CHAPTER 2

REVIEW OF LITERATURE

2.1 Proteinaceous waste

2.1.1 Keratinaceous waste- feather waste Vast quantity of chickens are being utilised every day in the society that produces a large amount of feathers waste in poultry industries. So far, feathers are known to have been chemically and physically prepared to be used as feather meal as well as digestible nutritional protein for animal feed. Keratin is a major constituent of feathers, possessing almost 90% of feather weight [54]. Keratin-consisting materials have always been plentiful in the nature but restricted in practical usages, mainly because of their insolubility and non-degradability by the ordinary proteolytic enzymes. New developments of keratinase production have attracted many attentions to apply keratinase in poultry industry. Feathers waste in poultry industries present a high-quality supply of keratins. This valuable source of keratin could be used either as a source of fertilizer glues and films, or many selected amino acids, and proteins which are applied in animal feed industry [55]. Obviously, large amounts of keratinase for industrial scale processes are essentially needed which is not cheap. Many researches show bacteria able to produce keratinase. However, best host cell for overproduction of keratinase will remain unknown. Several efforts have been done to overproduce keratinase as demands increasing. It is now certain that many different species such as bacteria, actinomycetes, and fungi are able to produce keratinase. However, the level of production and providing conditions are still remaining and are yet to be discovered [56]. 2.1.2 Chicken Feather Waste Poultry industry is continuously producing increasing amount of poultry meat and noticeable quantities of organic residues such as feather, bone meal, blood, offal and so on.

Chicken feathers, making up about 5% of the body weight of poultry, is a considerable waste product of the poultry industry being produced about 4 million tons per year world-wide [57, 58]. Disposal of waste feathers is a major concern for poultry industry and accumulation of this huge volume of the waste feathers results in environmental pollution and protein wastage.

Figure 2.1: Chicken feathers image [82]

Currently a minor quantity of waste feathers is used in other industrial applications such as clothing, insulation and bedding [59], producing biodegradable polymers [60] and enzymes [61] and also as a medium for culturing microbes. A higher quantity of pretreated feather is utilized to produce a digestible dietary protein feedstuff for poultry and livestock [62-66]. However, to decrease the risk of disease transmission via feed and food chain legislation on the recovery of organic materials for animal feed is becoming tighter (Commission of the European Communities, 2000), [67, 68].

2.2 Pretreatment methods for hydrolysis of poultry feathers

Because of the complex, rigid and fibrous structure of keratin, poultry feather is a challenge to anaerobic digestion. Its poorly degradable under anaerobic conditions. [60] However, application of appropriate pretreatments methods hydrolyzes feather and breaks down its tough structure to corresponding amino acids and small peptides [62, 69]. For more than half a century many studies have been performed and various pretreatment methods have been applied to improve the digestibility of feather meal, dietary animal protein

feedstuff and feather biogas potential [70, 71]. Feather meal treatment methods are usually categorized into two groups: hydrothermal treatments and microbial keratinolysis [62, 72]. 2.2.1 Hydrothermal pretreatment Hydrothermal pre-treatment includes thermo-chemical treatment methods (such as acidic hydrolysis and alkali hydrolysis), and also steam pressure cooking [62, 73]. These methods usually need high temperatures [71] or high pressure [74, 75] with addition of diluted acids such as hydrochloric acid [21] or alkali such as sodium hydroxide [62, 74]. Acidic solutions promote the loss of some amino acids such as tryptophan. [75]. Alkaline reactions are slow and degradation of some amino acids with hydroxide is less. Hence the use of bases is recommended. A stepwise diagram for the hydrolysis of protein rich material under alkaline condition is indicated in Fig. 2.2 [76].

Figure 2.2: Protein hydrolysis during thermo-chemical treatment [76]

As a whole, hydrothermal hydrolysis usually consumes high amount of energy and employs expensive equipment during lengthy processes (8 to 12 hrs), [77, and 64]. Thus, optimization of

the treatment conditions is an important issue from technological and economical points of view when applying this method. 2.2.2 Biological pretreatment Biodegradation of feathers is another alternative method. Some bacterial strains can produce keratinase proteases which have keratinolytic activity and are capable to keratinolyse feather keratin. These enzymes help the bacteria to obtain carbon, sulfur and energy for their growth and maintenance from the degradation of -keratin [78]. Various keratinases from different microorganisms such as Bacillus sp. Bacillus licheniformis [79-81] Burkholderia, Chryseobacterium, Pseudomonas, Microbacterium sp., Chryseobacterium sp., Streptomyces sp. has been isolated and studied to date [62, 78]. Microbial proteases are classified into acidic, neutral, or alkaline groups, depends on the required conditions for their activity and on the characteristics of the active site group of the enzyme, i.e. metallo-, aspartic- , cysteine- or sulphydryl- or serine-type. Alkaline proteases which are active in a neutral to alkaline pH, especially serine-types, are the most important group of enzymes used in protein hydrolysis, waste treatment and many other industrial applications. Alkaline protease from Bacillus subtilis was used for the keratinolysis of waste feathers [82]. Subtilisins are extracellular alkaline serine proteases, which catalyse the hydrolysis of proteins and peptide amides. Savinase is one of these enzymes; Alcalase, Esperase and Maxatase are others. These enzymes are all produced using species of Bacillus. Maxatase and Alcalase come from B. licheniformis, Esperase from an alkalophilic strain of a B. licheniformis, and Savinase from an alkalophilic strain of B. amyloliquefaciens [82]. An important advantage of enzyme treatment method is fully biodegradability of enzymes by themselves as proteins. Hence, unlike other remediation methods, there is no buildup of unrecovered enzymes or chemicals that must be removed from the system at the end of degradation process. Although enzymatic treatment is a promising technology; it has some limitations and disadvantages, as well. Currently, the main disadvantage of using alkaline proteases is the high cost of the enzymes production. Much of the cost of producing enzymes is related to high purification of enzymes solutions to avoid the side effects and side activities of the crude enzyme solution which is cheaper. Furthermore, in contrast with microbes which can reproduce themselves and increase their population to be able to consume a large quantity of substrate and survive in harsh environments, extracellular 9

enzymes like alkaline protease do not have reproducibility. Namely, increasing the enzyme population must be done through adding new enzymes from outside into the system. On the other hand, these alkaline proteases lose some reactivity after they interact with pollutants and could eventually become completely inactive. Hence they do not have the adaptability to the harsh environment even though they can survive in a wide range of environmental conditions. This means that the enzyme concentrations must be monitored and controlled during the process in order to optimize enzyme kinetics for site-specific conditions [82]. 2.2.3 Chemical-Biological pretreatment Keratins are insoluble macromolecule comprises super coiled long polypeptide chains with high degree of cross linked disulphide bonds between contiguous chains. According to the literatures disulfide bonds in keratin significantly decrease protein digestibility [83]. And for complete easy degradation of feather all enzymatic keratinolysis from any organism essentially needs to be assisted by a suitable redox [84]. Therefore, it has been suggested that some reductants, such as thioglycollate, copper sulphate, ammonia and sodium sulphite [85] and others, might cleavage the disulfide bonds in keratin and allows the proteases to have access to their peptide bond substrates [86], and consequently improve the degradability of feathers. For instance Ramnani et al., 2007 found that savinase is capable of near complete feather degradation (up to 96%) in the presence of sodium sulfite [84].

2.3 Keratin Structure

Chicken feathers are composed of over 90% of keratin protein, small amounts of lipids and water. Feathers keratin consists of high quantities of small and essential amino acid residues such as glycyl, alanyl and seryl as well as cysteinyl and valyl. Keratin is also the main protein components of hair, wool, nails, horn, and hoofs. Animal hair, hoofs, horns and wool contain keratin, and birds feather contains -keratin. The polypeptides in -keratin are closely associated pairs of helices, whereas -keratin has high proportion of pleated sheets. This conformation confers an axial distance between adjacent residues of 0.35 nm in -sheets, compared to 0.15 nm in -helices. The sheets have a far more extended conformation than the helices.

10

Keratins are insoluble macromolecule comprises a tight packing of supercoiled long polypeptide chains with a molecular weight of approximately 10 kDa. High degree of cross linked cystin disulphide bonds between contiguous chains in keratinous material imparts high stability and resistance to degradation [60, 62-64]. Hence, a keratinous material is a tough, fibrous matrix being mechanically firm, chemically unreactive, waterinsoluble and protease-resistant [78]. Such a molecular structure makes feathers poorly degradable under anaerobic digestion condition. Fig. 2.3 shows keratin molecular structure:

Figure 2.3: Keratin molecular structure [87]

2.4 Enzymes
According to a new technical market research report, WORLD MARKETS FOR FERMENTATION INGREDIENTS (FOD020C), the global market for fermentation products was nearly $16 billion in 2008, and is expected to increase to $22.4 billion by the end of 2013, for a compound annual growth rate (CAGR) of 7%. The amino acids segment has the largest share of the market at $5.4 billion in 2008, and an expected increase by 2013 to more than $7.8 billion, for a CAGR of 7.6%. The market for industrial enzymes was the second-largest segment at $3.8 billion in 2008, with an expected rise to $4.9 billion in 2013, for a CAGR of 8.9%. This report is an update of World Markets for Fermentation Ingredients (FOD020B), published in 11

2005. During the past four years, substantial changes of the industry setup have taken place. The most important products manufactured by fermentation are still the same crude antibiotics, organic acids, amino acids, enzymes, vitamins, polysaccharides, and carotenoids. For virtually all of these categories, markets expanded strongly, but the production landscape changed massively. The market value of crude-fermentation-derived antibiotics is estimated at $1.8 billion for the year 2008. This value is much less than the estimates of many analysts as it refers to the actual trade value of crude products and is supposed to reflect reality more accurately. Consumption increased by more than 10% per year during the past five years, but prices, after some erratic movements in 2006 and 2007, are back to the standard low levels. In contrast to expectations, expansion of consumption took place primarily in the veterinary and infeed sectors. The report reviews the global fermentation industry with emphasis on major fermentation-derived products used in food, feed, pharmaceutical, and technical applications. It provides the most up-to-date information on quantities manufactured, prices and market value developments, and on industry structures. It enables the reader to understand the industry in general, provides in particular insight into the inter-relationship between the ethanol and other carbohydrate-using industries [88].

Source: BCC Research [88]

Figure 2.4: Global Market for Fermentation Product, 2008 and 2013

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Enzyme demand worldwide to reach $7 billion in 2013, the world market for enzymes will recover from a difficult, 2009 to reach $7 billion in 2013 continued strong demand for specialty enzymes. With the environment and cost issues surrounding conventional chemical processes being subjected to considerable scrutiny, biotechnology rapidly is gaining ground due to the various advantages it offers over conventional technologies. Industrial enzymes represent the heart of biotechnology processes. The field of industrial enzymes now is experiencing major R&D initiatives, resulting both in the development of a number of new products and in improvement in the process and performance of several existing products. Currently, new and emerging applications are driving demand and the industry is responding with a continuous stream of innovative products. Significant future growth will require investments by all the participants in research and applications development. This BCC study examines current commercial applications worldwide, their markets and growth opportunities. It also looks into market penetrations of newer grades of enzymes and their applications and sales, as well as new developments and potential applications on the horizon. This report gives a clear, quantitative picture of the supply and demand scenario and highlights technological and investment opportunities in the field [89].

Figure 2.5: Global enzyme markets by application sectors, through 2009 ($ Millions), according to BCC (2008)

According to a new technical market research report, Enzymes for Industrial Applications (BIO030E) from BCC Research (www.bccresearch.com), the global market for industrial

13

enzymes will be worth $2.3 billion in 2007. This is expected to increase to over $2.7 billion by 2012, a compound average annual growth rate (CAGR) of 4%. The greatest growth rate is expected in the animal enzymes sector, with a CAGR of 6% between 2007 and 2012, helped in large part by the increased use of phytase enzymes to fight phosphate pollution. The market is broken down into applications of technical, food and animal feed enzymes. Of these, technical enzymes have over 50% of the market. Valued at nearly $1.1 billion in 2007, this segment is expected to be worth $1.4 billion by 2012, a CAGR of 3.5%. The animal feed enzyme segment is currently worth $280 million and will be worth $375 million in 2012, a CAGR of 4%. The higher growth in this sector will be helped in part by the increased use of phytase enzymes to fight phosphate pollution. New and emerging applications have helped drive demand for enzymes, and the industry is responding with a continuous stream of innovative products.

Table 2.1: Global enzymes market based on application sectors ($millions)

Global enzymes market based on application sectors ($millions) Application sector Technical Enzymes Food Enzymes Animal Feed Enzymes Total 2005 1,075 775 240 2,090 2006 1,105 800 260 2,165 2007 1,140 830 280 2,250 2012 1,355 1,010 375 2,740 CAGR% 2007-2012 3.5 4.0 6.0 4.0

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2.4.1 Proteases Proteases refer to a group of enzymes whose catalytic function is to hydrolyze peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Protease forms a large group of enzymes belonging to the class of hydrolases [90], ubiquitous in nature and performs a major role with respect to their applications in both physiological and commercial fields.These enzymes are widely distributed nearly in all plants, animals and microorganisms. In higher organisms about 2% of the genes codes are formed by these enzymes [91]. Traditionally the proteinases have been regarded as degradative enzymes which are capable of cleaving protein foods. They liberate small peptides and amino acids needed by the body. Also they participate in the turnover of cellular protein. Indeed, this is one of the best characteristic of the proteinases, such as the mammalian digestive enzymes trypsin, chymotrypsin, and pepsin and the lysosome enzymes cathepsin B and cathepsin D. Proteolytic enzymes have the ability to carry out selective modification of proteins by limited cleavage such as activation of zymogenic forms of enzymes, blood clotting and lysis of fibrin clots [92], and processing and transport of secretory proteins across the membranes. These properties add considerable interest to an already important group of enzymes. Additionally proteolytic enzymes have been used for a long time in various forms of therapy [93]. Their use in medicine is gaining more and more attention because several clinical studies are indicating their applications in oncology, inflammatory conditions, blood rheology control and immune regulation. These are also used in crucial biological processes such as regulation of metabolism, enzyme modification, photogenecity, complement system, apoptosis pathways, invertebrate prophenoloxidase activating cascade etc [94]. Furthermore, a study of proteolytic enzymes is valued because of their importance as reagents in laboratory, clinical, and industrial processes. Proteinases from both microbial and nonmicrobial sources, are extensively used in the food industry (baking, brewing, cheese anufacturing, meat tenderizing) [95], in the tanning industry, and in the manufacture of biological detergents [96]. Thus, there is an increasing interest in the proteinases and peptidases of both eukaryotic and prokaryotic microorganisms. Proteases execute a large variety of pharmaceutical functions; particularly their involvement in the life cycle of disease causing organisms has led them to become a potential target for developing therapeutic agents against fatal diseases such as cancer and AIDS [97]. The vast diversity of proteases, in contrast to the specificity of their 15

action, has attracted worldwide attention in an attempt to exploit their physiological and biotechnological applications [98, 99]. Though proteases are enzymes of meta-bolic as well as commercial importance, there is not much literature available on their biochemical and biotechnological aspects [99-103]. However, the earlier reviews are lack of satisfactory information, the biologicalconcept of proteases, which offers new possibilities and potentials for their biotechnological functions.

2.4.1.1 Types of Proteases Proteinases may be classified in a number of ways, for example, on the basis of the pH range over which they are active (acid, neutral, or alkaline), or their ability to hydrolyze specific proteins (keratinase, elastase, collagenase etc.), or their similarity to well characterized proteinases such as pepsin, trypsin, chymotrypsin, or the mammalian cathepsins. Hartley,1960 described the most satisfactory system of classification based on the presence of main catalytic amino acid residue in their active site:(1) Serine proteinases , with a serine and histidine; (2) Cysteine proteinases, with a cysteine; (3) Aspartic proteinases , with an aspartate group and (4) Metalloproteases , with a metallic ion (Zn++, Ca++ or Mn++) in their active site. However, currently proteases are classified on the basis of three major criteria: (a) depending upon site of action; (b) depending upon optimum pH and (c) miscellaneous. Proteases are grossly subdivided into two major groups, i.e., exopeptidases and endopeptidases, depending on their site of action. Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the substrate .In general, depending upon the optimum pH proteases are named as: (i) Acid proteases, active in the pH range 2 - 3.5; (ii) Neutral Proteases, active in the pH between 6.5 and 7.5 and (iii) Alkaline proteases, active in the pH between 7.5 and 10.5. A few miscellaneous proteases which do not precisely fit into the standard classification, e.g., TPdependent proteases which require ATP for activity [104]. Based on their amino acid sequences, proteases are classified into different families [105] and further subdivided into clans to accommodate sets of peptidases that have diverged from a common ancestor. Each family of ptidases has been assigned a code letter denoting the type of catalysis, i.e., S,

16

C, A, M, or U for serine, cysteine, aspartic, metallo, or unknown type respectively. 2.4.1.1.1 Exopeptidases The exopeptidases act only near the ends of polypeptide chains. Based on their site of action at the N or C terminus (Table 2.2), they are classified as amino- and Carboxypeptidase, respectively.

Table 2.2: Classes and active site of exopeptidases

*Open circles represent the amino acid residues in the polypeptide chain. Solid circles indicate the terminal amino acids. Solid triangle indicates site of peptide cleavage 2.4.1.1.1.1 Aminopeptidases Aminopeptidases act at a free N terminus of the polypeptide chain and liberate a single amino acid residue, a dipeptide, or a tripeptide (Table 2.2). They are known for removing the Nterminal Met that may be found in heterologously expressed proteins but not in many naturally occurring mature proteins. Aminopeptidases occur in a wide variety of microbial species including bacteria and fungi [106]. In general, aminopeptidases are intracellular enzymes, but there has been a single report on an extracellular aminopeptidase produced by Aspergillus oryzae [107]. The substrate specificities of the enzymes from bacteria and fungi are distinctly different in that the organisms can be differentiated on the basis of the profiles of the products of

17

hydrolysis [108]. Aminopeptidase I from Escherichia coli is a large protease (400 kDa). It has a broad pH optimum of 7.5 to 10.5 and requires Mg+2 or Mn+2 for optimal activity [109]. A leucine aminopeptidase was purified about 670 fold from germinated grains of barley (Hordeum vulgare). This leucine aminopeptidase is remarkably similar to mammalian leucine aminopeptidase (EC 3.4.1.1). The Bacillus licheniformis aminopeptidase has a molecular weight of 34.00 kDa. Its activity is enhanced by Co+2 ions. Similarly, another species of Bacillus genus i.e. B. stearothermophilus produces aminopeptidase. Structurally it is a made up of two subunits whose molecular mass is about 80 to 100 kDa [110] and is activated by Zn+2, Mn+2, or Co +2 ions. 2.4.1.1.1.2 Carboxypeptidases The Carboxypeptidase act at C terminals of the polypeptide chain and liberate a single amino acid or a dipeptide. Carboxypeptidases are divided into three major groups, serine carboxypeptidases, metallocarboxypeptidases, and cysteine carboxypeptidases, based on the nature of the amino acid residues at the active site of the enzymes. The serine carboxypeptidases isolated from Penicillium sp., Saccharomyces sp., and Aspergillus sp. are similar in their substrate specificities but differ slightly in other properties such as pH optimum, stability, molecular weight, and effect of inhibitors. Metallocarboxypeptidases from Saccharomyces sp. [111] and Pseudomonas sp. [112] require Zn+2 or Co +2 for their activity. The enzymes are also hydrolyze the peptides in which the peptidyl group is replaced by a pteroyl moiety or by acyl groups. 2.4.1.1.2 Endopeptidases Endopeptidases are characterized by their preferential action at the peptide bonds in the inner regions of the polypeptide chain away from the N and C termini. The presence of the free amino or carboxyl group has a negative influence on enzyme activity. The endopeptidases are divided into four subgroups based on their catalytic mechanism, (i) serine proteases, (ii) aspartic proteases, (iii) cysteine proteases, and (iv) metalloprotease. Table 2.3 summarizes general properties of four classes of endopeptidases [261]. To facilitate quick and unambiguous reference to a particular family of peptidases, have been assigned a code letter denoting the catalytic type, i.e., S, C, A, M, or U (Unknown Protease) followed by an arbitrarily assigned number.

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Table 2.3: Classes and some general properties of endopeptidases

Property

Serine

Cysteine

Aspartic

Metallo

Old Name Enzyme Nomenclature Active site component acid pH range Temperature range (0C) Molecular mass (kDa) Inhibitors Location Examples

Serine 3.4.21 Serine

Thiol 3.4.22 Cysteine

Carboxyl 3.4.23 Aspartic

Metallo 3.4.24 Zn++

7-9 20-80 20-135

PMSF, DIFP
Intra- and extra-cellular

3-7 25-70 20-65

2-6 40-70 30-60

5-9 40-60 20-60

EDTA
Intra-and extracellular

E-64, Iodoacetate Pepstatin

Lysosomes Lysosomes

Elastin, Plasmin

Cathepsins B & L

CathepsinD

Gelatinase

2.4.1.1.2.1 Aspartic proteases (EC 3.4.23) Aspartic proteases, commonly known as acidic proteases, depend on aspartic acid residues for their catalytic function. Acidic proteases have been grouped into three families, namely, pepsin (AP1), retropepsin (AP2), and enzymes from Para retroviruses (AP3) [113]. The members of families AP1 and AP2 are known to be related to each other, while those of family AP3 show some relatedness to AP1 and AP2. Most aspartic proteases show maximal activity at low pH (pH 3 to 4) and have isoelectric points in the range of pH 3 to 4.5. Their molecular masses are in the range of 30 to 45 kDa, the exception being larger enzymes in Podospora anserine [114]. The members of the AP1 family have a bilobal structure with the active-site cleft located between the lobes [115]. The active-site aspartic acid residue is situated within the motif Asp-Xaa-Gly, in which Xaa can be Ser or Thr. The aspartic proteases are generally inhibited by pepstatin [265]. They are also sensitive to diazoketone compounds such as diazoacetyl-DL-norleucine methyl ester (DAN) and 1, 2-epoxy-3-(p -nitrophenoxy) propane (EPNP) in the presence of copper

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ions. Microbial acid proteases exhibit specificity against aromatic or bulky amino acid residues on both sides of the peptide bond, which is similar to pepsin, but their action is less stringent than that of pepsin. Microbial aspartic proteases can be broadly divided into two groups, (i) pepsinlike enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and (ii) renninlike enzymes produced by Endothia and Mucor sp. An interesting property of many of the fungal acid proteases i.e. protease of unidentified species of Penicillium is able to activate bovine trypsinogen [116]. Morihara and Oka, 1973 [117] reported a relationship between this trypsinogen kinase activity and the ability of proteinases to hydrolyze specific oligopeptides at bond involving the carboxyl group of lysine, although lysine and arginine containing bonds in the insulin B chain are not cleaved. It is interesting to note that proteinases from the protozoan Tetrahymena pyriformis [118] and Dictyostelium discoideum [119] have trypsinogen kinase activity. Amino acid sequence analysis [120] and X-ray crys-tallographic analysis [121, 122, 123] of the proteinases of Rhizopus chinensis, Penicillium janthinellum, Penicillium roqueforti and Endothia parasitica have revealed a considerable degree of homology between the fungal proteinases and mammalian aspartic proteinases including pepsin and rennin, suggesting that they all evolved from a common ancestral gene [124].

2.4.1.1.2.2 Serine proteases (EC 3.4.21) Serine proteases are characterized by the presence of a serine group in their active site. These are numerous and widespread among viruses, bacteria, and eukaryotes, suggesting that they are vital to the organisms. Serine proteases are found in the exopeptidases, endopeptidase, oligopeptidase, and omega peptidase groups. Based on their structural similarities, serine proteases have been grouped into 20 families, which have been further, subdivided into about six clans with common ancestors [125]. The primary structures of the members of four clans, chymotrypsin (SA), subtilisin (SB), carboxypeptidase C (SC), and Escherichia D-Ala-D-Ala peptidase A (SE) are totally unrelated, suggesting that at least four separate evolutionary origins for serine proteases. Clans SA, SB, and SC have a common reaction mechanism consisting of a common catalytic triad of the three amino acids, serine (nucleophile), aspartate (electrophile), and histidine (base). Although the geometric orientations of these residues are similar, the protein folds are quite different, forming a typical example of a convergent evolution. The catalytic

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mechanisms of clans SE and SF (repressor LexA) are distinctly different from those of clans SA, SB, and SC, since they lack the classical Ser-His-Asp triad. Another interesting feature of the serine proteases is the conservation of glycine residues in the vicinity of the catalytic serine residue to form the motif Gly-Xaa-Ser-Yaa-Gly. Serine proteases are recognized by their irreversible inhibition by 3, 4-dichloroisocoumarin (3, 4-DCI), diisopropylfluorophosphate (DFP), phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone (TLCK). Some of the serine proteases are inhibited by thiol reagents such as pchloromercuribenzoate (PCMB) due to the presence of a cysteine residue near the active site. Serine proteases are generally active at neutral and alkaline pH, with an optimum between pH 7 and 11. They have broad substrate specificities including esterolytic and amidase activity. Their molecular masses range between 18 and 35 kDa, for the serine protease from Blakeslea trispora, which has a molecular mass of 126 kDa [126]. The isoelectric points of serine proteases are generally between pH 4 and 6. Serine alkaline proteases that are active at highly alkaline pH represent the largest subgroup of serine proteases. Serine proteases are inhibited by DFP or a potato protease inhibitor but not by tosyl-L-phenylalanine chloromethyl ketone (TPCK) or TLCK. Their substrate specificity is similar to but less stringent than that of chymotrypsin. These enzymes hydrolyze a peptide bond which has tyrosine, phenylalanine, or leucine at the carboxyl side of the splitting bond. The optimal pH of alkaline proteases is around pH 10, and their isoelectric point is around pH 9. Their molecular masses are in the range of 15 to 30 kDa. Although alkaline serine proteases are produced by several bacteria such as Arthrobacter, Streptomyces, and Flavobacterium sp. [127], subtilisins produced by Bacillus sp. are the best known. Alkaline proteases are also produced by S. cerevisiae [128] and filamentous fungi such as Conidiobolus sp. [129] and Aspergillus and Neurospora sp. [130]. Subtilisin of Bacillus origin represents the second largest family of serine proteases. Two different types of alkaline proteases, subtilisin Carlsberg and subtilisin Novo or bacterial protease Nagase (BPN9), have been identified. Subtilisin Carlsberg produced by Bacillus licheniformis was discovered in 1947 by Linderstrom, Lang, and Ottesen at the Carlsberg laboratory [131]. Subtilisin Novo or BPN9 is produced by Bacillus amyloliquefaciens. Subtilisin Carlsberg is widely used in detergents. Both subtilisins have a molecular mass of 27.5 kDa but differ from each other by 58 aminoacids. These have similar

21

properties such as an optimal temperature of 60C and an optimal pH of 10. Both enzymes exhibit broad substrate specificity and have an active-site triad made up of Ser 221, His 64 and Asp 32. The active-site conformation of subtilisin is similar to that of trypsin and chymotrypsin despite the dissimilarity in their overall molecular arrangements. The serine alkaline protease from the fungus Conidiobolus coronatus has a distinct different structure from subtilisin Carlsberg in spite of their functional similarities [132].

2.4.1.1.2.3 Cysteine proteases (EC 3.4.22) Cysteine proteases occur in both prokaryotes and eukaryotes. About 20 families of cysteine proteases have been recognized. The activity of all cysteine proteases depends on a catalytic dyad consisting of cysteine and histidine. The order of Cys and His (Cys-His or His-Cys) residues differ among the families [127]. Generally, cysteine proteases are active only in the presence of reducing agents such as HCN or cysteine. Based on their side chain specificity, they are broadly divided into four groups: (i) papain-like, (ii) trypsin- like with preference for cleavage at the arginine residue, (iii) specific to glutamic acid, and (iv) others. Papain is the best known example of cysteine protease. Cysteine proteases have neutral pH optima, although a few of them, e.g., lysosome proteases, are maximally active at acidic pH. These are susceptible to sulfhydryl agents such as PCMB but are unaffected by DFP and metal-chelating agents. Clostripain, produced by the anaerobic bacterium Clostridium histolyticum, exhibits a stringent specificity for arginyl residues at the carboxyl side of the splitting bond and differs from papain in its obligate requirement for calcium. Streptopain, the cysteine protease produced by Streptococcus sp., shows a broad specificity, including oxidized insulin B chain and other synthetic substrates. Clostripain has an isoelectric point of pH 4.9 and a molecular mass of 50 kDa, whereas the isoelectric point and molecular mass of streptopain are pH 8.4 and 32 kDa, respectively.

2.4.1.1.2.4 Metalloproteases (EC 3.4.24) Metalloproteases are the most diverse types of the catalytic proteases [114]. This type of enzymes characterized by the requirement for a divalent metal ion for their activity. These include

22

enzymes from a variety of origins such as collagenases from higher organisms, hemorrhagic toxins from snake venoms, and thermolysin from bacteria [135, 136]. About 30 families of metalloprotease have been recognized, of which 17 (M1) contain only endopeptidases, 12 (M2) contain only exopeptidases, and 1 (M3) contains both endo and exopeptidases. Based on the specificity of their action, metalloproteases are divided into four groups, (i) neutral, (ii) alkaline, (iii) Myxobacter I, and (iv) Myxobacter II. The neutral proteases show specificity for hydrophobic amino acids, while the alkaline pro-teases possess a very broad specificity. Myxobacter protease I is specific for small amino acid residues on either side of the cleavage bond, whereas protease II is specific for lysine residue on the amino side of the peptide bond. All of them are inhibited by chelating agents such as EDTA but not by sulfhydryl agents or DFP. A few examples of metalloproteinases have been reported in fungi, and most have been shown to be zinc-containing enzymes. Gripon et al., [135] have suggested that the enzymes of Penicillium caseicolum and Penicillium roqueforti and the neutral proteinase of Aspergillus oryzae and Aspergillus sojae represent a distinct group of enzymes for which they suggest the name acid metalloproteinase. These have lower pH optima and molecular weights i.e. pH 5 and 19,000 respectively, and a different specificity with the oxidized insulin B chain from the thermolysin like neutral metalloproteinases. The Penicillium proteinases are also insensitive to phosphoramidon, a specific neutral metalloproteinase inhibitor. The basidiomycete Tricholoma columbetta produces a low molecular weight neutral proteinase [136] which has some resemblance to the metalloproteinase of another basidiomycete, Armillaria mellea [137].

2.4.1.2 Distribution of proteases Proteases are widely distributed in each part of biological source. Due to this, it belongs to one of the subtype of digestive enzyme. Plant kingdom occupies the topmost rank (43.85 %) for finding proteases, followed by bacteria (18.09 %), fungi (15.08 %), animals (11.15 %), algae (7.42 %) and viruses (4.41 %). Isolated proteases only contribute 27 to 67 % of biological origin irrespective of either animal, microbial or plant proteases while remaining proteases are not well studied. Cysteine protease abundantly occurs (34.92 %) in plants. Microbes have ability to secrete large quantities of serine (13.21 %) and aspartic (8.81%) proteases. Recently, glutamic acid protease of microbial origin has been recorded. Serine, cysteine and aspartic proteases are 23

commonly found in animals (Fig. 2.2). Plant proteases are virtually present in every part of plants viz., root, stem, leaf, flower, fruit, seed, gum and latex. Plant latex is the richest source of protease. About 43.91 % of plant proteases have not been fully characterized. Very rare finding is recorded on asparaginyl protease. On the other hand, usually cysteine and serine endoproteases occurred in plants. Aspartic protease and aminopeptidases are rarely found in plants. Figure 2.6 summarizes the distribution of protease enzymes in biological sources.

2.4.1.2.1 Plant Proteases Crude preparation of the enzyme has a wide specificity due to the presence of various proteinase and peptidase isozymes. The performance of the enzyme depends on the plant source, the climatic conditions for growth and the methods used in its extraction and purification; for example, if the fruit is healthy, then enzyme found is more active. Milk clotting, a property of proteolytic enzyme was recorded in the latex of Carica papaya [138]. Carica papaya latex is rich in proteolytic enzymes, commercially called papain [139]. Papain is a traditional plant protease and has a long history of use [140]. It is extracted from the latex of unripe papaya fruits, which are grown in subtropical areas of west and central Africa and Asia (Tanzania, Uganda, Zaire, Sri Lanka, Thailand and India). The papain is active between pH 5.0 and 9.0 and is stable up to 80 or 90C. It is widely used in industry as a meat tenderizer and has also other uses in the pharmaceutical, detergent, veterinary and food industry. Method of crystallization of papain was established by Monti et al., 2000 [141]. Milk clotting enzyme is present in the fruit of Withania coagulans [142]. Proteolytic enzyme was successfully separated from the lattices of Ficus carica and Ficus glabrata [143]. Kramer and Whitaker, 1964 determined the properties of proteolytic enzyme, purified from the latex of Ficus carica [144]. Proteolytic activity was found in some plant latex including Calotropis procera, Calotropis gigantea, Cryptostcgia grandiflora, Carica papaya and Ficus carica [139]. Ginger rhizome is reported for new source of proteolytic enzyme [145] and the extracted proteolytic enzyme named, Zingibain, showed more activity at its optimum pH 5.0 and optimum temperature at 60C. The amino acid sequence was evaluted for tryptic peptides of the thiol proteinase i.e. actidin which is found in the fruit of Actinidia chinensis [146]. Proteolytic activity was reported in green asparagus, kiwi fruits and miut; optimum temperature for activities of Green Asparagus and Miut were 40-45C and that of kiwi

24

fruit was 60C [147]. Germinated finger millet (Eleusine coracana) seed-lings showed proteolytic activity against hemoglobin and albumin [148]. A Benzyloxycarbonylalanylcitruline p-nitroanilide is a powerful substrate for papain, bromelain, ficin and many other plant cysteine proteinases [149]. A latex serine protease i.e. Euphorbian P of Euphorbia pulcherrima had molecular mass about 74kDa, pI 4.7 and optimum pH 7.0 [150]. Two serine centered proteolytic enzymes, namely euphorbian d 1 and d 2 are separated from Elaeophorbia drupifera latex on the basis of their Mr of 117 K and 65 K respectively [151]. A new protease is well characterized in developing maize endosperm and high lysine opaque-2 maize endosperm which plays an important role in modification of storage protein [152]. Ficin E is well characterized serine protease of Ficus elastica latex having molecular mass about 50 kDa, pI 3.7 and optimum pH 6.0 [153]. An important and popular milk product i.e. cheese was prepared by incorporating vegetable rennet i.e. protease of Calotropis procera [154]. Curcain is a new protease of Jatropha curcas latex whose molecular mass about 22 kDa and isoelectric point is 5.8 [155]. This isolated enzyme of J. curcas plays an important role in the healing process of skin injury i.e. wound [156]. Bromelain is a proteolytic enzyme derived from stem and the juice of pineapples (Ananas comosus). This enzyme is an alternative to papain and is used to tenderize meat. In modern therapy, bromelain is consumed as a digestive and demonstrates anti-edematous, antiinflammatory, anti-thrombotic and fibrinolytic activities [157]. Total 17 proteases were purified from the lattices of 8 different species of Euphorbiaceae family having molecular mass in between 33-117 kDa [158]. Isolated aminopeptidase of oat leaf shows optimum activity at pH 8.4 by using azocasein and rubisco as substrate [159]. Three protease fractions were obtained by purification of a wild thistle (Cynara cardunculus) extract with ammonium sulphate precipitation. Optimum pH and temperature on the proteolytic activity of the crude extract was found to be 5.7 and 37C respectively [160]. Milk clotting activity of some plants found in Pakistan including Opuntia phylloclades, Cereus triangularis, Euphorbia caducifolia, Calotropis procera, Carica papaya, Ficus bengalensis, Ficus elastica and Euphorbia hista was determined by using skimmed milk powder as a substrate [161]. Immunochemical properties of Protease A of dormant cotton seed was established using double immunodiffusion technique through immunological affinity in between trypsin and protease A [162]. Multiple forms of the cysteine proteinases ananain and comosain were derived from pineapple stem [159]. An attempt has been

25

made by Terp et al., 2000 [162] and Fontanini and Jones, 2002 [163] to characterize hordolisin and subtilisin -like serine protease called SEP - 1 from barley. Amino acid sequences of these proteases are matched with other plant subtilases. Thus, both enzymes belong to the cucumisin like group. A type of serine protease of tomato flesh of Lycopersicon esculentum, showed optimum activity at pH 7 and 45C temperature with Km value of 0.48 per cent by using casein as a substrate [164]. The ovules of flowering plants are difficult to obtain in the amounts sufficient for extraction and purification of proteolytic enzymes. For this reason studies have been focused primarily on the characteristics of proteases of the ovary pistil style and stigma. A proteolytic enzyme was extracted from the latex of Ficus hispida having optimum temperature and pH 40C and 7.0 respectively with the isoelectric point of 4.4 to 4.7 [165]. A high molecular weight (80kDa) cucumisin like serine protease, isolated from the latex of Euphorbia supine by two steps chromatography [166]. Tropical squash seeds of Cucurbita ficifolia possess serine protease, which hydrolyses casein and Suc-Ala-Ala-Pro-PhepNA at pH 10.5; oxidized insulin Bchain is also cleaved by this enzyme on several of its peptide bonds. This enzyme is optimally active at 9.2 pH and 55C temperature [167]. A new subtilisin like protease called plantagolisin identified from leaves extract of Plantago maior by using affinity chromatography on bacitracin sepharose and ion exchange chromatography on Mono Q in FPLC. This was obtained at pH 11 and 70C temperature [168]. A high molecular weight (60 kDa) proteolytic enzyme occurred in leaf extract of Calotropis procera which had optimum temperature 70C [169]. Roots of higher plants (Allium porrum, Zea mays and Helianthus indicus) could secrete protease and maintain the proteolytic activity for a long time. The culture medium of aseptically cultivated seedlings of these plants showed highest proteolytic activity at pH 7 [170]. An acidic serine protease occurred in the root extract of gramineae member i .e Zea mays, whose molecular mass is about 54 kDa [171]. The seeds of Albizzia lebbeck and Helianthus indicus show milk clotting activity. This milk clotting protease is extracted by using ammonium sulphate precipitation [172]. Two examples of novel cysteine proteases are well characterized from germinating cotyledons of soybean. Molecular masses and isoelectric point of both enzymes are 26.178 kDa, 26.429 kDa, pI 4.4 and pI 4.7 respectively [112]. A usual thermostable aspartic protease of Ficus racemosa latex exhibits a broad spectrum pH range between pH 4.5 - 6.5 and maximum activity at 60 0.50C. This enzyme has 44.50 0.50 kDa molecular weight [173]. Pedilanthin, a novel protease

26

identified to homogeneity of Pedilanthus tithymaloids latex, which had 63.1 kDa molecular mass. This enzyme had optimum pH 8.5 and temperature optima at 68C [174]. A low molecular weight (25kda) alkaline serine protease of Holarrhena antidysentrica seeds had optimum activity at pH 7.5 which exhibited its highest activity at 350C using 1% casein as a substrate. The Km and Vmax values are 1.1mg ml-1 and 38971 Units min -1 mg-1 respectively [175]. A high molecular weight serine protease (134.3kDa) named as indicain has been identified to homogeneity from the latex of Morus indica using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The pH and temperature optimum of this enzyme is 8.5 and 80C respectively. The extinction coefficient and isoelctric pH of this enzyme was 41.24 and pI 4.8 respectively. The molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residue [176]. A good potential of industrial application of bromelain was recorded in textile, brewing and fermentation industry [177]. This enzyme has been clinically fully analyzed by Taussin and Batkin, 1988 [178]. A new cysteine protease as a vegetable source for milk clotting enzyme was puri -fied from the root latex of Jacaratia corumbensis and enzyme showed pH and temperature optima in between pH 6.5 - 7.0 and 55C respectively with 33 kDa molecular mass [179]. A new papain like endopeptidase i.e. asclepain c -II has been isolated and well characterized from the petiole latex of Asclepias curassavica. This enzyme displayed molecular mass of 23.59 kDa, pI > 9.3, maximum proteolytic activity at pH 9.4 - 10.2 and showed poor thermostability [180]. An alkaline chymotrypsin like serine protease i.e. dubiumin of Solanum dubium seeds showed optimum enzyme activity at 70C temperature. This enzyme had 66 kDa molecular mass and pI is 6 [181]. Benghalensin, a serine protease is identified to homogeneity from the latex of Ficus benghalensis by a single step procedure using anion exchange chromatography. This enzyme had 47 kDa molecular mass, pI is 4.4, optimum pH is 8 and optimum temperature is 55C. The molecular structure of enzyme consists of 17 tryptophan, 31 tyrosine and 09 cysteine residue [182]. A comparative study in between proteolytic activity, milk clotting activity and gelatinolytic activity is given in latex of twenty one laticiferous plants, belonging to seven different latex bearing families of Khandesh region of Maharashtra, India. Highest milk clotting activity was reported in the latex of Euphorbia nivulia. The decreasing order of potential proteolytic activity are in the order of Euphorbianivulia > Carica papaya > Calotropis procera > Ficus carrica

27

based on Tyrosine Unit [183]. Shivaprasad et al., 2009 reported the thrombin like activity of cysteine protease, Pergularain eI of Pergularia extensa latex having molecular mass 23.35 kDa and the N-terminal amino acid sequence as L -P -H -D-V [184]. The latex of Aslepiadaceae member i.e. Calotropis gigantea possess fibrinogenolytic, fibrinolytic and proteolytic activity. Inhibition pattern shows that the isolated protease enzyme belongs to cysteine protease family [185]. Procerain B is a novel cysteine protease of the latex of Calotropis procera, enzyme shows broad optimum pH and temperature range i .e pH 6.5 to 8.5 and 40 to 600C temperature respectively. This enzyme had 25.70 kDa molecular weight and pI is 9.52 [186]. Crinumin is a chymotrypsin like serine protease isolated from the latex of Crinum asiaticum. This enzyme is characterized for its physiological and chemical properties. It has a wide range of pH stability (4.5 to 11.5 and optimum at pH 8.5), optimum temperature at 700C, 67.7 kDa molecular mass, 6.9 extinction coefficient, and number of 13 tryptophan, 24 tyrosine and 15 cysteine residues with 7 isulphide bridges [187]. A number of subtilases have been isolated from various cucurbitaceous plants. Cucumisin a protease enzyme of sarcocarp of melon fruit, Cucumis melo) has been characterized completely [188] and supported by Uchikoba et al., 1995 [189]. However, protease D, though it is present in same part of this plant need to be characterized more [190]. A high molecular weight serine protease (78 kDa) called kiwano protease is present in similar part i.e. sarcocarp of Cucumis metuliferus. It had maximum activity at pH 8.0 and 300C temperature [206]. A protease obtained from the sarcocarp of wax gourd (Benincasa hispida). The first 33 residues amino acids in the N terminus were sequenced, and it was shown that first 25 residues of amino acids matched with cucumisin. Its molecular weight is 67.00 kDa. It ad optimum pH and temperature is 9.0 and 600C respectively [191]. Interestingly cucumisin like serine protease is present in the sarcocarp of snake gourd (Tricosanthes bracteata) [192].

28

Figure 2.6: Per cent wise distribution of proteases (A) Distribution of proteases; (B) Plant protease; (C) Microbial proteases; (D) Animal proteases and (E) Plants part used for isolation and characterization of proteases

2.4.1.2.2 Animal Proteases The most common and known proteases of animal origin are pancreatic trypsin, chymotrypsin, pepsin and rennin [193, 194]. These are prepared in pure form in bulk quantities. However, their production depends on the availability of livestock for slaughter, which is governed by political and agricultural policies. Trypsin is one of the three principal digestive proteinases. In the digestive process, trypsin acts with the other proteinases to break down dietary protein molecules

29

to their component peptides and amino acids. Trypsin continues the process of digestion (that begins in the stomach) in the small intestine where a slightly alkaline environment (about pH 8) promotes its maximal enzymatic activity. This enzyme is active only against the peptide bonds in protein molecules that have carboxyl groups donated by arginine and lysine [195]. Trypsin is the most discriminating of all the proteolytic enzymes in terms of the restricted number of chemical bonds that it will attack [196]. Chymotrypsin is a proteolytic, or protein digesting enzyme, active in the mammalian intestinal tract. It catalyzes the hydrolysis of peptide bonds in which the carboxyl groups are provided by one of the three aromatic amino acids i.e. phenylalanine, tyrosine or tryptophan. Pure chymotrypsin is an expensive enzyme and is used only for diagnostic and analytical applications [197]. Now a days chymotrypsin tablets are available, that are used as safe painkillers. Pepsin is an enzyme produced in the mucosal lining of the stomach that acts to degrade protein. In the laboratory studies, pepsin is most efficient in cleaving bonds involving the aromatic amino acids, phenylalanine, tryptophan, and tyrosine. Pepsin is an aspartyl protease and resembles human immunodeficiency virus type 1 (HIV-1) protease, responsible for the maturation of HIV-1 [198]. Rennet is a pepsin -like protease , having the property of clotting , or curdling of milk . Rennet is obtained from the inner lining of fourth or true stomachs (abomasum) of milk-fed calves [199]. It is used extensively in the making of cheese and junket. The specialized nature of the enzyme is due to its specificity in cleaving a single peptide bond in casein to generate insoluble para casein and C-terminal glycopeptide [14]. Acid proteinase activity has been detected in both African (Trypanosoma brucei) and Latin American (Trypanosoma cruzi) species of trypanosomes. Although it was suggested initially that a T. brucei rhodesiense proteinase isolated from trypomastigotes was like cathepsin D, i.e. an aspartic proteinase [200], it has now been shown that the major proteinase from T. brucei bloodstream forms must be a cysteine proteinase, since activity is stimulated by both ethylene glycolbis - (P -aminoethyl ether) N, N -tetraacetic acid and cysteine, inhibited by p -chloromercuribenzoate, and unaffected by pepstatin [201]. The enzyme had an optimum pH around 4 for acid denatured hemoglobin hydrolysis [202]. Proteinase activity has also been reported in Trypanosoma rangeli [203], The partially purified protease of flagellate parasite obtained from genitourinary tract of cattle i.e. Tritrichomonas foetus [204] which is responsible for the hydrolysis of denatured hemoglobin, azocasein, and a -N -benzoyl -L -argininamide. It

30

has a molecular weight of between 17,500 and 20,000, having optimum pH between 6.5 and 7.0. Its activity was blocked by a number of cysteine proteinase inhibitors but not by pepstatin suggesting that the isolated enzyme belongs to cysteine type protease. Proteinases are also present in phytoflagellates. Proteolytic activity in malarial parasites was first reported by Moulder and Evans, 1946 [209] for Plasmodium gallinaceum, a chicken parasite. The first attempt was made for characterization of protease in Plasmodium berghei and Plasmodium knowlesi species [210]. They also reported soluble proteolytic activity with pH optima in between 4 and 8 for hemoglobin hydrolysis. Peptidase activity has been detected in Tetrahymena, particularly in the cytoplasm and on the outer cell surface of Tetrahymena thermophila [211]. Proteolytic activity in the rabbit brain homogenate was determined by using haemoglobin and casein as a substrate [212]. Cathepsin D, a specific aspartic protease occurs in brain of diabetic rat induced by alloxan monohydrate injection. Also proteolytic activators are found in cerebral extract of the same rat [159]. Cathepsin L- like proteinase is characterized from goat brain [213]. Purified enzyme shows the 5.0 optimum pH and 50C optimum temperature. The effect of guar gum, lignin and pectin on proteolytic enzyme was investigated in gastrointestinal tract of rat [214]. A novel protease Cathepsin P of mouse placenta has been well characterized [215]. Chymosin, the major component of rennet (milk clotting enzyme) is an acid protease, which is isolated from abomasal tissue of goat kid (Capra hircus). The purified chymosin had a molecular mass of 36 kDa with maximal activity at 30C at pH 5.5 [216]. Plasmin and plasminogen derived activities were measured in bovine and human milk with a chromogenic tripeptide H-D-valyl-L-lysine-p-nitroanilide substrate [217]. A thrombin like serine proteinase occurred in the snake venom i.e. Bothrops asper. The purified enzyme has a molecular mass about 27 kDa [218]. Various kinds of proteolytic enzymes were purified from animal source [219]. Astrup (1951) investigates the activation of proteolytic enzyme in blood (plasmin and fibrinolysin) by an interaction between its proenzyme and the insoluble tissue activator (fibrinokinase) [220]. Blackwood et al., 1962 [221] reported the changes occurred in the proteolytic enzyme systems of rat tissues in response to heterogeneous growth of human ovarian tumors. A digestive protease enzyme occurred in larval guts of fifth instar stage of Spilosoma obliqua (Lepidoptera: Arctiidae). This protease was purified using ammonium sulfate fractionation, ion-exchange chromatography, and hemoglobin-sepharose affinity

31

chromatography. The purified protease had a molecular mass of 90 kDa and a pH optimum of 11. The purified protease optimally hydrolyzed casein at 50C [173]. Two cysteine peptidases coined Metamorphosis Associated Cysteine Peptidase (MACP-I and MACP-II) are identified to homogeneity from 40-46 h after puparium formation stage of insect (Ceratitis capitata) which is a key moment of metamorphosis. Both enzymes showed sensitivity against a specific inhibitor of papain-like cysteine peptidases i.e. Ep-475. MACP -I is a single chain protein with 80 kDa molecular mass and it includes several isoforms with pI values of pH 6.256.35, 6.7, and 7.2. This enzyme has an optimum pH of 5 and its pH stability ranges from pH 4 to 6. The molecular weight and N-terminal sequence (VNIESDTADQ) suggest that MACP-I is a novel enzyme. On the other hand cathepsin B -like cysteine protease i.e. MACP -II) belongs to acidic protease family. The molecular weight of this enzyme is 30 kDa and pI is 5.85. N-terminal sequence of this enzyme is LPEQFE -P -QF [174]. A bacterium species i.e. Bacillus sp. PN 51 is isolated from bat feces. An alkaline serine protease is characterized from this Bacillus sp. This enzyme has highest protease activity at 600C at pH 10. Enzyme activity of this enzyme is strongly inhibited by PMSF and chymostatin, suggesting that the purified enzyme belongs to serine family of protease [175]. 2.4.1.2.3 Microbial Proteases 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. Microbial proteases account for approximately 40% of the total worldwide enzyme sales. Proteases from microbial sources are preferred to the enzymes from plant and animal sources since they possess almost all the characteristics desired for their biotechnological applications. 2.4.1.2.3.1 Bacterial Protease Milk clotting enzyme (MCE) is produced by Egyptian Bacillus sphaericus NRC 24. MCE is obtained by fractional precipitation with acetone, followed by ion exchange chromatography by using DEAE Sephadex A 25 and finally by Sephadex G 100 column.

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The pH 6.5 is the optimum pH of MCE at 550C [176]. Protease enzyme of mutated strain of Bacillus subtilis shows proteolysis phenomenon in the manufacturing of Canadian cheddar cheese. This is advantageous in the formation of softer curd [177]. A microbial rennet i.e. milk clotting protease enzyme is partially purified from cell free supernatant of Bacillus cereus [178] Extracellular alkaline protease isolated from Gram positive Bacillus firmus MTCC 7728, showed maximum activity t pH 9 and 400C temperature. Maximum proteolytic activity was observed after 48 h growth, when growth is reached stationary phase [179]. A new acidic protease is obtained from the culture medium of thermotolerant Gram positive Bacillus badius MTCC 7727. This enzyme exhibits optimum activity at pH 5 and 400C temperature [180]. A neutral protease is partially purified from Bacillus subtilis. The optimal conditions for protease production was an optimum substrate concentration 0.5%, optimum incubation period 30 h, optimum temperature and pH is 400C and 7 respectively. This protease enzyme is partially purified by ammonium sulphate precipitation and Sephadex G 200 filtration. Purified protease enzyme had a maximum activity at pH 7 [181]. A halotolerant strain of Bacillus subtilis FP -133 is isolated from fish paste. This strain is identified for its intracellular protease production. The molecular mass of this intracellular protease is about 59 kDa and the enzyme (protein) consists of four subunits, each with a molecular mass of 14 kDa [182]. A solid state fermentation is employed for the production of alkaline protease by a thermophilic strain of Bacillus subtilis DM-04 using agro industrial waste product and kitchen waste material viz., mustard oil cake, wheat bran, rice bran, Imperata cylindrical grass, banana leaves, potato peels and used tea leaves. The crude protease enzyme shows optimum activity at 400C under alkaline condition [183]. A newly isolated halotolerant Bacillus aquimaris VITP 4 is used for the production of extracellular protease. The optimum pH and temperature for production of enzyme is pH 7.5 and 37C respectively [184]. A thermophillic neutral protease is characterized from thermophilic Bacillus strain HS 08. Molecular mass and optimum pH and temperature of this enzyme is 30.9 kDa, pH 7.5 and 650C temperature respectively. Azocasein is the best substrate for enzyme activity [185]. The growth and protease production by Bacillus sp. (SBP - 29) was examined and maximum protease activity achieved by using soybean meal as substrate; enzyme has optimum

33

temperature and pH optima is 60C and 9.5 respectively [186]. Surfactants, laundry detergent and organic solvent resistant alkaline protease occurred in cell free supernatant of Bacillus sp. HR - 08. The Zymogram analysis of the crude extract revealed the presence of five extracellular proteases. One of the protease enzymes is partially purified by three step procedure including ammonium sulphate precipitation, DEAE sepharose ion exchange chromatography and Sephacryl S 200 gel filtration. The purified protease has an optimum temperature and pH of 600C and 10 respectively and molecular mass of this enzyme is 29 kDa [187]. An alkaline protease occurred in cell free broth of Bacillus circulans BM 15 strain. This enzyme showed optimum activity at pH 7 and 400C; having molecular mass about 30 kDa [188]. An alkaline serine protease from Bacillus circulans has been characterized in detail for its robustness and ecofriendly application potential in leather processing and detergent industries. Molecular mass of the purified enzyme is around to 39.5 kDa and it exhibits optimum activity at 70C under alkaline pH environment [189]. A bacterial strain Bacillus licheniformis is isolated from Tihamet Aseer, Saudi Arabia. Protease of this candidate displays proteolytic property at optimal pH and temperature 9.0 and 55C respectively [190]. Thermostable alkaline protease was purified from Bacillus licheniformis MIR 2 9 [191]. Another strain of Bacillus i.e. Bacillus licheniformis NH 1 is able to produce detergent stable and thermostable alkaline serine protease. The protease had optimal activity at 68C temperature and pH is 10.5 [192]. A novel organic solvent stable alkaline protease is identified in cell free supernatant of a new strain i .e Bacillus licheniformis YP 1 A, which is isolated from crude oil contaminant soil. This enzyme retained more than 95% of its initial activity after preincubating at 40C for 1 h in presence of 50% (V/V) organic solvents such as DMSO, DMF and cyclohexane. This protease is active in a broad range of pH from 8 to 12 with the optimum pH 9.5 and optimum temperature as 600C [193]. Twelve strains of Bacillus licheniformis were isolated from traditionally fermented African locust bean (iru) for the production of protease. One of the strain of Bacillus licheniformis LBBL-11 exhibits highest proteolytic activity. Maximum protease production by using this strain occurred after 48 h of growth which is the end period of exponential phase of growth. The protease from this Bacillus sp. had optimum pH is 8 at 600C [194]. Highly thermostable protease was purified and characterized from broth culture of Bacillus

34

steareothermophillus TLS33 [195] and Bacillus pumilus [196]. An alkalophilic strain of Bacillus pumilus MK 6-5 is able to produce thermostable alkaline protease. This enzyme is purified by using ammonium sulphate precipitation, ion exchange and gel filtration chromatography. Inhibition profile of this enzyme exhibited by PMSF suggested that the protease belonged to serine protease [196]. A low molecular weight (34.60 kDa) alkaline serine protease is identified to homogeneity using ammonium sulphate precipitation and gel filtration from Bacillus pumilus CBS. The N-terminal sequence of first 21 amino acids (aa) of the purified enzyme is AQTVPYGIPQIKAPAVHAQGY. The highest homology of 98.1% is observed with BPP-A protease of Bacillus pumilus MS-1. This protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine proteases superfamily. The optimum pH is 10.6 while the optimum temperature is 650C [198]. An alkalophilic strain of bacterium i.e. Bacillus cereus is able to produce an extracellular alkaline protease, which is suitable for commercial laundry detergent. This enzyme shows maximum activity against casein at pH 10.5 and 500C temperature and 28 kDa is the molecular mass of the enzyme [199]. The Bacillus cereus MCM B-326 was isolated from buffalo hide and characterized for production of extracellular protease. Maximum protease production occurred at pH 9 and 300C under shake culture condition by using starch soybean meal medium. This enzyme is used in leather processing unit due to its dehairing principle [200]. An extracellular bleach stable protease producing strain was isolated from marine water sample and identified as Bacillus mojavensis A21. The A21 alkaline protease is purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange chromatography, with a 6.43 -fold increase in specific activity and 16.56 % recovery. The molecular weight of the purified protease is 20 kDa. The enzyme is highly active over a wide range of pH from 7 to 13, with an optimum pH at 8.5. The N-terminal amino acid sequence of the first 20 amino acids of this purified protease is DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with bacterial peptidases, suggesting that the isolated enzyme is a new and novel protease [201]. A comparative account on hydrolytic activities of exogenous protease in four strains of Pseudomonas sp. viz., Pseudomonas sp. C 61, Pseudomonas sp. C 20, Pseudomonas

35

fragi (ATCC 4973) and Pseudomonas fluorescens (NRRL-B-1244) is given . Molecular mass of isolated protease from C 61, C 20 and P. fluorescens is 46.80, 49.20 and 46.10 kDa respectively. While P. fragi produces two proteases whose molecular mass is about 49.20 and 34.20 kDa [202]. Gram negative bacteria i.e. Pseudomonas aeruginosa exhibits the proteolytic activity [203]. Temperature and nutrient factors affect the proteinase production by P. fluorescens and P. aeruginosa in broth and milk [204]. A protease enzyme is identified in cell free supernatant of Pseudomonas aeruginosa PD 100. Ammonium sulphate precipitation, Sephadex G 50 filtration and CM -sephadex chromatography is employed for the purification of protease. This protease has molecular mass about 36 kDa, pI is 6.2 and optimum pH 8 at 600C. It finds potential application for waste treatment, used in detergent and leather industry [205]. A potent bacterium for extracellular protease production was identified as Pseudomonas sp. RAJR 044. Mutant of this strain JNGR 242 shows highest yield of protease productivity i.e. 2.5 times higher than parent one. The purified protease enzyme of both strains i.e. parent and mutant a single homogenous band on SDS-PAGE gel corresponding to 14.4 kDa. Inhibition study demonstrates that the isolated protease belongs to serine protease family [206]. A solvent tolerant, thermostable and alkaline metalloprotease occurs in cell free broth culture of alkalophilic Pseudomonas aeruginosa MTCC 7926. The purified protease has an optimum temperature and pH of 25650C and 6-11 respectively and molecular mass is 35 kDa. Purified metalloprotease showed industrial interest in dehairing of animal skin, anti-staphylococcal activity and processing of X -ray film for recover of silver [207]. Forty three bacterial strains are isolated from crude oil contaminated samples by using toluene and cyclohexane enrichment medium. Out of these, ten bacterial species show highest protease activity. Among them one of the isolate i.e. PT 121 is identified as Pseudomonas aeruginosa. Protease of PT 121 shows highest enzyme activity and it acts as a catalyst for aspartame precursor Cbz-Asp-Phe-NH2 synthesis in presence of 50%dimethylsulphoxuide [208]. Watanabe, 1988 characterizes the proteolytic properties in more than 100 strains of oral isolates of Mycoplasma salivarium with respect to aminopeptidase and carboxypeptidase activity [209]. Petit and Guespin, 1992 [210] describe extracellular proteolytic activity of protease which occurs in submerged growth of a Gram negative soil

36

bacterium Myxococcus xanthus DK 1622. This enzyme shows the milk clotting activity and it belongs to aspartic protease family on the basis of inhibition by pepstatin [210]. Protease activity is first time detected in Streptomyces rimosus TM-55 after 12 h of growth in submerged culture. The optimum pH of this enzyme is 6.5 and temperature is 400C [211]. Alkaline protease is identified from Streptomyces gulbergensis. Purified enzyme shows optimum pH is 9.0 and 450C temperature. This enzyme is applied in washing surgical instruments [212]. A comparative account is given in total 157 stock cultures of lactic acid bacteria for their ability to produce extracellular proteinase with milk clotting activity. One of the cultures is identified as Enterococcus faecalis TUA 2495 L, which has highest milk clotting activity. The estimated molecular mass of the purified enzyme of this strain is 35 kDa and pI is 5.4 [213]. A bacterial strain i.e. Listeria monocytogenes was isolated from degraded cow meat. It exhibits proteolytic activity against casein, gelatin and egg albumin at pH 9 and 30C temperature [214]. A novel extracellular alkaline protease enzyme is identified in cell free supernatant of marine bacterium eredinobacter turnirae. Optimum temperature and pH for maximum enzyme activity of this protease is 500C and 9 respectively and molecular mass is 40 kDa [215]. Protease activity was detected in total 317 isolated mesophilic Streptomycetes sp., were obtained from several areas around Egypt [216]. A low molecular weight (18.4 kDa) glutamic acid specific protease is identified from Streptomyces griseus [217]. A novel thermostable and detergent stable subtilisin like serine protease called as aqualysin I is identified from the culture medium of Thermus aquaticus YT - 1. This enzyme is purified by applying FPLC system with a mono-S-column. It maintains the stability in presence of urea and Tween 20 [218]. A novel aspartic protease is present in lactic acid bacteria (Oenococcus oeni), which is isolated from Argentinean wine by using ammonium sulphate precipitation and sephadex G-100 filtration. Structurally this enzyme has two identical subunits whose molecular mass is about 33.10 and 17.00 kDa [219]. Keratinase is a type of protease enzyme, which is identified from Chrysobacterium sp. Kr6 growing on poultry feathers [220]. Keratinolytic metalloprotease is purified from bacterium Microbacterium sp. strain kr 10 by sequential liquid chromatography on Sephadex G-100

37

and Q-Sepharose column. Estimated molecular mass of this enzyme is 42 kDa. The enzyme had pH and temperature optima of 7.5 and 500C respectively [221]. 2.4.1.2.3.2 Fungal Protease The fungal proteases are active over wide pH range (pH 4 to 11) and exhibit broad substrate specificity. Aspergillus oryzae produces acid, neutral, and alkaline proteases. However, these have a lower reaction rate and worse heat tolerance than the bacterial enzymes do. Fungal enzymes can be conveniently produced in a solid-state fermentation process. Milk clotting enzyme i.e. microbial rennet is identified from Mucor pussilus [222]. A solid state fermentation [223] and submerged fermentation [224] are employed for the production fungal rennet by thermophillic strain of Mucor miehei. This enzyme is used in cheese production which satisfies the characteristics of organoleptic quality [223]. The solid state fermentation is an appropriate system for rennin production. A report includes ability of protease production in twelve species of genus Mucor. All the strains could produce protease enzyme after 120h period of incubation at 280C. Highest proteolytic activity is recorded in Mucor racemosus Fres. F. chibinensis [225]. The production of a rennin like milk clotting enzyme is possible by Penicillium citrinum 805 using corn-steep water as a medium. The enzyme has maximum activity at 600C and at pH 6 [226]. Protease enzyme of Candida albicans shows proteolytic activity against proteins isolated from human saliva [227]. A good protease produced by Aspergillus fumigatus when grown on glucose-peptone-gelatin medium, pH 5 and 300C for four days [228]. The fungus Acremonium typhinum produces a novel endoprotease during symbiotic endophytic infection of the grass, Poa ampla. This enzyme belongs to thiol containing serine protease family [229]. The wheat bran is most suitable substrate for the production of alkaline protease produced by Trichoderma koningii [230]. Penicillium expansum produced an alkaline protease in culture broth which had maximum activity at pH 10.5 and 350C temperature, whose molecular mass is 20.5 kDa. Purification of this enzyme is achieved by acetone precipitation and column chromatography on Sephadex G100 and DEAE Sephadex A-50 [231]. The structure of aspartic proteinase is estimated from zygomycetes fungus Rhizomucor pusillus. Structurally this enzyme contains two

38

asparagine linked high mannose type oligosaccharide chains at Asn 79 and Asn188 [232]. A milk clotting enzyme was identified from Penicillium oxalicum through fractional precipitation with ethanol. This enzyme showed optimum activity at pH 4.3 and 650C [233]. New extracellular subtilisin like, high molecular weight (75 kDa) alkaline protease (PoSI) is isolated from white rot fungus Pleurotus ostereatus culture broth. Isoelectric point of this enzyme is 4.5 [234]. A rice pathogenic fungus i.e. Sarocladium oryzae secretes proteinase enzyme in the culture medium. This enzyme showed the proteolytic activity against azocasein as a substrate at pH 9.0 [235]. Acetone precipitation, ion exchange chromatography and gel filtration methods are used for the purification protease enzyme from this fungal strain. Estimated molecular mass of the enzyme is 23 kDa and it shows maximum activity at pH 8 and 400C temperature [236]. Similarly a new milk clotting protease produced by solid state fermentation by Aspergillus oryzae LS1 by using wheat bran as a substrate [237]. Five different kinds of agricultural wastes viz., corn cob, oat husk, sugar cane bagasses, corn husk and cassava peel used in submerged fermentation for protease production by using Penicillium janthinellum [238]. The rice bran is used as a substrate for the production of neutral metalloprotease by solid state fermentation using Rhizopus microsporus NRRL 3671 [239] and a local strain of Aspergillus oryzae (Ozykat-1) [240]. This enzyme showed maximum activity at a temperature 600C and pH 7. Maximum yield protease could achieve by using wheat bran as a substrate by a highly potent, local strain of Aspergillus awamori: Nakazawa MTCC 6652 [242] by using a modified form of solid state fermentation. Extracellular bleach stable an alkaline serine protease produced by Aspergillus clavatus ES 1. This enzyme showed maximum activity at pH 8.5 and 50C temperature [243]. 2.4.1.2.3.3 Viral proteases Viral proteases have gained importance due to their functional involvement in the processing of proteins of viruses that cause certain fatal diseases such as AIDS and cancer. Serine, aspartic, and cysteine peptidases are found in various viruses [97]. All of the virus-encoded peptidases are endopeptidases; there are no metallopeptidases. Retroviral aspartyl proteases that are required for viral assembly and replication are

39

homodimers and are expressed as a part of the polyprotein precursor. The mature protease is released by autolysis of the precursor. An extensive literature is available on the expression, purification, and enzymatic analysis of retroviral aspartic protease and its mutants [319]. Recent research is focused on the three-dimensional structure of viral proteases and their interaction with synthetic inhibitors with a view to designing potent inhibitors that could combat the relentlessly spreading and devastating epidemic of AIDS. Crystal and three dimensional structure of aspartyl protease of the HIV-I, has been determined to 3 A0 resolution; the structure suggested a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation [320]. Three dimensional structure of the protease has been elucidated from human cytomegalovirus (hCMV) at 2.27A0 resolution [321]. 2.4.1.2.3.4 Algal Protease Higher level of proteolytic activity occurred in multicellular algae than in unicellular algae [322]. Matrix metallo protease inhibitor has been identified as a potential therapeutic candidate from brown algae Ecklonia cava [323]. A comparative account on proteolytic activity is given in total 47 species of macroalgae including 9 species of chlorophyta, 22 species of rhodophyta and 16 species of phaeophyta [324]. A fibrinolytic enzyme, serine protease family member is identified from a marine green alga, Codium divaricatum and it is abbreviated as C. divaricatum protease (CDP). Its molecular is 31 kDa and it showed maximum activity at pH 9 [325]. Similarly another fibrinolytic trypsin like serine protease enzyme is identified from Codium latum, a member of marine green alga, and it is abbreviated as C. latum protease (CLP) [326].

2.4.1.3 Mechanism of action of protease and Physicochemical properties of proteases The mechanism of the action of proteases has been an interesting subject to researchers. Purification of proteases to homogeneity is a prerequisite for studying their mechanism of action. Vast numbers of purification procedures for proteases, involving affinity chromatography, ionexchange chromatography, and gel filtration techniques, have been well documented in the literature. Primary methods of purification of plant proteases often include precipitation 40

(either by using solvent i.e. acetone or salt i.e. ammonium sulfate), ion exchange chromatography and gel filtration; secondary, yet more specific, techniques include affinity chromatography, chromatofocusing and hydrophobic interaction chromatography. After the purification enzymes are subjected to characterization by considering these tools, some physicochemical and biochemical properties of few earlier reported plant proteases listed in Table 2.1, mostly include plant proteases studied during the past four to five decades. 2.4.1.3.1 Serine Proteases Peptide bond hydrolysis is a very common process. A wide variety of enzymes can perform proteolytic reactions. Members of one large family of protease are called serine proteases because of the important serine group at the active site. All of the serine proteases contain three residues at their active site: a serine, a histidine, and an aspartate. These comprise the characteristic catalytic triad. In the numbering scheme for chymotrypsin (a numbering scheme which is typically used in studies of any of the mammalian serine proteases), the residues are Ser 195, His 57, and Asp102. Serine proteases increase the rate of peptide bond hydrolysis by ~1010 times compared to the uncatalyzed reaction. Performing this feat requires a specific structure. As mentioned above, the serine proteases all have three residues that are critical for catalysis: a serine, a histidine, and an aspartic acid. These are conserved in all of the serine proteases, and are superimposable in the crystal structures of these proteins. The side-chain of the amino acid residue of substrate peptide can bind to the recognition site on the enzyme. Serine195 performs a nucleophilic attack on the substrate. Histidine 57 abstracts a proton from Ser 195 during the process. The result of the nucleophilic attack is a covalent bond between the Ser 195 side-chain oxygen and the substrate. The negative charge that develops on the peptide carbonyl oxygen is stabilized by hydrogen bonds formed from two protease backbone amide protons. This region of the protein is called the oxyanion hole, because it stabilizes the negative charge on the oxygen. The oxyanion hole is critical for catalysis [327]. Histidine 57 donates a proton to the substrate amide nitrogen, allowing release of the C-terminal part of the substrate as a free peptide (peptide 1). The final step is an attack by water on the ester bond between the peptide and the Ser195 oxygen. This forms the second product of peptide with a normal carboxyl group, and regenerates the serine hydroxyl. The second peptide

41

then dissociates from the enzyme to allow another catalytic cycle to begin [Fig. 2.7]. As it is apparent in the series of drawings below, serine 195 is the residue that performs the actual catalysis, while the other residues seem to be important for positioning of the serine and for stabilizing the intermediate states. Aspartate102 is relatively far from the substrate; it helps position histidine 57, and raises the pKa of histidine 57 to allow the histidine to act as a base. This is important to the catalytic process: mutation of aspartate102 to asparagine decreases the kcat by ~10,000-fold [328]. Serine proteases are very useful as model enzymes for studying catalytic processes, because they use a variety of techniques for accelerating the reaction rate. They use acid-base catalysis; the histidine abstraction of the serine proton, and the histidine donation of the proton to allow release of the first peptide. Serine proteases also use charge stabilization (the oxyanion hole) to lower the energy of the transition state and use covalent catalysis to assist in the reaction.

Figure 2.7: Mechanism of action of Serine Protease

Serine proteases are very useful as model enzymes for studying catalytic processes, because they use a variety of techniques for accelerating the reaction rate. They use acid-base catalysis; the histidine abstraction of the serine proton, and the histidine donation of the proton to allow release of the first peptide. Serine proteases also use charge stabilization (the oxyanion hole) to lower the energy of the transition state and use covalent catalysis to assist in the reaction.

42

2.4.1.3.2 Aspartic Proteases Aspartic proteases are a family of eukaryotic protease enzyme that utilizes an aspartate residue for catalysis of their peptide substrates. In general they have two highly conserved aspartates in the active site and are active at acidic pH. Suguna et al., 1987 proposed a general mechanism for peptide cleavage by aspartyl protease [Fig. 2.8]. While a number of different mechanisms for aspartyl proteases have been proposed, the most widely accepted is a general acid base mechanism involving coordination of water molecule between the two highly conserved aspartate residues. One aspartate activates water by abstracting a proton, enabling the water to attack the carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate. Rearrangement of this intermediate leads to protonation of the scissile amide.

Figure 2.8: Mechanism of action of Peptide cleavage by Aspartic Protease

2.4.1.3.3 Metalloproteases Metalloproteases constitute a family of enzymes from the group of proteinases, classified by the nature of the most prominent functional group in their active site. The mechanism of the action of metalloproteases is slightly different from that of the above described proteases. These enzymes depend on the presence of bound divalent cations. Kester and Matthews, 1977 suggested an acid base catalysis for metalloproteases [Fig. 2.9], by taking interaction between water molecule and the Zn+2 ions [329].

43

Figure 2.9: Mechanism of action of Metallo Protease

Manzetti et al., 2003 provided evidence that a coordination between water and the zinc, where in a histidine from the HExxHxxGxxH-motif participates in catalysis by allowing the Zn+2 ions to assume a quasi-penta coordinated state, via its dissociation from it [330]. In this state, the Zn+2 ion is coordinated with two oxygen atoms from the catalytic glutamic acid, the substrates carbonyl oxygen atom and the two histidine residues and can polarize the glutamic acids oxygen atom, proximate the scissile bond, and induce it to act as reversible electron donor. This forms an oxyanion transition state. At this state water molecules act on the dissociated scissile bond and complete the hydrolyzation of the substrate. 2.4.1.3.4 Cysteine proteases Cysteine proteases catalyze the hydrolysis of carboxylic acid derivatives through a doubledisplacement pathway involving general acid-base formation and hydrolysis of an acyl-thiol intermediate. The mechanism of action of cysteine proteases is thus very similar to that of serine proteases.

Figure 2.10 : Mechanism of action of Cysteine Protease

44

A striking similarity is also observed in the reaction mechanism for several peptidases of different evolutionary origins. The plant peptidase papain can be considered the archetype of cysteine peptidases and constitutes a good model for this family of enzymes. They catalyze the hydrolysis of peptide, amide ester, thiol ester, and thiono ester bonds [331]. The initial step in the catalytic process (Figure, where Im and + Him refer to the imidazole and protonated imidazole respectively) involves the noncovalent binding of the free enzyme and the substrate to form the complex. This is followed by the acylation of the enzyme, with the formation and release of the first product, the amine R-NH2. In the next deacylation step, the acyl-enzyme reacts with a water molecule to release the second product, with the regeneration of free enzyme [Fig. 2.10]. The enzyme papain consists of a single protein chain folded to form two domains containing a cleft for the substrate to bind. The crystal structure of papain confirmed the Cys25- His159 pairing [332]. The presence of a conserved asparagine residue (Asn175) in the proximity of catalytic histidine (His159) creating a Cys-His-Asn triad in cysteine peptidases is considered analogous to the Ser-His-Asp arrangement found in serine proteases. Studies on the mechanism of action of proteases have revealed that they exhibit different types of mechanisms based on their active-site configuration. The serine proteases contain a Ser-His-Asp catalytic triad, and the hydrolysis of the peptide bond involves an acylation step followed by a deacylation step. Aspartic proteases are characterized by an Asp-Thr-Gly motif in their active site and by acidbase catalysis as their mechanisms of action. The activity of metalloproteases depends on the binding of a divalent metal ion to a HExxHxxGxxH-motif. Cysteine proteases adopt a hydrolysis mechanism involving a general acid-base formation followed by hydrolysis of an acyl-thiol intermediate.

2.4.1.4 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 arrangement, morphogenesis in development, inflammation, tumor growth

45

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, where as intracellular proteases play a critical role in the regulation of metabolism. Some of the major activities in which the proteases participate are described below. 2.4.1.5 Protein Turnover 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 [333]. The turnover of intracellular proteins in eukaryotes is also affected by a pathway involving ATP-dependent proteases [334]. Evidence for the participation of proteolytic activity in controlling the protein turnover was demonstrated by the lack of proper turnover in protease-deficient mutants.

2.4.1.6 Sale of Proteases and other enzymes The present estimated value of the worldwide sales of industrial enzymes is $1 billion [335]. Hydrolytic enzymes contribute 75 % of total enzyme sale including protease, carbohydrase and lipase. Proteases represent one of the three largest groups of industrial enzymes and account for about 59% [Fig. 2.11] of the total worldwide sales of enzymes.

46

Other Protease , 21%

Amylase, 18%

Rennin, 10%

Carbohydrase, 10% Lipase, 3%

Trypsin, 3% Pharmaceutical Enzyme, 10% Alkaline Protease, 25% Figure 2.11: Distribution of enzyme sales. Yellow portion indicates the total sales of proteases.

2.4.2 Keratinase Keratinase is a protease capable of digesting keratins in chicken feathers as well as animal wool and hair. Proteases are by and large classified into two major groups based on their cleavage habits. First group is called Endopeptidases which cleaves non-terminal peptide bonds inside polypeptide chains. Second group so-called Exoproteases breaks down peptide bond at the amino termini (aminopeptidases) or at the carboxy termini (carboxypeptidases) of their substrates. Proteases are further categorized based on functional groups of their active sites. Four major groups are: serine proteases, cysteine proteases, aspartic proteases, and metalloproteases. Keratinases are mostly known to be endopeptidase which is a member of serine protease family [103]. Keratins are less likely to be digested by enzyme such as trypsin, pepsin, and papain [104] because the stiff packing of the protein chain in -helix and -sheet structures resists andmechanically stabilizes the keratin to microbial degradation. However, keratin can be degraded by a number of species of saprophytic and parasitic fungi, a few actinomyces and Bacillus species [105]. Keratins proteolysis like the other proteins is effectively directed by proteases. Nevertheless, keratinases are known to have an effect on their hydrolysis [94]. Keratinases have already been 47

purified from several microorganisms such as fungi, a few bacteria, and some Streptomyces species [105]. Keratinase belongs to a group of proteinase enzymes that have high level of activity on insoluble keratin, playing a crucial role in hydrolyzing feather, hair, wool, collagen and casein in removing barriers in waste water treatment systems. Not only have these enzymes been applied in sewage systems but have also recently emerged in many applications including food, textile, medicine, and cosmetics industries. In fact, using of keratinases in skin medications to get rid of acne and psoriasis as well as removing of human callus in medical applications is well known. It is also utilized for the erection of a vaccine for dermatophytosis therapy [94, 103]. More interestingly, keratinases are well identified in leather industry to have been employed in dehairing process of animal skins instead of treating them with sodium sulfide [327]. The majority of known keratinases are endopeptidases belonging to the serine protease family. Amino acid sequences of several Bacillus keratinases are known to show striking sequence homology to Carlsberg subtilisin (E.C. 3.4.21.62), a well-described member of the serine protease family. All three catalytic active sites (Asp32, His64, Ser221) characteristic to subtilisins can be identified in the primary sequence of keratinases. Subtilisins and related extracellular proteases bear a triad of 'pre', 'pro' and 'mature' regions. The N-terminal 'pre' part serves as a signal sequence directing the translocation of the newly synthesized precursor molecules through the cell membrane. The adjacent 'pro' region acts as an intramolecular chaperone that promotes the correct fold of the protease domain and is a prerequisite for the protease maturation. In the last step of maturation, the enzyme is activated via an autocatalytic removal of the 'pro' region. Kinetic parameters of Bacillus licheniformis KK1 keratinase and Carlsberg subtilisin were determined and compared using a set of para-nitroaniline (pNA) conjugated oligopeptides as substrates [210]. Both enzymes showed similar kinetics with most of the oligopeptide substrates, preferentially cleaving next to hydrophobic and aromatic residues. The nearly identical protein sequence and the similar biochemical characteristics suggest a tight relationship between keratinases and subtilisins isolated from Bacillus strains. 2.4.2.1 Sources of microbial keratinases: Diversity among keratinase-producing microorganisms Keratinases are very widespread in the microbial world and they can be identified from

48

microorganisms of the three domains: Eucarya, Bacteria, and Archaea. These microorganisms have been isolated from the most distinct sites, from Antarctic soils to hot springs, including aerobic and anaerobic environments. Therefore, microbial keratinases present a great diversity in their biochemical and biophysical properties. The characteristics of some microbial keratinases are summarized in Table 2.4. In natural environments, keratinolytic fungi are involved in recycling the carbon, nitrogen, and sulfur of the keratins. Their presence and distribution seem to depend on keratin availability, especially where humans and animals exert strong selective pressure on the environment. A number of studies focused on the keratinolytic potential of dermathophytic fungi such as Trichophyton and Microsporum, mainly due to their medical and veterinary implications. Although some studies on the biotechnological potential of such genera are available [26], little commercial interest was attracted by this group because of their potential pathogenicity. Among nondermatophytic fungi, keratinases showing attractive biochemical properties were reported to be produced by Aspergillus, Trichoderma, Doratomyces, Myrothecium, Paecilomyces, Scopulariopsis, and also Acremonium, Alternaria, Beauveria, Curvularia, and Penicillium. Besides the biotechnological interest, these investigations may help in understanding the role of fungi in the degradation of complex keratinous substrates in thenature. Several keratinases have been isolated from a diversity of bacteria. Bacillus spp. appears as the prominent keratinase producer. Diverse strains of Bacillus licheniformis and Bacillus subtilis are described as keratinolytic [350, 70, 351, 73, 352], but other species such as Bacillus pumilus and Bacillus cereus also produce keratinases [324, 296, 198]. Furthermore, B. licheniformis [350] is the source of Versazyme, the first thermo-resistant commercial keratinase developed by Shih and coworkers at Bioresource International, Inc. Some thermophilic and alkaliphilic strains of Bacillus have also been described to show keratin-degrading activity, such as Bacillus halodurans AH-101, Bacillis pseudofirmus AL-89, and B. pseudofirmus FA30-01 [353]. Besides, microorganisms belonging to the same genus (Bacillus) can produce different keratinases (Table 2.4). In this regard, the exploitation of microbial diversity might provide keratinases with suitable properties for biotechnological uses. For instance, the keratinolytic potential and eratinolytic enzymes from novel mesophilic Bacillus species isolated from the Amazon basin

49

have been recently characterized in our laboratory [354], presenting interesting features for diverse potential applications. Keratinase producers have been also described among actinomycetes, mainly from the Streptomyces genus. These microorganisms, isolated from several different soil sites, are associated with the hydrolysis of a wide range of keratinous substrates like hair, wool, and feathers. For example, two highly keratinolytic actinomycete strains, Streptomyces flavis 2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic), were isolated from Antarctic soil. Thethermophilic species Streptomyces gulbarguensis [30], Streptomyces thermoviolaceus, and Streptomyces thermonitrificans have also been isolated from soils. Besides these thermophilic strains, some mesophilic Streptomyces have also been characterized like Streptomyces pactum DSM 40530 [33], Streptomyces graminofaciens [31] and Streptomyces albidoflavus K1-02 [54]. In addition to these Bacillus sp. and actinomycetes, keratinase production has been associated to an increasing number of bacteria. Since keratin degradation is facilitated at high temperatures and pH, and thermostable hydrolases are employed in various industrial processes, the thermophilic and alkaliphilic microorganisms are of great interest. Fervidobacterium pennavorans, Fervidobacterium islandicum, Meiothermus ruber H328, Clostridium sporogenes, and strains of Thermoanaerobacter sp. were isolated from extreme environments like hot springs, geothermal vents, solfataric muds, and volcanic areas. Some alkaliphilic strains such as Nesternkonia sp. and Nocardiopsis sp. TOA-1 have been also characterized, showing keratinase activity in strongly alkaline pH. The investigation of keratinolytic bacteria isolated from soils has revealed a high and undescribed diversity. For example, a single soil site revealed several keratinolytic isolates related to Bacillus, Cytophagales, Actinomycetales, and Proteobacteria. Several feather-degrading bacterial strains have been isolated from soil, poultry wastes, and other sources, and characterized as mesophilic keratinase producers. These include some Gram-positive, such as Lysobacter NCIMB 9497, Kocuria rosea, and Microbacterium sp. kr10 [271], and a few Gram-negative, such as Vibrio sp. [60], Xanthomonas maltophilia, Stenotrophomonas sp., Chryseobacterium sp. [362, 70], and Serratia sp. Many Archaea grow in environments usually lethal to most cells, including extremes in temperature, pH, salt content, and pressure. Thus, Archaea are valuable resource of proteases for fundamental

50

microbiology and enzymology studies, also possessing the potential for biotechnological applications. Archaea displaying keratinolytic activities were recently revealed through the in situ enrichment of thermophilic prokaryotes with hydrolytic activities in hot springs (68-87C and pH 4.1-7.0). One isolate, identified as 1507-2, grew on -keratin at 70 C and pH 6.0, and was found to be an archaeon of the Crenarchaeota phylum, representing a cluster of the socalled unknown Desulfurococcales. In the same investigation, a 220-kDa thermostable keratinase showing broad pH (6.0 to 10.0) and temperature (30 to 80C) ranges of activity, with an optimum at pH 7.0 and 66C, was found in the culture supernatant of strain 1523-1 growing on keratin.

Table 2.4: Diversity of keratinolytic microorganisms and some biochemical properties of their keratinases
Microorganism Catalytic type Molecular mass (kDa) Optimal pH Optimal T (C) Reference

Bacteria Bacillus sp. SCB-3 B. cereus DCUW B. licheniformis FK14 B. licheniformis K-508 B. licheniformis MSK103 B. licheniformis PWD-1 B. licheniformis RPk B. pumilis B. subtilis KD-N2 B. subtilis KS-1 B. subtilis MTCC (9102) B. subtilis RM-01 Clostridium sporogenes Chryseobacterium sp. kr6 Chryseobacterium indologenes TKU014 Metallo Metallo Fervidobacterium islandicum AW-1 Fervidobacterium pennavorans Kocuria rosea Kytococcus sedentarius Lysobacter sp. NCIMB 9497 Microbacterium sp. kr10 Nesternkonia sp. AL-20 Nocardiopsis sp. TOA-1 Stenotrophomonas maltophilia

Metallo Serine Serine Thiol Serine Serine Serine Serine Serine Serine Metallo Serine Metallo Metallo P2: 40 P3: 40 Serine Serine Serine Serine Metallo Metallo Serine Serine Serine

134 80 35 42 26 33 32 65 30.5 25.4 69 20.1 28.7 64 P1: 56 P2: 7-8 P3: 8-9 >200 130 240 30-50 148 42 23 20 35.2

7 8.5 8.5 8.5 9-10 7.5 9.0 8.0 8.5 7.5 6 9 8 8.5 P1: 10 P2: 40 P3: 40-50 9 10 10 7-7.5 7.5 10 >12.5 7.8

40 50 60 52 60-70 50 60 65 55 40 45 55 50 P1: 30-50

Lee et al. 2002 Ghosh et al. 2008 Suntornsuk et al. 2005 Rozs et al. 2001 Yoshioka et al. 2007 Lin et al. 1992 Fakhfakh et al. 2009 Kumar et al. 2008 Cai et al. 2008b Suh and Lee 2001 Balaji et al. 2008 Rai et al. 2009 Ionata et al. 2008 Riffel et al. 2007 Wang et al. 2008a

100 80 40 40-50 50 50 70 60 40

Nam et al. 2002

Friedrich and Antranikian 1996

Bernal et al. 2006a Longshaw et al. 2002 Allpress et al. 2002 Thys et al 2006 Gessesse et al. 2003 Mitsuiki et al. 2004 Cao et al. 2009

51

Streptomyces sp. S7 Streptomyces sp. strain 16

Serine-metallo Serine Serine Serine Serine Serine Serine Serine Serine Serine Serine Metallo Serine Serine Serine Serine Serine Serine Serine Serine

44 KI: 203.2 KII: 100.8 KIII: 31.8 KIV: 19.2 18 30 46 40 150 135 36 60 30-33 22 33 36-39 21 38-41 38 34 37

11 KI: 9 KII: 9 KIII: 9 KIV: 9 6-9.5 7-10 9 8 9.3 8 8 6.5-9 8 8-9 8.3 8.0 8.0 8-9 4.5 5.5 7.8 8.0

45 KI: 50 KII: 50 KIII: 50 KIV: 60 40-70 40-75 45 55 60 85 60 45 50 50 37 60-65 40 50-60 50 40 -

Tatineni et al. 2008 Xie et al. 2010

Streptomyces albidoflavus Streptomyces pactum Streptomyces gulbagensis DAS 131 Streptomyces thermoviolaceus Thermoanaerobacter sp. 1004-09 Thermoanaerobacter keratinophilus Xanthomonas maltophilia Fungi Aspergillus fumigatus Aspergillus oryzae Doratomyces microsporum Myrothecium verrucaria Paecilomyces marquandii Scopulariopsis brevicaulis Trichoderma atrvoviride F6 Trichophyton mentagrophytes Trichophyton schoenleinii Trichophyton sp. H-2 Trichophyton vanbreuseghemii

Bressollier et al. 1999 Bckle et al. 1995 Syed et al. 2009 Chitte et al. 1999 Kublanov et al. 2009a
Riessen and Antranikian 2001

De Toni et al. 2002 Santos et al. 1996 Farag and Hassan 2004 Gradisar et al. 2005
Moreira-Gasparin et al. 2009

Gradisar et al. 2005 Anbu et al. 2005 Cao et al. 2008 Tsuboi et al. 1989 Qin et al. 1992 Anbu et al. 2008 Moallaei et al. 2006

2.4.2.2 Biochemistry of keratinases 2.4.2.2.1 General biochemical characteristics The properties of microbial keratinases may be diverse depending on the producer microorganism. These enzymes are predominantly extracellular, although cell-bound and intracellular enzymes have been described [371]. Some general biochemical characteristics of selected keratinases are presented in Table 2.5. Most of the microbial keratinases are alkaline or neutral proteases showing optima pH ranging 7.5-9.0. However, some enzymes are optimally active outside this range, even at extreme alkalophilic pH or at slightly acidic pH [325]. A feature showed by several keratinases is the stability over a wide pH range [371]. This property is remarkable for the keratinase of Nocardiopsis TOA-1, which is stable over a pH range of 1.5 to 12.0 for 24 h at 30C. Increased stability has been recently achieved by recombinant keratinases, such as the B. licheniformis MKU3 keratinase expressed in Pichia pastoris X33 [43].

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The temperature optima of keratinases may also be very variable, often depending on the source and origin of the isolate (Table 2.5). The enzyme of the thermophilic F. pennavorans has optimum temperature at 80C while the mesophilic Stenotrophomonas maltophila DHHJ showed maximum activity at 40C. In some exceptional cases, as for F. islandicum AW-1, the optimum of 100C has been reported Nam et al. 2002. The molecular masses of several keratinases have been determined. Despite they range from 18 to 240 kDa for S. albidoflavus and K. rosea [54], respectively, most keratinases have less than 50 kDa (Table 2.5). The majority of keratinases are monomeric enzymes; however, multimeric keratinases are also reported Nam et al. 2002; Xie et al. 2010. Higher molecular masses are often associated to keratinases with metalloprotease character or those from thermophilic organisms (Table 2.5). The effect of metal ions and inhibitors on keratinolytic activity has been also investigated. The results of several studies have been recently compiled [331], showing as a general trend that the presence of divalent metal ions like Ca2+, Mg2+, and Mn2+ often stimulate the keratinases. This positive effect might be related to the maintenance of the active enzyme conformation, and to the stabilization of the enzyme-substrate complex [361]. Additionally, metal ions may protect the enzyme against thermal denaturation [367, 327]. In subtilisin-related keratinases, for instance, interaction of calcium with specific Ca2+-binding sites or autolysis sites may explain the improved thermostability in the presence of such metal ions [41]. On the other hand, transition and heavy metals like Cu2+, Ag+, Hg2+, and Pb2+ generally caused inhibition of keratinolytic enzymes. Organic solvents, detergents, and reducing agents have diverse effects on different keratinases. However, a tenden-cy observed in several investigations is the stimulation of the keratinolytic activity by reducing agents. Such effect is usually attributed to the reduction of cysteine bridges in the keratinous substrate rather than direct effects on the enzyme [72].

2.4.2.2.2 Structure and catalysis of keratinase The primary sequence of some keratinases was determined or deduced from its coding genes. This information allows comparing keratinases with other previously characterized proteases. The N-terminal sequence of B. licheniformis PWD-1 keratinase is identical to Carlsberg

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subtilisin, which is a typical member of the serine-type proteases. Its coding gene kerA presents elevated similarity and homology with the subtilisin Carlsberg and the deduced amino acid sequence differs by only five amino acids [324]. Further investigation revealed that the deduced sequence of kerA exactly agrees with those of substilisins Carlsberg (NCIMB6816), NCIMB10689 and ATCC12759, except for an arginine instead of lysine at position 144 and a valine instead of alanine at position 222 [209]. Since V222 is close to S220 at the catalytic site, this change may be related to the enhanced keratin hydrolysis. Keratinase KerRP from B. licheniformis RPk, consisting of 274 amino acids, showed 99% homology with kerA, and 98% with subtilisin Carlsberg, differing from kerA by 2 amino acids (K144 and A222 in KerRP and R144 and V222 in kerA) and from subtilisin Carlsberg by four amino acids (S102, A128, K144, and N211 in KerRP and T102, P128, R144, and S211, respectively, in subtilisin Carlsberg), but conserving the active site residues D32, H63, and S220 [41]. Likewise, a keratinase from B.licheniformis MKU3, conserving the amino acid residues that form the catalytic triad, showed 99% similarity with both kerA and subtilisin Carlsberg. Additionally, the amino acid sequence of a keratinase from B. licheniformis MSK103 showed homology of approximately 87% with kerA. The complete nucleotide sequencing of the gene encoding for a high-molecular-mass extracellular feather-degrading protease (Vpr) from B. cereus DCUW allowed the analysis of its structural domains: initial amino acids (1-25) encoded for a putative N-terminal extracellular signal sequence for secretion, amino acids 62-158 encoded for a subtilisin N sequence that is signature for N-terminal processing, amino acids 172-583 encoded for a catalytic peptidase S8 domain, and amino acids 606-917 encoded a Vpr-specific protease-associated domain. Similar to B. licheniformis, the main keratinolytic activity from B. subtilis is often associated with serine protease activity. The gene aprA cloned from a featherdegrading strain of B. subtilis showed significant similarity and homology with subtilisins. Despite the limited information on purified keratinases from B. subtilis, the subtilisin-like character appears to predominate. A keratinolytic protease purified from B. subtilis KS-1 showed an Nterminal sequence similar to that of other serine proteases of B. subtilis [69]. A novel keratinase from B. subtilis S14, showing no activity on collagen and an excellent dehairing activity, presents an identical N-terminal sequence to subtilisin E [28]. This enzyme also

54

showed an internal sequence VAVIDSGLDSSHPDLNVR that is identical to that of a bacterial alkaline subtilisin and to that of nattokinase [28]. The thermophilic feather-degrading bacterium F. penna-vorans produces the keratinase fervidolysin. The primary sequence of fervidolysin, deduced from the coding gene fls, showed high homology with the subtilisin-like proteases. Although the N-terminal sequence of fervidolysin is very distinct to that of subtilisins, its active site region was similar to subtilisin-like serine proteases. More detailed information on the configuration of fervidolysin was obtained from the 1.7 resolution crystal structure [298]. Fervidolysin is composed of four domains: a catalytic domain, two beta-sandwich domains, and the propeptide domain. The architecture of the catalytic domain closely resembles that of a subtilisin, and a calcium-binding site was observed within the catalytic domain, which exactly matches that of the subtilisin E-propeptide domain complex [298]. The N-terminal sequences of some fungal keratinases have been also described. These also present significant similarity with serine proteinases. The comparison of their N-terminal sequences indicates a closer relationship with those of subtilisins than that of keratinases from actinomycetes (Fig. 2.12). Indeed, the keratinases from Streptomyces appear to be close to Streptomyces griseus proteinase B (SBPG), the major component of pronase [367]. More recently, information on protein sequences of keratinolytic metalloproteases became available. A kerati-nolytic metalloprotease purified from Chryseobacterium sp. kr6 belongs to the M14 family of peptidases, also known as the carboxypeptidase A family [362]. This is the first keratinolytic enzyme associated to this family. The enzyme exhibit an O-glycosylation site DS* in the peptide 5 (KGSSADS*PNSEEK), that is usually found in proteins secreted by the related species Chryseobacterium meningosepticum. Flavastacin, an extracellular metalloprotease from C. meningosepticum presents a heptaglycoside linked to the DS* site, which may be associated with protection against autoproteolysis. Peptide sequences of three keratinases from C. indologenes were recently determined by mass spectrometry showing no significant homology to any other reported microbial peptide [360]. The complete DNA and amino acid sequences of a keratinolytic metalloprotease from Pseudomonas aeruginosa was also recently reported [324], and its N-terminal sequence showed only little homology with other microbial keratinases (Fig. 2.12). The catalytic type of many keratinases has been determined by using specific

55

substrates and inhibitors. The specificity of some keratinases is illustrated in Fig. 2. The chymotrypsin-like keratinase from S. albidoflavus exhibited specificity for aromatic and hydrophobic amino acid residues, as demonstrated by using synthetic peptides [367]. S. pactum DSM 40530 produces a keratinolytic serine protease that showed substrate specificity and stereospecificity to p-nitroanilide (pNA) derivatives of aromatic and basic amino acids lysine and arginine (L-enantiomers), but hydrolysis of benzoyl-D-Arg-pNA was not detected. The keratinase NAPase of Nocardiopsis sp. TOA-1, composed by 188 amino acids and conserving the catalytic triad of the active site of serine proteases (H35, D61, and S143), showed preference for aromatic and hydrophobic residues at the P1 position of synthetic pNa substrates; structural similarities suggest that APase might be categorized as a chymotrypsin-type serine protease of Streptomyces strains. An alkaline protease from the feather-degrading Nesterenkonia sp. AL20 also exhibited higher activity with tetrapeptides with hydrophobic residues located at P1 site. B. subtilis S14 keratinase showed preference for Arg at P1, small amino acids like Ala and Gly at P2, and Gln or Glu at P3 [28]. Besides the well-characterized serine-type keratinases from B. licheniformis, other keratinolytic proteases with atypical properties have been associated to this species. B. licheniformis strain HK-1 produces a keratinolytic protease that was partially inhibited by EDTA, 1,10-phenanthroline (Zn2+ specific chelator), PMSF, and Zn2+ [81]. The keratinolytic B. licheniformis K-508 secreted an unusual trypsin-like thiol protease that is strongly active towards benzoyl-Phe-Val-Arg-pNA and is not inhibited by PMSF [82]. The purified keratinase of K. rosea was strongly inhibited by 4-(2-aminoethyl) benzene-sulfonyl fluoride, soybean trypsin inhibitor, and chymostatin, suggesting that it belongs to the serine protease family. The keratinolytic metalloprotease from the Gram-negative bacterium Lysobacter sp. was strongly active towards carboxybenzoyl-Phe-pNA. The keratinase produced by Chryseobacterium sp. Kr6 appears to belong to the metalloprotease type since it was inhibited by EDTA and 1, 10-phenanthroline and lacks hydrolysis of the substrate benzoyl-L-ArgpNA [362]. The enzyme was strongly active on L-Leu-(amino-4-methylcoumarin) (AMC) and N-tBoc-Ile-Glu-Gly-Arg-AMC, and also active on L-Phe-AMC and L-Val-AMC, showing preference for hydrophobic and positive amino acids. Activation by Ca2+ and inhibition by excess Zn2+ of Gram-negative keratinases [366, 362] resembles typical bacterial metalloproteases like

56

Figure 2.12: Substrate specificity of some keratinases. KerA from B.licheniformis PWD-1 (Evans et al. 2000), keratinase K-508 from B. licheniformis (Rozs et al. 2001), keratinase S14 from B. Subtilis (Macedo et al. 2008), SAKase from S. albidoflavus K1-02 (Bressollier et al. 1999), keratinase Sp from S. pactum (Bckle et al. 1995), SFase-2 from S. fradiae ATCC 14544 (Kitadokoro et al. 1994), keratinase AL20 from Nesterenkonia sp. (Bakhtiar et al. 2005), NAPase from Nocardiopsis sp. (Mitsuiki et al. 2004), keratinase Pm from P. marquandii, and keratinase Dm from D. microsporus (Gradisar et al. 2005), keratinase Tv from Trichophyton vanbreuseghemii (Moallaei et al. 2006), keratinase 1004-09 from Thermoanaerobacter sp. (Kublanov et al. 2009a), keratinase kr6 from Chryseobacterium sp. (Silveira et al. 2009), keratinase L from Lysobacter sp. (Allpress et al. 2002), keratinase kr10 from Microbacterium sp. kr10 (Thys and Brandelli 2006), keratinase Pa from P. aeruginosa (Lin et al. 2009), and fervidolysin from F. pennivorans (Kluskens et al. 2002). Superscript letter the enzyme showed higher affinity for Suc-Ala-Ala-Pro-Leu-pNa. Superscript letter b the enzyme specificity was also high on Suc-Ala-Ala-Pro-Phe-pNA. Superscript letter c the enzyme was also highly active on N-t-Boc-Ile-Glu-Gly-Arg-AMC. Superscript letter d obtained from the 3D modeling of the catalytic site (Kluskens et al. 2002)

thermolysin and extracellular proteases from Pseudomonas species. Keratinolytic proteases are

57

largely classified as serine or metallo proteases and, independent of their catalytic type, it seems that hydrophobic and aromatic aminoacid residues are preferably cleaved at the P1 position (Fig. 2.12), resembling the specificity of chymotrypsin. The presence of Arg at P1 appears to be preferentially cleaved only by some keratinases from Bacillus sp. [82, 28] and Chryseobacterium sp., a preference also showed by trypsin. In this sense, the specificity of keratinases towards keratinous materials may arise from the amino acid composition of keratins, which contains about 50-60% of hydrophobic and aromatic residues [353, 59]. Additionally, the nature of amino acids at the vicinity of the cleaved bond was showed to influence the specificity for the P1 position, which might indicate the presence of an extended active site [367, 28]. For instance, substitution of Ala residue of Suc-Ala-Ala-Ala-pNA by Pro at the P2 position increased the kcat/Km value of NAPase from Nocardiopsis sp. TOA-1 by 37fold. The preference for longer substrates at both sides of the scissile peptide bond suggests the suitability of eratinases for the conversion of native and complex substrates. The cleavage of peptide bonds in the compact keratin molecules is difficult due to the restricted enzyme-substrate interaction on the surface of keratin particles and accessibility to splitting points. Therefore, the hydrolyzing ability of keratinolytic proteases may be due to its ability and specificity to bind onto compact substrates, and a more exposed active site. NAPase from Nocardiopsis sp. TOA-1 showed a strong adsorption capability (more than 70% of added enzyme) towardskeratin, obeying a Langmuir-type adsorption isotherm, which was likely due to the presence of an efficient binding pocket for keratin. In the Vpr protease from B. cereus DCUW, the C-terminal domain of the enzyme has the ability to noncovalently bind specific substrates like feather keratin via protein-protein interactions. Keratinase adsorption to fibrous keratin was previously showed to occur through electrostatic interactions [367].

2.4.2.2.3 Hydrolysis of native proteins Keratinases are mostly endo-proteases showing a broad spectrum of activity [270], usually hydrolyzing soluble proteins (such as casein) more effectively than insoluble proteins (such as keratins; Lin et al. 1992; Suh and Lee 2001; Brandelli 2005; Suntornsuk et al. 2005; Syed et al. 2009) [324, 363, 270, 361, 30]. Only few microbial keratinases show higher hydrolysis of keratins than soluble proteins [325]. Besides some exceptions, purified keratinolytic enzymes are

58

often ineffective to hydrolyze native keratin [367, 371, 362], a behavior that is mainly attributed to the high levels of disulfide bonds in the keratin molecules. Keratin hydrolysis by microorganisms is reported to not simply rely on the production of keratinolytic proteases. Release of thiol groups during microbial growth on keratinous materials supports the essential role of the reduction of disulfide bonds for efficient keratin degradation [276, 328]. Production of intra- and/or extracellular disulfide reductases [80, 217], release of sulfite and thiosulfate [331], and also a cell-bound redox system [331, 86] are reported to lead to sulfitolysis. In fungi, keratinolysis also seems to involve the mechanical attack of the keratinous substrates through mycelial pressure and/or penetration [265]. Since the main efforts are focused on the characterization of keratinolytic proteases, components responsible for the reduction of disulfide bonds are probably removed during the purification procedures. Thus, the keratinolysis process seems to involve, at least, sulfitolysis and proteolysis. The importance of disulfide bonds to the recalcitrance of the keratin structure is emphasized by the stimulation of keratin hydrolysis by purified keratinases through the addition of reducing agents that promote sulfitolysis [369, 271, 72, 181]. The cut of disulfide bonds changes the conformation of keratins and more sites for keratinase action are exposed. Production of free thiol groups is not observed during the hydrolysis of keratinous substrates, indicating that the breakdown of disulfide bonds is not accomplished by keratinases themselves [324, 270, 361, 181]. Therefore, a suitable redox environment is necessary for effective keratin degradation by keratinases, and even by other proteases [331]. pidermal soft -keratins such as that from stratum corneum, which possess a low level of disulfide bonds in comparison to hair and wool hard -keratins, are generally more susceptible to hydrolysis by keratinases [270]. Another observed trend is the higher hydrolysis of feather -keratins when compared to hard keratins from hair and wool [270, 29]. Cysteine residues, responsible by formation of disulfide bonds, are present at higher concentrations in wool 10.5-17% [59, 330] than in feather -keratin 4.2-7.6% [353, 31]. In -keratin, the polypeptide chains are closely associated pairs of helices, whereas -keratin has high proportions of -pleated sheets. These structural features provide a more extended conformation for -keratins in comparison to -keratins [61], which could result in the enhanced keratinase accessibility to the former. In this context, a keratinase

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from Chryseobacterium sp. kr6 was reported to hydrolyze keratinous substrates in the following order: stratum corneum from human sole > porcine skin > chicken feathers > chicken nails > wool > hair keratin [270]. Other microbial keratinases are also reported to hydrolyze both and -keratins [72]. A keratinase from B. pseudofirmus FA30-01 was able to degrade feather, but was poorly active towards wool and hair [327], similar to that observed for B. subtilis P13 [29] and B. subtilis RM-01 keratinases [233]. Contrarily, keratinases from Doratomyces microsporus and Paecilomyces marquandii hydrolyzed -keratins from skin, nail, and hair, but not keratins from chicken feathers. Therefore, other properties, such as fibril structure and porosity [61], might dictate the differential hydrolysis of these substrates. Although some inferences on the keratinolysis mechanism can be drawn, the elucidation of this complex process needs to be supported by conclusive experimental data. The hydrolysis of feather keratin by free and immobilized keratinase from B. licheniformis PWD-1 yielded a major peak of 18 kDa in size-exclusion HPLC, which increased with the reaction time from 1 to 10 h [70]. A single peak was also observed during hydrolysis of feather keratin by Chryseobacterium sp. kr6 keratinase [270]. Such pattern might indicate that feather keratin was hydrolyzed at specific cleavage site(s) [70]. In summary, the multiplicity of catalytic mechanisms observed for microbial keratinases (including serine, thiol, and metalloproteases) could have an important effect in the natural environment, where the synergistic action of keratinolytic microorganisms and its enzymes and metabolites may degrade the recalcitrant structure of native keratin more easily than a pure culture. After the initial attack by keratinases and disulfide reductases, other less specific proteases may also act on the substrate, resulting in extensive keratin degradation.

2.4.2.3 Production of keratinases The biotechnological application of keratinolytic proteases requires the production of these enzymes in sufficient amounts for commercial purposes. Keratinase production is usually induced by keratin [26, 27] and, thus, a keratinous substrate (chicken feathers, feather meal, hair) is often added to the cultivation medium. Such keratin-rich materials are produced in high amounts by agroindustrial activities and are normally discarded as wastes. Therefore, this microbial technology connects the production of valuable products (keratinases, microbial biomass, protein hydrolysates) from low-

60

cost substrates with an alternative and efficient way of waste management [270, 359, 328]. However, the addition of a keratinous substrate is not always required for keratinase production [352, 298]. Other non-keratinous substrates, such as soy flour, soybean meal [29], skim milk, shrimp shell powder [360], gelatin [271], casein, and cheese whey [60], have been reported to act as inducers of keratinase production. Furthermore, in some cases, keratinase production appears to be constitutive [82, 298]. Recently, peptide limitation in culture media induced the sequential production of collagenase, elastase, and keratinase by B. cereus IZ-06b and IZ-06r. The keratinolytic activities produced on keratinous substrates, in comparison to readily assimilable substrates, may result from nitrogen limitation rather than keratin induction. In this case, keratinous substrates would act only as indirect inducers. Supplementation of keratin-containing media with different carbon and/or nitrogen sources might result in higher levels of keratinase production. For instance, the addition of glucose [80, 26], sucrose [27], starch [327, 30], molasses [352], and bagasses; and additional nitrogen sources, such as urea, peptone, tryptone, yeast extract, ammonium chloride, and sodium nitrate are reported to enhance enzyme yields [331, 27]. Conversely, the addition of supplementary substrates carbohydrates; inorganic and/or organic nitrogen sources often decrease enzyme production by some microorganisms, mainly due to catabolite repression mechanisms [270, 276]. Therefore, the effect of different growth substrates on keratinase production is highly variable, depending on the microorganism, the substrate and the carbon and nitrogen concentration, implicating that the medium composition should be determined on a case-by-case basis [270]. Besides the composition of the culture medium, incubation temperature, pH, and aeration are among the important variables investigated in view to obtain high keratinase yields Maximum keratinase activities are usually achieved in the late exponential or stationary growth phases [271, 371, 327, 385]. In this sense, keratinase production was bserved to be growth-associated in B. licheniformis FK 14 [361]; similar results were observed with Chryseobacterium sp. kr6 [270] and Streptomyces gulbargensis DAS 131 [30]. Nevertheless, Serratia sp. HPC 1383 showed the highest proteolytic activity in the initial phase of growth (24 h) on feather meal medium, whereas maximum biomass was achieved after 96 h. Development of mutant and recombinant microbial strains is also investigated, representing useful techniques to enhance keratinase production and keratin degradation [72, 251]. In the specific case of the opportunistic pathogen P. aeruginosa, cloning and

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heterologous expression of its keratinase gene also represents a viable alternative to ensure safety [324]. The gene kerA, which encodes a B. licheniformis keratinase, is expressed specifically for feather hydrolysis [324]; therefore, the presence of feather keratin as the sole carbon and nitrogen source in the culture medium may result in preferential expression of the keratinolytic protease. This gene has been cloned and expressed in heterologous microorganisms such as Escherichia coli and B. subtilis, but the keratinase yields are lower than the wild strain [360]. However, increased keratinase yield was achieved by chromosomal integration of multiple copies of the kerA gene in B. licheniformis and B. subtilis. The kerA gene was also cloned for extracellular expression in P. pastoris, resulting in a recombinant enzyme that was glycosylated and even though active on azokeratin. The vast majority of investigations report keratinase production through submerged cultivations. Only recently the production of keratinolytic enzymes through solid-state processes has been demonstrated [265]. The potential of keratinase production by immobilized microorganisms was also reported [62]. 2.4.3 Applications of Proteases and Keratinases Keratinases from microorganisms have attracted a great deal of attention in the recent decade, particularly due to their multitude of industrial applications such as in the feed, fertilizer, detergent, leather and pharmaceutical industries. Currently, the most promising application of keratinases/ keratinolytic microorganisms is the production of nutritious, cost-effective, environmentally benign feather meal for poultry. Other applications of keratinases have yet to be thoroughly explored before commercialization. The following section will discuss some of the prospective applications of keratinases that are rapidly gaining importance. All proteolytic enzymes have characteristic properties with regard to temperature, pH, ion requirement, specificity, activity and stability. These biochemical parameters determine the application of protease in industry apart from other factors, which include the cost of production and development, markets and the economy of application. Proteases have a huge variety of applications, mainly in the detergent and food industries. In view of the recent trend of developing ecofriendly technologies, proteases are envisaged to have extensive applications in leather treatment and in several bioremediation processes. Proteases are used extensively in the

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pharmaceutical industry for preparation of medicines such as ointments for debridement of wounds, etc. 2.4.3.1. 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. The use of proteases in laundry detergents accounts for approximately 25 per cent of the total worldwide sales of enzymes. Rhm Company in Germany isolated the first enzyme for industrial use, in 1914 [360]. They used a trypsin enzyme isolated from animals pancreas that degrades proteins. It proved to be so powerful compared to traditional washing powders that the original small packages size made the German housewives suspicious; the product had to be reformulated and sold in larger packages. The real breakthrough of enzymes occurred with the introduction of microbial proteases into washing powders. The first commercial bacterial Bacillus protease was marketed in 1956, under the trade name BIO-40 by the Swiss Company Gebrder Schnyder. In 1962, Novo Nordisk, in Denmark, was the first company to mass-produce an alkaline protease suitable for wash conditions; they introduced Alcalase, produced by Bacillus licheniformis and commercially named BIOTEX. This was followed by Maxatase, a detergent made by GistBrocades [336]. The biggest market for detergents is in the laundry industry, amounting to a worldwide production of 13 billion tons per year. The ideal detergent proteases have broad substrate specificity to facilitate the removal of a large variety of stains (food, blood, grass, and body secretions). Activity and stability at high pH and temperature, and compatibility with other chelating and oxidizing agents added to the detergent are among the major prerequisites for the use of proteases in detergents. The key parameter for the best performance of a protease in a detergent is its pI (ionic strength). It is known that a protease is more suitable for this application if its pI coincides with the pH of the detergent solution. Esperase and Savinase T (Novo Industry) produced by alkalophilic Bacillus sp., are two commercial preparations with very high isoelectric points (pI 11.0) hence, they can withstand higher pH ranges. Due to the present energy crisis and the awareness for energy conservation, it is desirable to use proteases that are active at lower temperatures. A combination of lipase, amylase, and cellulose is expected to enhance the performance of protease in laundry detergents. All detergent proteases currently

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used in the market are serine proteases produced by Bacillus strains. Fungal alkaline proteases are advantageous due to the ease of downstream processing to prepare a microbe-free enzyme. An alkaline protease from Conidiobolus coronatus was found to be compatible with commercial laundry detergents used in India, it retained 43 per cent of its activity at 50C for 50 min., in the presence of 25 mM Ca+2 and 1 M glycine [337]. Proteolytic enzymes have dominated the detergent market since ancient times. In fact, approximately 89% share of detergent enzymes is captured by alkaline proteases, with Novo Nordisk and Genencor International being the major suppliers [371]. Nonetheless, there is always a need for newer enzymes with novel properties that can further widen the scope of enzyme-based detergents. Keratinases have the ability to bind and hydrolyze solid substrates like feather. This is an important property of detergent enzymes as they are required to act on protein substrates attached to solid surfaces, making them attractive additives for hard-surface cleaners. They could also help in the removal of keratinous soils that are often encountered in the laundry, such as collars of shirts, on which most proteases fail to act. An extended application of keratinases in detergents is their use as additives for cleaning up of drains clogged with keratinous wastes [371].
Table 2.5: Protease in industry Industry Leather Food processing Baking Enzyme Trypsin, Other protease Several proteases Neutral protease Calf rennet and other trypsin, chymotrypsin, ficin, Fungal protease, Chymosin Alkaline protease Papain Papain Thermolysin Trypsin, Chymopapain, Carboxypeptidase, Trypsin Application Bating of leathers, Dehairing and dewooling of skins Modification of protein rich material i.e. soy protein or wheat gluten Dough conditioners Coagulation of milk protein (cheese production), production of enzyme modified cheese; whey processing Replacement of calf rennet Active component of calf rennet; also production by genetically engineered microbes developed Extensive use in laundry detergents for protein stain removal Meat tenderization Removal of turbidity Reverse hydrolysis in aspartame synthesis Removal of dead tissues and dissolution of blood clots Treatment of certain types of hemia Conversion of hog insulin, Production of human insulin

Dairy

Detergent Meat Beverages Confectionery

Pharmaceutical

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2.4.3.2 Leather Industry Leather processing involves several steps such as soaking, dehairing, bating and tanning. The major building blocks of skin and hair are proteinaceous. Leather industry contributes to one of the major industrial pollution problems, and effluent disposal; the pollution causing chemicals, sodium sulfide, salt, solvents, lime, etc. arise mainly from the pre-tanning processes of leather processing [338]. In order to overcome the hazards caused by the tannery effluents, the use of enzymes as a viable alternative to chemicals has successfully resorted to in improving leather quality and in reducing environmental pollution [339, 340]. Proteases are used for selective hydrolysis of noncollageneous constituents of the skin and for removal of nonfibrillar proteins such as albumins and globulins, in the several pretanning operations. The purpose of soaking is to swell the hide [341]. Traditionally, this step was performed with alkali. Currently, microbial alkaline proteases are used to ensure faster absorption of water and to reduce the time required for soaking by 10-20 hours [342, 343]. The use of non-ionic and, to some extent, anionic surfactants to accelerate the process is compatible with the use of enzymes. The conventional method of dehairing and dewooling consists of development of an extremely alkaline condition followed by treatment with sulfide to solubilize the proteins of the hair root (the severe alkaline condition was a health hazard for the workers). At present, alkaline proteases with hydrated lime and sodium chloride are used for dehairing, resulting in a significant reduction in the amount of wastewater generated. Earlier methods of bating were based on the use of animal feces as the source of proteases; these methods were unpleasant, unhygienic and unreliable, and were replaced by methods involving pancreatic trypsin [339]. Currently, trypsin is used in combination with others Bacillus and Aspergillus proteases for bating. The selection of the enzyme depends on its specificity for matrix proteins such as elastin and keratin, and the amount of enzyme needed depends on the type of leather (soft or hard) to be produced. Increased usage of enzymes for dehairing and bating not only prevents pollution problems but is also effective in saving energy. Novo Nordisk manufactures three different proteases, Aquaderm, NUE, and Pyrase, for use in soaking, dehairing, and bating, respectively (Novo Nordisk website). Leather processing technology involves a series of operations, amongst which pre-tanning contributes to the major amount of pollution (approximately 70%). Sodium sulfide, lime and

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solid wastes generated as a result of pre-tanning are mainly responsible for increased biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total dissolved solids (TDS) [317]. Biocatalytic leather processing involves the use of a mixture of enzymes, among which proteases, lipases and carbohydrases are well exploited for various pre-tanning stages [317]. In addition, keratinolytic proteases lacking collagenolytic and having mild elastolytic activities are increasingly being explored for the dehairing process. They would help in the selective breakdown of keratin tissue in the follicle, thereby pulling out intact hairs without affecting the tensile strength of leather [28]. A few reports that indicate that keratinases could be useful depilating agents are available [372, 54]. In fact, a keratinase from B. subtilis S14 Macedo et al. 2005 [28] was reported to completely eliminate the need for toxic sodium sulfide. Thus, sulfide-based hair-destroying dehairing processes that pose an environmental threat by increasing the BOD could be replaced by keratinase-based cleaner hair-saving dehairing technology. 2.4.3.3 Food industry The use of proteases in the food industry dates back to antiquity. They have been routinely used for various purposes such as cheese making, baking, preparation of soy hydrolysates, and meat tenderization. 2.4.3.3.1 Dairy industry The major application of proteases in the dairy industry is in the manufacture of cheese. The milk-coagulating enzymes fall into four main categories: (a) animal rennets, (b) microbial milk coagulants, (c) vegetable rennet and (d) genetically engineered chymosin [14]. The first commercial application of agricultural biotechnology approved by the FDA (Food and Drug Administration) in 1990 was the development of fermentation-derived chymosin, an enzyme used in cheese production to coagulate milk. Because natural chymosin must be extracted from the lining of the stomachs of slaughtered 04-day-old calves, supplies were limited. Advances in biotechnology have enabled scientists to create an unlimited, cheaper, and more consistent supply of chymosin by using a genetically engineered microorganism to produce the enzyme through fermentation. This technology is now used in over 90 per cent

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of all cheese manufacturing industries in U.S., creates chymosin that is 40 to 50 per cent less expensive than the natural enzyme (Tomorrows Bounty webpage). The microbial enzymes exhibited two major drawbacks, i. e., (a) the bitterness in cheese, after storage due to the presence of high levels of nonspecific and heat-stable proteases; and (b) a low yield. The exhaustive research on this matter has resulted in the production of enzymes that are completely inactivated at normal pasteurization temperatures and contain very low levels of nonspecific proteases. 2.4.3.3.1.1 Milk Milk is a translucent white liquid produced by the mammary glands of mammals. Approximate composition of milk can be given (Fig. 2.13):

87.3% water (range of 85.5% - 88.7%) 3.9 % milkfat (range of 2.4% - 5.5%) 8.8% solids-not-fat (range of 7.9 - 10.0%):

protein 3.25% (3/4 casein) lactose 4.6% minerals 0.65% - Ca, P, citrate, Mg, K, Na, Zn, Cl, Fe, Cu, sulfate, bicarbonate, many others acids 0.18% - citrate, formate, acetate, lactate, oxalate enzymes - peroxidase, catalase, phosphatase, lipase gases - oxygen, nitrogen vitamins - A, C, D, thiamine, riboflavin, others

The nitrogen content of milk is distributed among caseins (76%), whey proteins (18%), and nonprotein nitrogen (NPN) (6%)

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Figure 2.13: Milk Composition as percent total volume

2.4.3.3.1.2 Casein Casein is the predominant phosphoprotein that accounts for nearly 80% of milk proteins. The principal casein fractions are alpha (s1) and alpha (s2)-caseins, -casein, and kappa-casein. The distinguishing property of all caseins is their low solubility at pH 4.6. The common compositional factor is that caseins are conjugated proteins, most with phosphate group(s) esterified to serine residues. These phosphate groups are important to the structure of the casein micelle. Calcium binding by the individual caseins is proportional to the phosphate content. Casein consists of a fairly high number of proline peptides, which do not interact. There are also no disulfide bridges. As a result, it has relatively little tertiary structure. Because of this, it cannot denature. It is relatively hydrophobic, making it poorly soluble in water. It is found in milk as a suspension of particles called casein micelles which show some resemblance with surfactant-type micelle in a sense that the hydrophilic parts reside at the surface. The caseins in the micelles are held together by calcium ions and hydrophobic interactions.

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Figure 2.14: Casein micelle and casein submicelle

Figure 2.15 : - casein

2.4.3.3.1.2 Casein Types

Table 2.6: Four different types of bovine casein exist, each with several genetic variants.

Type Mol. wt. pI Phosphates/mole E1% g protein/L in skim milk -s1 22,068-23,724 4.2-4.76 8-10 10.0-10.1 12-15 -s2 25,230 10-13 3-4 23,944-24,092 4.6-5.1 4-5 4.5-4.7 9-11 19,007-19,039 4.1-5.8 1 10.5 2-4

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2.4.3.3.1.2.1 -s1 Casein -s1 Casein is the most prevalent form of casein in bovine milk. It has been reported to exhibit antioxidant and radical scavenging properties. It has also been reported to be involved in the transport of and casein from the endoplasmic reticulum to the Golgi apparatus. It has two hydrophobic regions, containing all the proline residues, separated by a polar region, which contains all but one of eight phosphate groups. It can be precipitated at very low levels of calcium.

Figure 2.16: -s1 Casein

2.4.3.3.1.2.2 -s2 Casein: Proteolytic fragments of -s2 Casein have been shown to exhibit antibacterial activity. Specifically the 39 amino acid casocidin-1 peptide fragment has been shown to inhibit E. coli and Staph. carnosis growth. It has concentrated negative charges near N-terminus and positive charges near C-terminus. It can also be precipitated at very low levels of calcium.

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Figure 2.17: -s2 Casein

2.4.3.3.1.2.3 -Casein: -Casein and its fragments have been implicated in a number of biological functions. The casoparan peptide has been reported to activate macrophage phagocytosis and peroxide release. Casohypotensin and casoparan may be involved in bradykinin regulation. Casohypotensin has also been shown to be a strong inhibitor of endo-oligopeptidase A, a thiol-activated protease capable of degrading bradykinin and neurotensin, and hydrolyzing enkephalin-containing peptides to produce enkephalins. -Caseins are also a source of casomorphin peptides which exhibit opioid activity binding to opioid receptors. Casomorphins may be the hydrolysis product of dipeptidyl peptidase IV. It has highly charged N-terminal region and a hydrophobic Cterminal region. Very amphiphilic protein acts like a detergent molecule. Self association is temperature dependant; will form a large polymer at 20 C but not at 4 C. Less sensitive to calcium precipitation.

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Figure 2.18: -Casein

2.4.3.3.1.2.4 -Casein It is a mammalian milk protein involved in a number of important physiological processes. Casein's orientation on the surface of the casein micelle functions as an interface between the hydrophobic interior caseins and the aqueous environment. During clotting of milk, hydrolysis by chymosin or rennin releases the water soluble fragment, para-k-casein and the hydrophobic caseinomacropeptide. Very resistant to calcium precipitation, stabilizing other caseins. Rennet cleavage at the Phe105-Met106 bond eliminates the stabilizing ability, leaving a hydrophobic portion, para-kappa-casein, and a hydrophilic portion called kappa-casein glycomacropeptide (GMP), or more accurately, caseinomacropeptide (CMP).

Figure 2.19: -Casein

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2.4.3.3.1.3 Rennet Rennet is usually a natural complex of enzymes produced in any mammalian stomach to digest the mother's milk, and is often used in the production of cheese. Rennet contains many enzymes, including a proteolytic enzyme (protease) that coagulates the milk, causing it to separate into solids (curds) and liquid (whey). The active enzyme in rennet is called chymosin or rennin but there are also other important enzymes in it, e.g., pepsin or lipase. There are non-animal sources for rennet that are suitable for vegetarian consumption.

Figure 2.20: preparation of Rennet

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2.4.3.3.1.4 Renin/Chymosin Renin belongs to a family of enzymes referred to as aspartic proteases, which also includes the enzymes pepsin, cathepsin, and chymosin. Renin is a mono specific enzyme that displays remarkable specificity for its only known substrate, angiotensinogen.

Figure 2.21: 3D network - Casein

Chymosin causes cleavage of a specific linkage - the peptide bond between phenylalanine and methionone in the - Casein. If this reaction applies to milk, the specific linkage between the hydrophobic (para-casein) and hydrophilic (acidic glycopeptides) group of casein inside milk would be broken, since they are joined by phenylalanine and methionine. The hydrophobic group would unite together and would form a 3D network to trap the aqueous phase of the milk. The resultant product is calcium phosphocaseinate. Due to this reaction, rennin is used to bring about the extensive precipitation and curd formation in cheese making.

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Figure 2.22: specific linkage the peptide bond between phenylalanine and methionone in the - Casein

Chymosin (rennin) is essential for the manufacture of good quality cheeses. Found in the fourth stomach of suckling calf's. Very expensive and inhumane to process now so it has been engineered into bacteria that mass produces. It has a very specific activity- Hydrolyzes only one bond in -casein, one of the many proteins that make up the milk casein protein complex (-, -, -casein). This breaks up the casein complex (micelle) and it aggregates leading to a clot, the first step in cheese production. Most other proteases can initiate a milk clot like chymosin but they would continue the casein hydrolysis producing bitter peptides and eventually breaking the clot.

Figure 2.23: Schematic representation of events in clotting of milk. The s-, -, and -caseins are shown by striped, stippled and white balls, respectively.

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2.4.3.3.1.5 Milk Clotting The milk-clotting process consists of 3 main phases: 1. Enzymatic degradation of -casein 2. Micellar flocculation 3. Gel formation Each step follows a different kinetic pattern, the limiting step in milk-clotting being the degradation rate of -casein. The kinetic pattern of the second step of the milk-clotting process is influenced by the cooperative nature of micellar flocculation whereas the rheological properties of the gel formed depend on the type of action of the proteases, the type of milk, and the patterns of casein proteolysis The overall process is influenced by several different factors, such as pH or temperature. The conventional way of quantifying a given milk clotting enzyme employs milk as the substrate and determines the time elapsed before the appearance of milk clots. However, milk clotting may take place without the participation of enzymes because of variations in physicochemical factors, such as low pH or high temperature. As early as 1970 milk-clotting enzyme from Mucor pusillus was isolated by Kei Arima [272]. Milk coagulation is a basic step in cheese manufacturing. For a long time calf rennet, the conventional milk-clotting enzyme obtained from the fourth stomach of suckling calves is the most widely used coagulant in cheese making all over the world to manufacture most of the cheese varieties. The worldwide reduced supply of calf rennet and the ever increase of cheese production and consumption have stimulated the research for milk-clotting enzyme (MCE) from alternative sources to be used as calf rennet substitutes. Various animals, plants and microbial proteases have been suggested as milk coagulants. However, attention has been focused on the production of milk-clotting enzymes (MCEs) from crude extract of spices since no work has been done till date and they are part of food since historic times. Work done on production of milk clotting enzymes from culture of Bacillus Subtilis natto [379] and from some Pakistani Plants [157] has been taken into account and the same procedures for different assays are followed.

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Protease, also known as proteolytic enzymes or proteinases, are enzymes that break down protein into amino acids so the body can use them. Protease can also destroy viruses and bacteria, because they are proteins. There are also commercial uses for protease. Rennet is a type of protease originating from the fourth stomach of calves. Rennet is used in the process of making cheese from milk. 2.4.3.3.2 Baking industry The safety of the source organism is an important consideration in the safety assessment for recombinant enzymes. Aspergillus oryzae, not considered to be pathogenic, is widely distributed in nature and is commonly found in foods. Enzymes from Aspergillus oryzae are extensively used in production of a variety of foods such as syrups, alcohol, fruit juices, brewing, chocolate syrup, baking and meat tenderizing [368]. The enzyme is used in the baking industry as a processing aid to strengthen gluten in dough systems. It causes a more elastic and stronger gluten network similar to that obtained by traditional oxidizing agents such as potassium bromate or ascorbic acid. The enzyme is active in the dough and the leavening of the unbaked bread, but normally inactivated by high temperatures during the baking. The enzyme is used as a processing aid only, and is not expected to be present in the final food. Any residue would be in the form of inactivated enzyme, which would be metabolized like any other protein [369]. 2.4.3.3.3 Soy sauce production Soybeans serve as a rich source of food, due to their high content of good-quality protein. Proteases have been used from ancient times to prepare soy sauce and other soy products. The alkaline and neutral proteases of fungal origin play an important role in the processing of soy sauce. Proteolytic modification of soy proteins helps to improve their functional properties. Treatment of soy proteins with alcalase at pH 8 results in soluble hydrolysates with high solubility, good protein yield, and low bitterness. The hydrolysate is used in protein-fortified soft drinks and in the formulation of dietetic feeds. 2.4.3.3.4 Brewing industry The brewing industry is a major user of proteases. In the production of brewing wort Bacillus

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subtilis protease are used to solublize protein from barley adjuncts, thereby releasing peptides and amino acids which can fulfill the requirement of the nitrogen supply. The proteolytic enzymes are used in chill proofing, a treatment designed to prevent the formation of precipitates during cold storage. In beer, hazes are formed due to the presence of proteinanceous substances which also precipitate the polyphenols and oligosaccharides. Hydrolysis of the protein components prevents aggregation of the insoluble complex. 2.4.3.3.5 Meat tenderization India is endowed with the largest buffalo population in the world. It accounts for 59.08 % of the world buffalo population. About 10.66 million buffaloes are slaughtered annually producing 1.47 million MT buffalo meat. They are slaughtered mainly for meat. The byproducts from slaughtered animals are also of good value. Buffalo tripe is one of the important edible offal and weighs about 4.36 to 5.45 kg per animal. Commercial exploitation of tripe for development of processed product manufacture is very limited because of its poor functional properties and inherent toughness due to high collagen content. It is essential to develop technologies for utilization of tripe into processed product manufacture by reducing its toughness. In order to improve tenderness of meat, a number of methods have been tried. Tenderization may be achieved by use of chemical or proteolytic enzymes. Proteolytic enzyme, papain is used to tenderize tough meat cuts. Papain is very powerful in hydrolyzing fibrous protein and connective tissue [370]. In general, uniform penetration of tenderizer enzyme has always posed problem during tenderization treatment [371]. 2.4.3.4 Synthesis of aspartame The use of aspartame as a noncalorific artificial sweetener has been approved by the Food and Drug Administration. Aspartame is a dipeptide composed of L-aspartic acid and the methyl ester of L-phenylalanine. The L configuration of the two amino acids is responsible for the sweet taste of aspartame. Maintenance of the stereospecificity is crucial, but it adds to the cost of production by chemical methods. Enzymatic synthesis of aspartame is therefore, preferred. Although proteases are generally regarded as hydrolytic enzymes, they catalyze the reverse reaction under certain kinetically controlled conditions. An immobilized preparation of

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thermolysin from Bacillus thermoprotyolyticus is used for the enzymatic synthesis of aspartame. Toya Soda (Japan) and DSM (The Netherlands) are the major industrial producers of aspartame. 2.4.3.5 Pharmaceutical industry The wide diversity and specificity of proteases are used to great advantage in developing effective therapeutic agents. Oral administration of proteases from Aspergillus oryzae (Luizym and Nortase) has been used as a digestive aid to correct certain lytic enzyme deficiency syndromes. Clostridial collagenase or subtilisin is used in combination with broad-spectrum antibiotics in the treatment of burns and wounds. An asparginase isolated from E. coli is used to eliminate aspargine from the bloodstream in the various forms of lymphocytic leukemia. Alkaline protease from Conidiobolus coronatus was found to be able to replace trypsin in animal cell cultures [372]. Curcain a plant protease was purified from the latex of Jatropha curcus was found to be active in wound healing agent. 2.4.3.6 Therapeutic uses The most obvious use of proteolytic enzymes is to assist digestion. Injection of some foreign proteases into human reduces tissue inflammation and pain. Use of proteolytic enzymes helped reduce the discomfort of breast engorgement in lactating women. Proteolytic enzymes were reported in reducing pain, swelling and inflammation caused by sugary and injury. 2.4.3.7 Photography industry The photography industry uses large quantity of silver in the light sensitive emulsion that it produces. When such film is processed, to recover the expensive silver, the procedure involves separating the silver containing gelatin from the film base. The aqueous solution that results contain both gelatin and silver, but the presence of protein hinders the separation of silver. Addition of proteolytic enzymes at a temperature of 500C and pH 8.0 rapidly degrades the gelatin and allows the silver particles to separate out. .

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2.4.3.8 Production of films, coatings and glues Recently there has been an increased interest in the production of biodegradable films, coatings and glues from keratinous waste products like hair, feathers, skin, fur, animal hooves, horns etc. for compostable packaging, agricultural films or edible film applications. Keratin structure is chemically modified and hydrolyzed to produce stable dispersions for such applications. Alternatively controlled hydrolysis of keratin using keratinases could offer an environmentfriendly technology. 2.4.3.9 Animal feed Feathers represent over 90% protein, the major component being -keratin, a fibrous and insoluble structural protein extensively cross-linked by disulfide, hydrogen and hydrophobic bonds. Owing to their insoluble nature, feathers are resistant to degradation by common microbial proteases, viz. trypsin, pepsin and papain. Thus, the several million tons of feathers generated annually by the livestock industry leads to troublesome environmental pollution and wastage of a protein-rich reserve [64, 330]. Until recent years, feathers were baked at high temperature and pressure and used as animal feed supplement in the form of feather meal. The hydrothermal treatment, in addition to being expensive, resulted in the destruction of certain essential amino acids, viz. methionine, lysine and tryptophan, yielding a product with poor digestibility and variable nutrient quality [360]. The drawbacks of the high-temperature treatment impel the use of microbial keratinases that serve as attractive alternatives to hydrolyze feather into nutritionally rich animal feed [64]. The application of keratinases/keratinolytic microbes for improvement of feather as poultry feed has been extensively reviewed by Onifade et al. 1998 [64]. The bulk of information on feather meal production using microorganisms is provided by Shih and coworkers at North Carolina University. It is well documented that supplementing feather meal/ raw feather with crude keratinase enzyme PWD1 modifies the structure of keratin, leading to improved digestibility and bolstered growth of poultry [370]. Feather meal is relatively inexpensive and is shown to be superior to soybean meal in terms of total cysteine, valine and threonine content, and the hydrolyzed meal can replace soybean meal at 7% dietary level. The crude enzyme can also serve as a nutraceutical product, leading to significant improvement in broiler performance. In addition, nutritional enhancement can be

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achieved by hydrolysis of feather meal/raw feather using keratinolytic microorganisms. Fermentation significantly increases the levels of essential amino acids (methionine, lysine and arginine), and the microbial biomass contributes as a rich source of protein. Feeding trials of the feather lysate produced by B. licheniformis PWD1 revealed growth curves identical to that observed with standard soybean meal [17]. In order to produce sufficient quantities of keratinase PWD1 for application in feather meal production, scaling up of the enzyme has been accomplished in a 150-l fermenter [70]. Furthermore, cloning, overexpression and bioimmobilization of the enzyme have been successfully carried out to meet the demands of the animal feed industries [324, 70]. The technology for production of keratinase PWD1 is licensed to BRI and is being developed under the trade name Versazyme. 2.4.3.10 In fertilizer The protein-rich concentrate feather meal generated for poultry feed can also be applied for organic farming as a semislow- release nitrogen fertilizer [356]. Organic farming relies on the use of nitrogen-rich organic amendments that serve the dual purpose of improving plant growth and intensifying microbial activity in soil. Traditionally, guano has been widely used as a fertilizer for organic farming. However, owing to high expenses, there is a need to search for more suitable alternatives. Feather meal being nitrogen rich (15% N), inexpensive and readily available source serves as a potential substitute to guano. It not only supplies nitrogen to plants and promotes microbial activity, but also structures the soil and increases water retention capacity. The microbially hydrolyzed feather meal can further edge over the steamed meal as fertilizer due to its high nutritive value, easy production and economic feasibility. 2.4.3.11 Degradation of prion proteins Prions are proteinaceous particles responsible for fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE) that include the dreaded mad cow disease, scrapie, kuru and CreutzfeldJakob disease. Infectivity by prions is accompanied by the conversion of harmless PrPc to infectious PrPsc, facilitated by PrPsc itself. These -keratin-rich PrPsc forms wad together into dementia- causing clumps. Shih and coworkers at BRI have reported that the broadspectrum keratinase PWD1 (Versazyme) is capable of completely

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degrading prions from brain tissue of bovine spongiform encephalopathy (BSE)- and scrapieinfected animals in the presence of detergents and heat treatment. The enzymatic breakdown of prions would most importantly help revive the use of animal meal as feed, which faced much criticism by the European Union despite its high nutritive value due to risk of TSE. It would also prove useful for decontaminating medical instruments, lab equipment and interchangeable items like contact lenses and dentistry tools. 2.4.3.12 Miscellaneous Besides their industrial and medicinal applications, proteolytic enzymes play an important role in basic research. Proteases are used to clarify the structure function relationship, in selective cleavage of proteins for sequence determination, or peptide mapping and synthesis. Protein engineering and directed evolution strategies are exploited to create and screen for novel protease variants that can be used in protein hydrolysis and synthesis applications Other potential applications of keratinases include the anaerobic digestion of poultry waste to generate natural gas for fuel, modification of fibers such as silk and wool, in medicine and pharmaceuticals for elimination of acne or psoriasis, elimination of human callus for preparation of vaccines for dermatophytosis and additives in skin-lightening agents as they stimulate keratin degradation. 2.4.4 New Technology A number of reports on the homology of proteases are available in literature. Studies of DNA and protein sequence homology are important for a variety of purposes and have therefore become routine in computational molecular biology. They serve as a prelude to phylogenetic analysis of proteins and assist in predicting the secondary structure of DNA and proteins. The nucleotide and amino acid sequences of a number of proteases have been determined, and their comparison is useful for elucidating the structure-function relationship [105]. The homology of proteases with respect to the nature of the catalytic site has been studied [114]. Accordingly, the enzymes have been allocated to evolutionary families and clans. It has been suggested that there may be as many as 60 evolutionary lines of peptidases with separate origins. Some of these contain members with quite diverse peptidase activities, and yet there are some striking

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examples of convergence [97]. Liggieri et al., [92] purified Asclepain c I from the latex of Asclepias curassavica which is 87% homologous with Funastrain c II [89], 86% with Asclepain f [76] and 75% with cysteine proteinase I purified from Carica candamarcensis [373]. Papain likes endopeptidases Morrenain b I was purified from the latex of Morrenia brachystephana [85]. It shows 73% sequence homology with plant protease araujiain h II [374], 64% with cathepsin K purified from Mus musculus [375] and 60% with cathepsin L-like protease isolated from Leishmania major [361]. Endopeptidase named asclepain f, purified from the latex of Asclepias fruticosa [76] has 81% sequence homology with plant protease of Morrenia odorata and 76.2 % with asclepain B isolated from Asclepias syriaca [376]. Takagi et al, [302] found that the thermostable proteases of Bacillus stearothermophilus and Bacillus thermoproteolyticus are 85% homologous and the thermo labile proteases of Bacillus subtilis and Bacillus amyloliquefaciens are 82% homologous, whereas the thermostable protease of Bacillus stearothermophilus is only 30% homologous to the thermo labile protease of Bacillus subtilis. Koide et al., 1986 compared the amino acid sequences of intracellular serine proteases from Bacillus subtilis with those of subtilisin Carlsberg and subtilisin BPN and showed that they were 45% homologous [94]. The amino acid sequence of an extracellular alkaline protease, subtilisin J, is highly homologous to that of subtilisin E and shows 69% identity to that of subtilisin Carlsberg, 89% identity to that of subtilisin BPN, and 70% identity to that of subtilisin DY. The amino acid sequence of subtilisin J is completely identical to that of the protease from Bacillus amylosacchariticus except for two amino acid substitutions, Thr130 to Ser130 and Thr162 to Ser162, in addition to one amino acid substitution in the signal peptide and two in the propeptide region. The probable active-site residues of subtilisin J, i.e., Asp32, His64, and Ser221, are identical to those of other subtilisin from Bacillus. Therefore, it was concluded that the alkaline protease from Bacillus stearothermophilus is a subtilisin. Similarly, the various Bacillus serine alkaline proteases, such as bacillopeptidase F, subtilisin, Epr, and ISP-1, show considerable homology and conserved amino acids around the active site residues, i.e., Ser, Asp, and His [197]. Alkaline proteases from various species of Aspergillus also show a high degree of homology [241]. Alp from Aspergillus oryzae shows considerable homology (29 to 44%) to

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the members of the subtilisin family with conserved active-site residues. However, Alp exhibits little homology to mammalian serine proteases such as trypsin and chymotrypsin [311]. Studies of the homology of proteases have shown that the residues involved in the substrate and metal ion binding, catalysis, disulfide bond formation and active-site formation are conserved. Analysis of sequence homology is used in deciphering the structure-function relationship of proteases.

2.5 Media Optimization

When developing an industrial fermentation, designing fermentation medium is of critical importance because medium composition can significantly affect product concentration, yield and volumetric productivity. For commodity products, medium cost can substantially affect overall process economics. Medium composition can also affect the ease and cost of downstream product separation, for example in the separation of protein products from medium containing protein. There are many challenges associated with medium design. Designing the medium is a laborious, expensive, open-ended, often time-consuming process involving many experiments. In industry, it often needs to be conducted frequently because new mutants and strains are continuously being introduced. Many constraints operate during the design process, and industrial scale must be kept mind when designing the medium. A medium design campaign can involve testing hundreds of different media. One of the more difficult aspects of the medium design process is dealing with this flow data. In reality, often the information generated from design experiments is difficult to assess because of its sheer volume. Beyond about 20 experiments with five variables it very difficult for a researcher to maintain medium component trends mentally, especially when more than one variable changes at a time. Data capture and data mining techniques are crucial in this situation.

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Table 2.7: Summary of medium design strategies [349]

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2.5.1 Artificial neural network An artificial neural network (ANN), usually called "neural network" (NN), is a mathematical model or computational model that tries to simulate the structure and/or functional aspects of biological neural networks. It consists of an interconnected group of artificial neurons and processes information using a connectionist approach to computation. In most cases an ANN is an adaptive system that changes its structure based on external or internal information that flows through the network during the learning phase. Modern neural networks are non-linear statistical data modeling tools. They are usually used to model complex relationships between inputs and outputs or to find patterns in data.

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These networks are also similar to the biological neural networks in the sense that functions are performed collectively and in parallel by the units, the term Artificial Neural Network (ANN) tends to refer mostly to neural network models employed in statistics, cognitive psychology and artificial intelligence. Neural network models designed with emulation of the central nervous system (CNS) in mind are a subject of theoretical neuroscience (computational neuroscience). 2.5.2.1 Tools Used The project requires the knowledge of the following tool which was used to carry out the analysis. 2.5.2.1.1 MATLAB 7.0 MATLAB 7.0 is a numerical computing environment and fourth generation programming language. Developed by The Math Works, MATLAB allows matrix manipulation, plotting of functions and data, implementation of algorithms, creation of user interfaces, and interfacing with programs in other languages. Although it is numeric only, an optional toolbox uses the MuPAD symbolic engine, allowing access to computer algebra capabilities. An additional package, Simulink, adds graphical multidomain simulation and Model-Based Design for dynamic and embedded systems. In this project we are using the neural network tool box of the software to model our networks for the validation of literature work.

2.5.1.1 Back propagation Neural Network 2.5.1.2.1 Introduction Backpropagation was created by generalizing the Widrow-Hoff learning rule to multiple-layer networks and nonlinear differentiable transfer functions. Input vectors and the corresponding target vectors are used to train a network until it can approximate a function, associate input vectors with specific output vectors, or classify input vectors in an appropriate way as defined by us. Standard backpropagation is a gradient descent algorithm, as is the Widrow-Hoff learning rule, in which the network weights are moved along the negative of the gradient of the performance function. The term backpropagation refers to the manner in which the gradient is computed for 87

nonlinear multilayer networks. Properly trained backpropagation networks tend to give reasonable answers when presented with inputs that they have never seen. Typically, a new input leads to an output similar to the correct output for input vectors used in training that are similar to the new input being presented. This generalization property makes it possible to train a network on a representative set of input/target pairs and get good results without training the network on all possible input/output pairs.

Figure 2.24: working of BPNN

2.5.2.2.2 Algorithm The network learns a predefined set of input output example pairs by using a two phase propagate adapt cycle. The networks begin with learning a predefined set of input-output example pairs by using a two phase propagate-adapt cycle. After an input pattern has been applied as a stimulus to the first layer of networks units, it is propagated through each upper layer until an output is generated. The output pattern is then compared to the desired output, and an error signal is computed for each output unit.

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A part of error signal was transmitted from output layer to each node in intermediate layer that contributes directly to the output.

The number of passes decides how many times our data is processed through the hidden unit before giving the final predicted value and error from the desired output.

2.5.2.2.3 Procedure 1. The input and output feature was defined and the other parameters were set
2.

The network are then initiliazed by the inputs,their maximum values, minimum values, the wieghts were set.

3. The network was then simulated by providing the inputs and output layer. 4. The results of simulation are stored in simulation results network1_outputs and network1_errors 5. The others were parameters were set. The epoch value was our variable which we have recorded from 10 to 300.rest parameters remained same. 6. The network outputs obtained were then exported to ms.excel to calculate the mean square error and correlation coefficient of a particular epoch value 7. The mean of RMSE and corr coeff were taken corresponding to epoch values 10 to 300.

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