Dessertation

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.

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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 structurefunction 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.

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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-chlorometacresol, 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 sizeexclusion 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 Indole test Nitrate reduction test Methyl red test Urease test Voges proskauer test Starch hydrolysis test Citrate utilization test Catalase test

RESULT Positive Positive Positive Negative Positive Positive Positive 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 1 2

OF BSA (µg/ml) X-axis 200 400

AT 660 (nm) Y-axis 0.125 0.24

3 4 5 CRUDE ENZYME

600 800 1000

0.353 0.47 0.602 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 4 8 12 16 20

(nm) Y-axis 0.2 0.40 0.612 0.82 1.02 0.634

1 2 3 4 5 CRUDE ENZYME

1.2 1 0.8 0.6

Line 1

O.D. values 0.4 0.2 0 0

4

8

12

16

Conc. Of tyrosine (µg/ml) Figure 4.1.3

55

20

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

1 2 3 4 5

X-axis 30 36 42 48 54

Y-axis 0.13 0.14 0.06 0.07 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

1 2 3 4 5

X-axis 7 7.5 8 8.5 9

Y-axis 0.02 0.04 0.06 0.04 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.

6. REFERENCES

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