then the cloth was soaked in 2% formaldehyde for 30 minutes and
washed with water to remove the excess formaldehyde. Then the cloth was cut
into equal pieces and they were incubated with the lyophilized protease at
30°C for different incubation period 5 minutes-40 minutes. After incubation
time, each piece was rinsed with water for 2 min and then dried. The same
procedure was done for control expect incubation with the enzyme.

48
Dehairing of skin
Goat’s skin was cut to 5 cm² pieces and incubated with the lyophilized
protease at 42ºC. The skin was checked for removal of hair at different
incubation time ranging from 1 hour- 8 hours.

4. RESULTS AND DISCUSSION

The proteolytic bacteria isolated from fish waste was identified as

Bacillus species, based on various morphological, staining and biochemical
characteristics.
The protease enzyme that was produced by Bacillus species was
assayed. The quantification, enzyme assay, characterization (viz., pH and
temperature) ,purification, determination of molecular mass, antimicrobial
activity ,antibiotic sensitivity test and application studies on protease enzyme
was carried out.
In the present study, the protease enzyme obtained from Bacillus
species was produced, purified and characterized. The crude enzyme extracts
were ammonium sulphate saturated. Then the purified enzymes were used
for dehairing on goat skin.
BIOCHEMICAL TEST

49
 Table 4.1 Biochemical test
TEST RESULT
Indole test Positive
Nitrate reduction test Positive
Methyl red test Positive
Urease test Negative
Voges proskauer test Positive
Starch hydrolysis test Positive
Citrate utilization test Positive
Catalase test Positive

CARBOHYDRATE FERMENTATION TEST

Glucose Positive
Fructose Positive
Arabinose Positive
Lactose Negative
Mannitol Positive
Sucrose Negative
Xylose Positive

50
51
Figure 4.1

52
4.1. PRODUCTION OF ENZYME

Bacillus subtilis grown in enzyme production medium for 2 days for

the production of protease enzyme. The test organism grew well in the
medium by producing the enzymes. The protein level of the crude enzyme
were estimated by Lowry’s method, the results are presented in Table 5.1 and
they found to be 700 µg/ml (plot 5.1) is the standard plot for Lowry’s method.
The crude enzyme was tested for protease level. The results of protease assay
are presented in Table 5.2 and they found to be 14.2 µg of tyrosine/ml (plot
5.2) is the standard plot for protease assay.

ESTIMATION OF PROTEASE BY LOWRY’S METHOD

Table 4.1.1
CONCENTRATION OPTICAL DENSITY
S.NO OF BSA (µg/ml) X-axis AT 660 (nm) Y-axis
1 200 0.125
2 400 0.24
3 600 0.353
4 800 0.47
5 1000 0.602
CRUDE ENZYME 0.396

53
 O.D.
values

Conc. Of BSA(µg/ml)

54
 PROTEASE ASSAY
Figure 4.1.2

ENZYME ASSAY BY KUNITZ METHOD

Table 4.1.2
S.NO CONCENTRATION OPTICAL
OF TYROSINE (µ DENSITY AT 620
g/ml) X-axis (nm) Y-axis
1 4 0.2
2 8 0.40
3 12 0.612
4 16 0.82
5 20 1.02
CRUDE 0.634
ENZYME

1.2

0.8

0.6 Line 1

O.D. values 0.4

0.2

0
0 4 8 12 16 20
Conc. Of tyrosine (µg/ml)
Figure 4.1.3

55
EFFECT OF TEMPERATURE
The temperature at which culture show maximum enzyme activity was
determined. The culture exhibited maximum enzyme activity at 37ºC.The
results were tabulated in Table 4.1.3. The graph was plotted and shown in plot
4.1.4.

Table 4.1.3
S.NO TEMPERATURE ºC OD at 620 nm
X-axis Y-axis
1 30 0.13
2 36 0.14
3 42 0.06
4 48 0.07
5 54 0.09

O.D. values

Temperature(°C)

Figure 4.1.4

EFFECT OF pH

56
 The pH level was changed to the medium to find the optimum range
at which there is a high enzyme activity. The culture exhibited maximum
enzyme activity at pH 8.2.The results were tabulated in table 4.1.4. The graph
was plotted and shown in plot 4.1.5.

Table 4.1.4

S.NO pH OD at 620 nm
X-axis Y-axis
1 7 0.02
2 7.5 0.04
3 8 0.06
4 8.5 0.04
5 9 0.03

O.D. values

pH

Figure 4.1.5

REMOVAL OF BLOOD STAIN

Alkaline protease showed high capability for removing proteins and
stains from cloth so it is used in detergent powder or solutions. The stained
cloth was destained by applying protease was observed.

57
DEHAIRING OF SKIN
Dehairing of goatskin was observed after 8 hours of incubation with
lyophilized protease.

Figure 4.1.6

DISCUSSION

The presence of both pathogenic as well as spoilage bacteria

often associated with fish/fish products indicates their presence in the fish
rather than as contaminants. The total number as well as species wise
distribution of various bacteria may vary from fish to fish depending on the
intrinsic or extrinsic factors. The intrinsic factors are those that are inherent
with the sample such as type of fish species, age, geographical location etc,
hence they cannot be controlled. Generally bacteria are abundant in the
environment in which fish live and it is impossible to avoid them being a
component of their diet. The bacteria ingested by the fish along with their diet
may adopt themselves to the environment of the gastrointestinal tract and
form a symbiotic association (Strom et al., 1990; Hansen et al., 1992). Fresh

58
water fishes have higher percentage of Gram positives such as Lactobacillus
sp, Sarcina sp, Corynebacterium sp, Bacillus sp and Lactococcus sp which
together comprised 50% of the total bacterial count. Among these almost all
are often associated with fish/fish product spoilage except Lactobacillus and
Lactococcus. Gram negatives bacteria such as Pseudomonas sp , Alcaligenes
sp , Aceintobacter sp and Aeromonas sp that cause fish spoilage were also
present, with the latter being a fish pathogen often seen fresh water fish
culture systems.

Many microorganisms such as Bacillus sp, fungi, Yeasts,

Actinomycetes been reported to produce extracellular alkaline
proteases(Pedersen et al., 1992).Some of the Gram-negative bacteria
producing alkaline proteases were identified as Pseudomonas
sp(Morihara,1963) and Vibrio metschnikovii strain RH530 (Kwon et al.,
1994) .

Several species of Bacillus sp have been reported to be predominant

proteolytic and are commercially used for their applications (Rebecca et al.,
1991) . It has been reported that a related species, Bacillus licheniformis,
produces very narrow zones of hydrolysis on casein agar despite being very
good protease in submerged cultures (Mao et al., 1992). Our results from the
present study are in coinciding with to this reported result. Usually alkaline
proteases and/or subtilisins are found to be more active against casein than
against hemoglobin or bovine serum albumin, since Bacillus sp protease is
also alkaline it was found to be active against only casein.
Alkaline proteases are generally produced by submerged
fermentation. In addition, solid state fermentation processes have been

59
exploited to a lesser extent for production of these enzymes (Chakraborty et
al., 1993., Malthi et al., 1991 and George et al., 1995). In our studies also
Bacillus sp produced protease by submerged fermentation, which is
coinciding with the reported results. The optimum incubation temperature for
cell growth and protease production was at 37°C as shown in the Figure 3.The
production of extracellular proteases during the stationary phase of growth is
characteristic of many bacterial species(Priest, 1997). At early stationary
phase, two or more proteases are secreted and the ratio of the amount of the
individual proteases produced also varied with the Bacillus strains (Priest,
1997 and Uehara et al, 1974). In other cases, the synthesis and secretion of the
protease was initiated during the exponential growth phase, with a substantial
increase near the end of the growth phase (Durham et al, 1987., Moon et
al,1991., Tsai et al, 1988., Takii et al, 1990., Manachini et al, 1988 and Ferrero
et al, 1996) and with maximum amounts of protease produced in the
stationary growth phase.

Ammonium sulphate found wide utility only in acidic and neutral pH

values and developed ammonia under alkaline conditions (Aunstrup, 1980).
Also, to prevent contamination of the final crude preparation, addition of
sodium chloride to the precipitate before dialysis has been suggested
(Aunstrup, 1980 and Shetty et al, 1993).

Bacillus sp are found as the most proteolytic among the isolates, in

the current study, is often being used commercially in bioremediation mixes in
aquaculture farms and hence stands good for exploitation for that purpose. In
commercial aquaculture, beneficial bacteria could be introduced by

60
incorporating them into compound fish diets the enzyme-producing
microorganisms isolated in the present study can be beneficially used as a
probiotic while formulating the diet for fish, especially in the larval stages
when the enzyme system is not efficient. (Sangbrita et al, 2006)

The foregone discussion concludes that the proteolytic bacteria

discussed so far, are a part of the natural flora of both marine and fresh water
fishes and their environment and these alkaline proteases are of considerable
interest in view of their activity and stability at alkaline pH. This describes the
proteases can resist extreme alkaline environments produced by a wide range
of alkalophilic microorganisms. Protease are well known biocatalysts that
perform a multitude of chemical reactions and are commercially exploited in
the detergent, food, pharmaceutical, diagnostics, and fine chemical industries.
Further, strain improvement using mutagenesis and/or recombinant DNA
technology can be applied to augment the efficiency of the producer strain to a
commercial status. The various nutritional and environmental parameters
affecting the production of alkaline proteases are delineated.

5. CONCLUSION
Proteolytic organisms associated with fish processing waste were
evaluated, characterized and identified. Organisms showed proteolytic activity
at various culture conditions such as change in incubation temperature and
pH. Optimum conditions for the proteolytic activity were found to be 37°C

61
and pH 8.2 and incubation period of 48hrs. Based on the activity of the
protease produced by the isolated Bacillus sp, it can be concluded that this
strain has the potential for producing an alkaline protease and hence has to be
further characterized to aid in recovery and scale up. They are used in the
laundry industry, where they help in removing protein based stains from
clothing . For an enzyme to be used as an detergent additive it should be stable
and active in the presence of typical detergent ingredients, such as surfactants,
builders, bleaching agents, bleach activators, fillers, fabric softeners and
various other formulation aids. Proteases are used in the dehairing process.
Recovery of hair of good quality and strength with a good saleable value.
Creation of an ecologically conducive atmosphere for the workers.
Enzymatically dehaired leathers have shown better strength properties and
greater surface area .Simplification of pre-tanning processes by cutting down
one step, viz. bating. A significant nature of the enzymatic dehairing process is
the time factor involved. The lime-sulphide process takes about 16 h, whereas
the enzymatic dehairing would be also completed within 12 hours.
Hence, any study on proteolytic microbes associated with
fish or fish by-products becomes important both from the point of view of
production and processing. Further, microbial proteases are an important
group of enzymes that can have application in various industries such as
leather processing, food processing, pharmaceutical, bioremediation process
and in textile industry to remove protein based stains.

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