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

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.

Peptidases

Peptidases hydrolyze peptide bonds within the protein chain,

previously called endopeptidase while proteases hydrolyse large polypeptides

into smaller molecules. (Adinarayana et al., 2004).

Protease families

Serine Threonine Cysteine AsparticMetallic groups

PROTEASES OR PEPTIDASES

ENDOPEPTIDASES EXOPEPTIDASES

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

6

protein catabolism, blood coagulation, cell growth and migration, tissue

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

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Proteolytic enzymes are involved in a great variety of physiological processes

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

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

8

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

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,

9

Streptomyces, Sermons and Escherichia genera.  Fungi produce several serine

proteinases. Cysteine proteinases are not so widely distributed as seen with

serine and aspartic proteinases.

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

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and agricultural policies (Hoffman 1974). Rennin (mainly chymosin) obtained

from the stomach (abomasums) of unweaned calves has been used in the

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,

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produced by Bacillus licheniformis; its commercial name was BIOTEX.

Maxatase, a detergent made by Gist-Brocades, followed this. The ideal

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.

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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

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time. Papain from the Kaves and unripe fruit of carica papaya has been used

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

16

substrates with base of fibrin, albumin and collagen suggesting usefulness for

different applications.

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

17

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

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.

19

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

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

20

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

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).

21

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).

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

22

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.

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))

23

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.

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.).

24

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

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

25

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

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.

26

Forminosano and simoncini (1964) proposed that Bacillus had an

effective unhairing power.

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.

27

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 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

29

Sodium chloride 5gm

Yeast extracts 1.5gm

Beef extract 1.5gm

Distilled water 1000ml

pH 7.5

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.

30

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

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.

31

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.

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

32

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.

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

33

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

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).

34

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

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 (g/ml).

35

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

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)

36

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.

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 37c for

5 minutes. Then 2ml of enzyme, whose activity was estimated, was added to

the tube labeled test. Again the tubes are incubated at 37c for 30 minutes.

37

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

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

38

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

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.

39

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.

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,

40

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

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

41

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.

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

42

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

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 4C.

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

1g of SDS was weighed and dissolved in 10ml of deionised water

and stored at 4C.

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

43

deionised water and it was filtered through Whatmann filter paper No.1 and

stored at room temperature.

6. Sample Buffer

Stacking buffer - 1.25ml

Glycerol - 1.0ml

-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

44

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.

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 - 6.0l (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

18l of deionised water, 12l of sample and 10l 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.

45

PREPARATION OF SDS PAGE

ASSEMBLING THE PLATES

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

46

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

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.

47

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

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

48

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.

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

49

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

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 g/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 g of tyrosine/ml (plot

5.2) is the standard plot for protease assay.

ESTIMATION OF PROTEASE BY LOWRY’S METHOD

TABLE 4.1.1

S.NO

CONCENTRATION

OF BSA (g/ml) X-axis

OPTICAL DENSITY

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

Conc. Of BSA(µg/ml)

O.D. values

PROTEASE ASSAY

Figure 4.1.2

54

ENZYME ASSAY BY KUNITZ METHOD

TABLE 4.1.2

S.NO CONCENTRATION

OF TYROSINE

(g/ml) X-axis

OPTICAL

DENSITY AT 620

(nm) Y-axis

1 4 0.2

2 8 0.40

3 12 0.612

4 16 0.82

5 20 1.02

CRUDE

ENZYME

0.634

0

0.2

0.4

0.6

0.8

1

1.2

0 4 8 12 16 20

Conc. Of tyrosine (µg/ml)

O.D. values

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

X-axis

OD at 620 nm

Y-axis

1 30 0.13

2 36 0.14

3 42 0.06

4 48 0.07

5 54 0.09

Temperature(°C)

O.D. values

Figure 4.1.4

56

EFFECT OF pH

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

X-axis

OD at 620 nm

Y-axis

1 7 0.02

2 7.5 0.04

3 8 0.06

4 8.5 0.04

5 9 0.03

pH

O.D. values

Figure 4.1.5

57

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.

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,

58

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

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

59

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

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).

60

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

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.

61

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

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.

62

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