PRODUCTION AND CHARACTERIZATION OF PROTEASE FROM TRICHODERMA ASPERELLUM ISOLATED FROM LAUNDRY WASTE WATER DISPOSING SITE

ABSTRACT

Protease represents one of the largest groups of industrial enzymes, and are widely used in the detergent, leather, food, pharmaceutical industrial as well as in bioremediation processes. However, increasing demand has intensified the search for protease with unique kinetic and thermodynamic properties which exhibit stability towards pH, temperature, substrate concentration and metal ions for biotechnological applications. Protease was isolated from Trichoderma asperellum (identified based on morphology and molecular characteristics) and partially purified using ammonium sulphate precipitation, dialysis and gel filtration. The partially purified enzyme was then characterized to determine the optimum and stable conditions required by the produced enzyme. The fermentation period for maximum enzyme production was on day

9. The pH optimization of protease production showed that pH 7 was more favourable for the enzyme production with activity of 387.778 μmol/min. The specific activity for the crude enzyme was found to be 308.29 U/mg protein. Ninety percent (90 %) ammonium sulphate saturation was found suitable to precipitate protein with highest protease activity. The partially purified enzyme had optimal pH and temperature of 9.5 and 50 oC, respectively. The enzyme was more stable at pH value of 6.0 to 7.0 and temperature of 4.0 to 60 oC using casein as substrate. . The Michealis- Menten constant, Km and maximum velocity, Vmax obtained from Lineweaver-Burk plot of initial velocity data at different substrate (casein) concentrations were found to be 1.152 mg/ml and

95.238 µmol/min. The enzyme activity was enhanced by Mn2+, Co2+ and Fe2+ whereas Ca2+, Mg2+ and Zn2+  had inhibitory effects on the enzyme. The kinetic parameters, such as the thermal inactivation rate constant (kd) increased after every 10 % rise in temperature, while the half-life (t1/2) and D-value decreased. The Z-value and activation energy for inactivation (Ea) was found to be 27.855 ºC and 75.857KJmol-1K-1 respectively. The thermodynamic parameters of inactivation such as the enthalpy, ΔHº was decreasing with increase in temperature. The Gibbs free energy, ΔGº and entropy, ΔSº were observed to be positive. The result of the study suggest that protease produced by Trichoderma asperellum has exceptional and great properties which could be applied in several industries especially in detergent and textile production.

CHAPTER ONE

INTRODUCTION

Enzymes are active catalysts, responsible for thousands of synchronized chemical reactions

involved in biotechnological process of living systems. Outstanding features of enzymes in comparison to chemical catalysts are their substrate specificity and specificity in promoting of biochemical reaction with their respective substrates ensuring synthesis of a specific bimolecular product without the concomitant production of by products (Chaplin et al., 1990). Enzyme has gradually been elongated in a variety of fields, such as food production, brewing, pharmaceuticals, medicine textiles, leather and detergents (Singh et al., 2014). Enzymes are vital component of detergents, and their use provides consumers with well-proven benefits, both in the washing process itself and in terms of the wider environment (Gomaa, 2013). These benefits include the possibility of working at low temperatures, saving water and energy, and removing the need for harsher chemicals that have been proven to have a negative effect on the environment. In addition, enzymes are biodegradable and leave no harmful residues. The composition of detergents depends on the application for which they are designed, so their enzymatic content may differ (Gomaa,

2013). Various enzymes such as amylase, protease and lipase have been reported to be applied in detergent industries (Gupta et al., 2002; Singh et al., 2014).

Protease represent one of the largest groups of industrial enzymes that are widely used in the detergent, leather, food and pharmaceutical industries as well as in bioremediation (Singh et al.,

2014). Protease are enzymes that breakdown protein by hydrolysis of peptide bonds (Rawlings et al., 2011), and they constitute two third of total enzymes used in various industries and its supremacy in the industrial market increases every year (Gupta et al., 2002). They are classified into seven broad groups according to the character, catalytic active site and condition of action (Dubey et al., 2007). They include serine, threonine, cysteine, aspartate, glutamic acid, asparagine peptide lyase and metalloprotease. Protease is one of the most common enzyme found in detergents that assist in the removal of protein-based stains (Barfoed, 1981). They convert their substrates into small, readily soluble fragments, which can be easily removed from fabrics. All the proteolytic enzymes found in detergents are mostly non-specific serine endoproteases (Maurer, 2004) with a preferred cleavage on the carboxyl side of hydrophobic amino acid residues, but capable of

hydrolyzing most peptide links. In addition, sources of protease are animals (chymotrypsin and trypsin), plants (papain and bromelain) and microorganisms (bacteria and fungi).

Microorganisms are the most common sources of enzymes used commercially owing to their physiological and biochemical properties, facile culture conditions and ease of cell manipulation (Dias et al., 2008). However, microbial proteases are known for their ability to secrete high levels of enzymes in their growth environment (Biesebeke et al., 2006). They are preferred to plant and animal sources to various advantages and can be applied in wide range of industrial sectors viz. food, feed, pharmaceutical, leather, detergent, textile and several others (Gomaa, 2013). A variety of microorganisms such as bacteria, fungi and yeast are known to produce protease (Madan et al.,2002).

In view of this, several studies have been carried out on the isolation of protease from various fungi using casein, peptone and yeast as protein source (Mabrouk et al., 1998; Rohban et al., 2009; Zhang et al., 2010; Zheng et al., 2011; Choudhary and Jain, 2012). However, there is a need to explore microorganism from laundry waste water disposal site that may produce proteolytic enzyme (using casein, peptone, beef extract, yeast extract and gelatin as protein source) with exceptional properties for biotechnology applications. This could be the key to unlocking the door to the availability of highly efficient multipurpose protease for application in detergent industry, simultaneously increasing the economic significance of microbial protease.

1.1 Proteases A protease, also called a peptidase or proteinase, is any enzyme that performs proteolysis, that is, protein catabolism by hydrolysis of peptide bonds (Rawlings et al., 2011). Proteases are the single class of hydrolytic enzymes that riveted the attention of wide spectrum population due to their diversified properties. Proteases (EC 3.4.21-24 and 99; peptidyl-peptide hydrolases) are enzymes that hydrolyze proteins via the addition of water across peptide bonds and catalyses peptide synthesis in organic solvents and in solvents with low water content (Sookkheo et al., 2000; Beg et al., 2003). The hydrolysis of peptide bonds by proteases as shown in Figure 1 is termed as proteolysis; the products of proteolysis are protein and peptide fragments, and free amino acids.

Figure 1: Catalysis of peptide bonds (Proteolysis) by proteases (Beg et al., 2003).

Figure 2:  Three dimensional (3D) structure of a protease complex, with its peptide substrate in black with catalytic residues in red (Rawlings et al., 2011).

Proteolytic enzymes are abundant in occurrence, found in all living organisms, and are important for cell growth and differentiation. There is renewed interest in the study of proteolytic enzymes, mainly due to the recognition that these enzymes not only play an important role in the cellular metabolic processes but have also gained significant attention in the industrial community (Gupta et al., 2002). Proteases account for about 65% of the total worldwide sale of enzymes (Rao et al.,

1998; Shankar et al., 2011; Sundarajan et al., 2011; Annamalai et al., 2014). Detergent, tanning and fiber sizing industries are fully exploiting the power of these enzymes (Rao and Pande 1999).

1.2 Classification of Proteases

According to the Nomenclature Committee of the International Union of Biochemistry and

Molecular Biology, proteases are classified in subgroup 4 of group 3 (Rao et al., 1998).

Proteases can be classified according to 3 major criteria. Such as;

a.   site of action,

b.   the catalytic mechanism,

c.   pH of optimal activity(Rao et al., 1998).

Proteases as shown in Tables below are broadly classified as endo- or exoenzymes on the basis of their site of action on protein substrates (Rao et al., 1998).

Table 1: Classification of Exopeptidases.

EC NO.

3.4.11

3.4.14

3.4.14

3.4.16-3.4.18

3.4.16

3.4.17

3.4.18

3.4.15

3.4.13

3.4.19

EC NO.

3.4.21

3.4.22

3.4.23

3.4.24

3.4.99

1.2.1 Exopeptidase

The exopeptidases act only near the ends of polypeptide chains. Based on their site of action at the

N or C terminus, they are classified as amino- and carboxypeptidases, respectively (Rao et al.,

1998).

1.2.1.1 Aminopeptidases

Amino peptidases act at a free N- terminus of the polypeptide chain liberating a single amino acid, a dipeptide or tripeptide. They are known to remove the N terminal Met that may be found in heterologously expressed proteins but not in many naturally occurring mature proteins. These enzymes occur in a wide variation of microbial species including, bacteria and fungi (Watson,

1976). Most of them require divalent ions like, Mg+2, Mn+2, Co+2, or Zn+2 for their optimal activity

(De-Marco and Dick, 1978). In general, aminopeptidases are intracellular enzymes, but there has been a single report on an extracellular peptidase produced by A. oryzae. (Rao et al., 1998).

1.2.1.2 Carboxy peptidases

Carboxy peptidases digest the protein and liberate single amino acid or a dipeptide from carboxy end. Three distinct groups of carboxypeptidases have been isolated. These are known as serine carboxypeptidases, metallo carboxypeptidass and cystein carboxy peptidases depending on the amino acid present at the active site of the enzymes. Serine carboxy peptidases isolated from different fungal sources have different pH optima, temperature sensitivity and molecular weight

but  similar  substrate  profiles.  Metallo  carboxy  peptidases  isolated  Pseudomonas  species  &

saccharomyces species require Zn2+ or Co2+ for their activity (Felix and Brouillet, 1966).

1.2.2    Endopeptidases

Endopepidases digest peptide bonds within the polypeptide chain away from the N or C termini. The endopeptidases are divided into four subgroups based on their catalytic mechanism, i.e. serine proteases, cysteine, aspartic protease and metalloproteases (Rao et al., 1998).

1.2.2.1 Serine Protease (EC.3.4.21)

Serine proteases are characterized by serine at the active site. They are wide spread and reported from bacteria, fungi and viruses. They are found in endopeptidases as well as exopeptidases. Three residues which forms the catalytic triad are essential in the catalytic process i.e. His (base), Asp (electrophile) and Ser (nucleophile) (Rawlings et al., 2011). The first step in the catalysis is the formation of an acyl enzyme intermediate between the substrate and the essential serine. Formation of this covalent intermediate proceeds negatively charged tetrahedral transition state intermediate and then the peptide bond are cleaved. During the second step or deacylation, the acyl-enzyme intermediate is hydrolyzed by a water molecule to release the peptide and to restore the Ser- hydroxyl of the enzyme. The deacylation, which also involves the formation of a tetrahedral transition state intermediate, proceeds through the reverse reaction pathway of acylation. A water molecule is the attacking nucleophile instead of Ser residue. The residues provide a general base and accept the OH group of the reactive Ser residue (Rao et al., 1998).

1.2.2.2. Cysteine Protease (EC.3.4.22)

Catalysis proceeds through the formation of a covalent intermediate and involves a cysteine and a histidine residue. The essential Cys and His play the same role as Ser and His respectively as in serine proteases. The nucleophile is a thiolate ion rather than a hydroxyl group. The thiolate ion is stabilized through the formation of an ion pair with neighboring imidazolium group of His. The attacking nucleophile is the thiolate-imidazolium ion pair in both steps (Rao et al., 1998).

1.2.2.3 Aspartic Protease (EC 3.4.23)

Aspartic acid proteases, commonly known as acidic proteases, are the endopeptidases that depend on aspartic acid residues for their catalytic activity. Acidic proteases have been grouped into three

families, namely, pepsin, retropepsin and enzymes pararetroviruses. Most aspartic proteases show maximal activity at low pH and have isoelectric points in the range of pH 3 to 4.5.Their molecular weights are in the range of 30 to 45 kDa. The aspartic proteases are inhibited by pepstatin (Rao et al., 1998).

1.2.2.4 Metalloprotease (EC 3.4.24)

The catalytic mechanism by metalloprotease involves the formation of a non-covalent tetrahedral intermediate after the attack of a zinc-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group (Rao et al., 1998).

1.2.3 Alkaline Proteases

Alkaline proteases exhibit optimal activity at a pH range between 8.0 and 11.0 are inactivated by serine active site inhibitors such as phenyl-methyl sulfonyl fluride (PMSF) and diisopropyl flurophosphate (DIFP). These enzymes are concentrated in laundry detergents and leather processing (Markland and Smith, 1971; Chu et al., 1992). Alkaline proteases secreted by both neutrophilic and alkalophilic bacilli are of interest because they represent a major source of commercially produced proteolytic enzymes (Horikoshi, 1971; Markland and Smith, 1971). They are mostly produced extracellularly (Kalisz, 1988) having a molecular weight ranging from 20,000 to 30,000 are stabilized by Ca+2  and have characteristically high isoelectric point (Keay et al., 1970; Markland and Smith, 1971). Two type of amino alkaline proteases have been identified and

characterized differing from each other by 58 amino acids (Aunstrup, 1980; Kalisz, 1998). They are subtilisin Carlsberg produced by Bacillus licheniformis and subtilism Novo, or Bacterial proteases Nagase (BPN), Synthesized by Bacillus amyloliquefaciens. Recently Sutar et al. (1992) and Phadatare et al. (1953) isolated fungus Conidiobulus coronatus having high alkaline proteases activity and Steele et al. (1992) isolated kurthia spiroforme from thermal spring which has alkaline protease activity. Tsuchiya et al. (1992) also reported the production of large amount of extracellular alkaline protease by alkalophilic Thermoactinomyces sp.

1.2.4 Other Classes of Protease

1.2.4.1 Threonine proteases: Threonine proteases are a family of proteolytic enzymes harbouring a threonine (Thr) residue within the active site. The prototype members of this class of enzymes

are the catalytic subunits of the proteasome, however the acyltransferases convergently evolved the same active site geometry and mechanism. Threonine proteases use the secondary alcohol of their N-terminal threonine as a nucleophile to perform catalysis (Cheng et al., 2005) The threonine must be N-terminal since the terminal amine of the same residue acts as a general base by polarizing an ordered water which deprotonates the alcohol to increase its reactivity as a nucleophile (Ekici et al., 2008).

1.2.4.2 Glutamic proteases: Glutamic proteases are a group of proteolytic enzymes containing a glutamic acid residue within the active site. This type of protease was first described in 2004 and became the sixth catalytic type of protease (Cherney et al., 2004). Members of this group of protease had been previously assumed to be an aspartate protease, but structural determination showed it to belong to a novel protease family (Sasaki et al., 2012; Takahashi, 2013). The first structure of this group of protease was scytalidoglutamic peptidase, the active site of which contains a catalytic dyad, glutamic acid (E) and glutamine (Q), which give rise to the name eqolisin. This group of proteases are found primarily in pathogenic fungi affecting plant and human (Oda, 2012).

1.2.4.3 Asparagine peptide lyases: Asparagine peptide lyase are classified according to their catalytic residue. The catalytic mechanism of the asparagine peptide lyases involves an asparagine residue acting as nucleophile to perform a nucleophilic elimination reaction, rather than hydrolysis, to catalyse the breaking of a peptide bond (Rawlings et al., 2011). The existence of this catalytic type of proteases, in which the peptide bond cleavage occurs by self-processing instead of hydrolysis, was demonstrated with the discovery of the crystal structure of the self-cleaving precursor of the Tsh autotransporter from E. coli (Tajima et al., 2010).

These enzymes are synthesized as precursors or propeptides, which cleave themselves by an autoproteolytic reaction (Tajima et al., 2010). The self-cleaving nature of asparagine peptide lyases contradicts the general definition of an enzyme given that the enzymatic activity destroys the enzyme (Finn et al., 2016). However, the self-processing is the action of a proteolytic enzyme, notwithstanding the enzyme is not recoverable from the reaction (Rawlings et al., 2011). All the proteolytic activity of the asparagine peptide lyases is only self-cleavages, then no further peptidase activity occurs (Finn et al., 2016). The main residue of the active site is the asparagine and there are other residues involved in the catalytic mechanism, which are different between the different families of asparagine peptide lyases (Dautin et al., 2007; Tajima et al., 2010).

1.2.4.4 Collagenase: Collagen is a fibrous protein and is a constituent of skin, bone, cartilage, tendon and other connective tissue. Its commercial importance in leather and in production of gelatin and glue has long been recognized (Levine et al., 2013). Collagen is converted into gelatin by boiling. Collagenases are enzymes capable of degrading collagen and are of two types, (i) low molecular weight serine collagenases (24-36 kDa) which are involved  in the production of hormones and pharmacologically active peptides and (ii) high molecular weight metallo- collagenases (30-150 kDa) containing zinc, which require calcium for stability and are involved in remodeling the extracellular matrix (Park et al., 2002; Ramundo and Gray, 2009).

1.2.4.5 Elastase: Elastin is a fibrous protein and together with collagen determines the mechanical properties of connective tissue. It imparts elasticity and allows the tissues to regain its original shape after stretching or contracting. Elastase is a protease, which breaks down elastin and has applications in food, pharmaceuticals and cosmetics industries (Chen et al., 2007). Fungal elastases have been reported from Aspergillus (Markaryan et al., 1994; Mellon and Cotty, 1995; Alp and Arikan, 2008) and entomopathogenic fungus Conidiobolus coronatus (Wieloch and Bogus, 2007). Mellon and Cotty (1996) purified and characterized elastase from Aspergillus flavus.

1.2.4.6 Keratinase: Keratinases are the proteolytic enzymes capable of hydrolyzing highly rigid, strongly cross-linked structural polypeptide, keratin which is recalcitrant to commonly known proteases such as trypsin, pepsin and papain (Gupta and Ramnani, 2006). Keratinases are widely distributed in nature and secreted by variety of organisms belonging to bacteria, actinomycetes and fungi. Traditionally keratinases have been in use for production of feather meal, fertilizers, glues etc. Their applications have been further extended to other areas such as detergent formulations, cosmetics, leather, medicine and animal feed. Keratinases are also finding applications in treatment of mad cow disease (degradation of prion) and biodegradable plastic (Gupta and Ramnani, 2006).

1.2.4.7 Microbial Rennins: Rennin, an aspartic acid protease is important enzyme in cheese manufacture. The enzymes possess high milk-clotting activity and low proteolytic activity, enabling them to be used as substitutes for calf chymosin in the cheese industry (Vaishali, 2013). Traditionally rennin is isolated from animal source (calf rennin) but increased demand and

religious and ethnic regulations against animal derived enzyme has generated interest in microbial rennis (Vaishali, 2013).

1.3 Mechanism of Action of Protease

Proteases are involved in digesting long protein chains into shorter fragments by splitting the peptide bonds that link amino acid residues. Some detach the terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases, carboxypeptidase A); others attack internal peptide bonds of a protein (endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, and elastase).

Catalysis is achieved by one of two mechanisms:

•   Aspartic, glutamic and metallo- proteases activate a water molecule which performs a nucleophilic attack on the peptide bond to hydrolyse it.

•   Serine, threonine and cysteine proteases use a nucleophilic residue (usually in a catalytic triad). That residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme (Rawlings et al., 2011).

Figure  3:  A  comparison  of  the  two  hydrolytic  mechanisms  of  action  used  for  proteolysis

1.4 Sources of Proteases

Proteases are physiologically necessary for metabolic activities of living organisms, they are abundant in nature. They are found in plant, animal and microbial sources.

1.4.1 Plant proteases:

Most common plant proteases are bromelain, ficin, and papain (Adulyatham and Owusu-Apenten,

2005; Lee et al., 1986). Plant genomes encode hundreds of proteases, largely of unknown function. Those with known function are largely involved in developmental regulation (Hoorn, 2008). Plant proteases also play a role in regulation of photosynthesis (Zelisko and Jackowski 2004).

1.4.1.1 Bromelain: Bromelain (EC 3.4.22.32) is a crude extract from the pineapple (Ananas comosus) plant. It is a mixture of sulfur-containing proteases. Bromelain is present in all parts of the pineapple plant but the stem is the most common commercial source. It is active between pH

5 to 9 and stable up to 70°C beyond which it is inactivated. It is used as meat tenderizer, anti- inflammatory agent and in debridement (Secor et al., 2005). The major mechanism of action of

bromelain appears to be proteolytic in nature, although evidence suggests an immune modulatory and hormone like activity acting via intracellular signaling pathways. Bromelain has also been shown to reduce cell surface receptors such as hyaluronic receptor CD44, which is associated with leukocyte migration and induction of pro-inflammatory mediators (Tochi et al., 2008).

1.4.1.2 Ficin: It is extracted from latex of ficus and is a sulfhydryl proteinase with cysteine at the active site (EC 3.4.22.3). It preferentially cleaves at tyrosine and phenylalanine residues. Ficin has proven to be a versatile low cost biocatalyst useful in peptide synthesis (Sekizaki et al., 2008).

1.4.1.3 Papain: It is a cysteine protease (EC 3.4.22.2) extracted from latex of papaya (Carica papaya). The crude enzyme has broad specificity due to mixture of several proteases. The enzyme is active between pH 5 to 9 and is stable up to 80-90°C in presence of substrates. It consists of 212 amino acids stabilized by 3 disulfide bridges. Its catalytic triad is made up of 3 amino acids – cysteine-25 (from which it gets its classification), histidine-159, and asparagine-158. It is extensively used in tenderization of the meat (to break down the tough meat fibers), preparation of highly soluble and flavoured protein hydrolysates, dissociate cells in the first step of cell culture preparations, to make single cell preparation and an ingredient in some toothpastes and mints as teeth-whitener (Kim et al., 2004).

1.4.2 Animal proteases:

Pepsin, chymotrypsin, trypsin and rennin are widely used animal proteases.

1.4.2.1 Chymotrypsin: It is found in the pancreatic extract of animals (EC 3.4.21.1). The enzyme cleaves peptides at the carboxyl side of tyrosine, tryptophan and phenylalanine although over time it also hydrolyzes other amide bonds, particularly those with leucine-donated carboxyls (Zelisko and Jackowski 2004). It is present in zymogen form and is activated on cleavage by trypsin into two parts that are still connected via an S-S bond. Pure chymotrypsin has main applications in analytical and diagnostic field. It is extensively used in deallergenizing of milk protein hydrolysates.

1.4.2.2 Pepsin: It is a digestive protease (EC 3.4.23.1) released by the chief cell in the stomach of almost all vertebrates that function to degrade food proteins into peptides. Pepsin is produced in its zymogenic form i.e pepsinogen, whose primary structure has additional 44 amino acids. This zymogen is activated by hydrochloric acid (HCl), which is released from parietal cell in the

stomach lining (Hoorn, 2008). HCl creates an acidic environment which allows pepsinogen to unfold and cleave itself in an autocatalytic fashion, thereby generating pepsin. Pepsin functions best in acidic environments between pH 1 to 2 and is inactivated above pH 6. Pepsin cleaves preferentially safer to N-terminal of aromatic amino acids such as phenyalanine and tyrosine. It is an aspartyl protease and has resemblance with HIV-1 protease. Pepsin is commonly used in the preparation of F (ab) 2 fragments from antibodies.

1.4.2.3 Rennin: It is an aspartic acid protease (EC 3.4.23.4), produced as an inactive precursor, pro-rennin in stomachs of all nursing mammals but more specifically in the fourth stomach of calves. The specialized nature of the enzyme is due to its specificity in cleaving a single peptide bond in k-casein to generate insoluble para-k-casein and C-terminal glycopeptide. It cleaves the peptide bond between phenyalanine and methionine, the specific linkage between the hydrophobic (para-casien) and hydrophilic (acidic glycopeptide) group of casein in milk, since they are joined by phenylalanine and methionine (Zelisko and Jackowski, 2004). The hydrophobic group would unite together and would form a three dimensional network to trap the aqueous phase of the milk resulting in the formation of calcium phosphocaseinate. This specificity is used to bring about the extensive precipitation and curd formation in cheese making.

1.4.2.4 Trypsin: It is a serine protease (EC 3.4.21.4) found in the digestive system and is responsible for the breakdown of food proteins. Trypsin has an optimal operating pH and temperature of about 8 and 37°C respectively and predominantly cleaves proteins at the carboxyl side of the lysine and arginine (Engelking and Larry, 2015). Trypsin is commonly used in proteomics, since it has a very well defined specificity. Trypsin is also used for the preparation of bacterial media, to dissolve blood clots, treat inflammation and to dissociate dissected cells. Trypsin has limited application in food because the protein hydrolysatses generated have bitter taste (Rawlings and Barrett, 1994). Based on the ability of protease inhibitors to inhibit the enzyme from the insect gut, trypsin is targeted for bio-control of insect pests.

1.4.3 Microbial Protease:

As the plant and animal proteases are unable to meet the current world demand of the enzyme an increased interest in microbial source has grown. Microbes are the preferred source of proteases owing to their great biochemical diversity and susceptibility to genetic manipulation. They can be cultured in enormous quantities within a short span of time to ensure an abundant supply of

proteases. Extracellular nature of microbial proteases simplify the downstream processing of enzyme and have a long shelf life, with less stringent storage requirements. Only non-toxic and non-pathogenic microbes are used for commercial production and referred to as “genetically regarded as safe” (GRAS).

1.4.3.1 Bacteria: Bacteria secrete proteases to hydrolyze the peptide bonds in proteins and therefore break the proteins down into their constituent amino acids. Bacterial and fungal proteases are particularly important to the global carbon and nitrogen cycles in the recycling of proteins, and such activity tends to be regulated by nutritional signals in these organisms (Sims, 2006). The net effect of nutritional regulation of protease activity among the thousands of species present in soil can be observed at the overall microbial community level as proteins are broken down in response to carbon, nitrogen, or sulfur limitation (Sims and Wander, 2002)

Bacteria contain proteases responsible for general protein quality control (e.g. the AAA+ proteasome) by degrading unfolded or misfolded proteins. A secreted bacterial protease may also act as an exotoxin, and be an example of a virulence factor in bacterial pathogenesis (for example, exfoliative toxin). Bacterial exotoxic proteases destroy extracellular structures. By complex cooperative action the proteases may proceed as cascade reactions, which result in rapid and effective amplification of an organism’s response to a physiological signal.

Bacterial neutral proteases have low thermo tolerance and have an affinity for hydrophobic amino acids. Some of them are metalloproteases with a requirement for divalent metal ions for activity, others are serine proteases. Bacterial alkaline proteases are characterized by optimal activity at high pH, broad substrate specificity with optimal temperatures around 60°C which make them ideal for use in detergent industry. Bacillus sp. is a potent producer of neutral and alkaline proteases among bacteria and prominent among them are Bacillus licheniformis, Bacillus subtilis, Bacillus amyloliquefaciens etc. (Rao et al., 1998; Kumar et al., 1999; Schallmey et al., 2004; Fujinami and Fujisawa, 2010).  Aureobasidium pullulans, Issatchenkia orientalis and Yarrowia lipolytica (Li et al., 2009) and mushrooms have also been reported (Zhang et al., 2010; Zheng et al., 2011).

1.4.3.2 Viruses: Viral proteases are involved in processing of proteins that cause fatal diseases like AIDS and cancer. Mature enzymes encoded within the human immunodeficiency virus type

1 (HIV-1) genome protease (PR); reverse transcriptase (RT) and integrase (IN) are derived from proteolytic  processing  of  a  large  polyprotein  (Gag-Pol).  The  viral  PR  catalyzes  Gag-Pol

processing, which is active as a homodimer (Olivares et al., 2007). Most of the viral proteases are endopeptidases rather than exopeptidases (Rawling and Barrell 1993). Due to the involvement of viral proteases in pathogenesis extensive studies on the three dimensional structure of viral proteases and their interaction with synthetic proteases inhibitors have been undertaken with a view to designing potent inhibitors that can combat the diseases like AIDS.

Viruses are known to elaborate serine peptidases, aspartic peptidases and cystine peptidases, all of which are endopeptidases (Rawlings and Barrett, 1993). Methanococcus jannaschii, a thermophilic methanogen isolated from deep-sea was shown to produce a hyperthermophilic and barophilic protease (Amoozegara et al., 2007).

1.4.3.3 Fungi: Fungi synthesize wide variety of proteases than do bacteria. Filamentous fungi can effectively secrete various hydrolytic enzymes and one of the main groups of secreted enzymes in fungi is protease. They usually show better results when cultured in solid-state fermentation as compared to bacteria (Pandey et al., 1999). Fungi are known to produce acid, neutral, alkaline and metallo proteases. A single organism can produce more than one type of protease (Lindberg et al., 1982). Fungal proteases are active over a wide pH range (pH 4 to 11) and exhibit broad substrate specificity (Rao et al., 1998). One of the first known representatives of proteases was proteinase K, an alkaline enzyme from Engyodontium album also known as Tritirachium album (Kotlova et al., 2007). Trichoderma sp. have also been reported to produce more than one type of protease. (Chinnasamy et al., 2011; Parma et al., 2015).

1.5 Trichoderma sp.

Trichoderma is a genus of fungi in the family Hypocreaceae that are present in nearly all soils and other diverse habitats (Harman et al., 2004). In soil, they frequently are the most prevalent culturable fungi. They are favored by the presence of high levels of plant roots, which they colonize readily. Some strains are highly rhizosphere competent, i.e., able to colonize and grow on roots as they develop (Howell et al., 2000). In  addition to colonizing roots, Trichoderma sp. attack, parasitize  and  otherwise  gain  nutrition  from  other  fungi.  Since Trichoderma sp.  grow  and proliferate best when there are abundant healthy roots, they have evolved numerous mechanisms for both attack of other fungi and for enhancing plant and root growth (Altomare et al., 1999). The genomes  of  several Trichoderma species have  been  sequenced  and  are  publicly  available. Different species of Trichoderma have been used for the production of protease using submerged

fermentation technique. Jarana et al. (2002) isolated and characterized a gene that encodes an extracellular aspartyl protease from Trichoderma harzianum CECT 2413. Parma et al. (2015) also studied production of lytic enzymes using Trichoderma strains during in-vitro antagonism with Sclerotiun rolfsii.

1.5.1 Taxonomy: According to Wikipedia (2010), Trichoderma sp. are classified scientifically as follows

Kingdom            Fungi Division             Ascomycota Class                  Sordariomycetes Order                 Hypocreales Family               Hypocreaceae Genus                Trichoderma

1.5.2 Genetics: Most Trichoderma strains have no sexual stage but instead produce only asexual spores (Samuels, 2006). However, for a few strains the sexual stage is known, but not among strains that have usually been considered for biocontrol purposes. The sexual stage, when found, is  within  the  Ascomycetes  in  the  genus Hypocrea.  Traditional  taxonomy  was  based  upon differences in morphology, primarily of the asexual sporulation apparatus, but more molecular approaches are now being used. Consequently, the taxa recently have gone from nine to at least thirty-three species (Azin et al., 2007).

Most strains are highly adapted to an asexual life cycle. In the absence of meiosis, chromosome plasticity is the norm, and different strains have different numbers and sizes of chromosomes. Most cells have numerous nuclei, with some vegetative cells possessing more than 100 (Samuels, 2006). Various asexual genetic factors, such as parasexual recombination, mutation and other processes contribute to variation between nuclei in a single organism (thallus). Thus, the fungi are highly adaptable and evolve rapidly. There is great diversity in the genotype and phenotype of wild strains.

While wild strains are highly adaptable and may be heterokaryotic (contain nuclei of dissimilar genotype within a single organism) (and hence highly variable), strains used for biocontrol in commercial agriculture are, or should be, homokaryotic (nuclei are all genetically similar or

identical) (Bae et al., 2011). This, coupled with tight control of variation through genetic drift, allows these commercial strains to be genetically distinct and non-variable. This is an extremely important quality control item for any company wishing to commercialize these organisms.

1.5.3    Identification and Characterization of Trichodema sp.

1.5.3.1 Morphological Identification: The most common method used to identify fungal species is by using morphological characteristics which is based on similarity of the observable morphological features such as colony appearances and shapes of conidia and vesicles. Most Trichoderma species have been described by using morphological features to distinguish species especially in earlier studies (Jarana et al., 2002; Parma et al., 2015). Colonies are transparent at first on media such as cornmeal dextrose agar (CMD) or white on richer media such as potato dextrose agar (PDA). Mycelium are not typically obvious on CMD, conidia (T. virens, T. asperellum, and T. flavofuscum) typically form within one week in compact or loose tuffs in shades of green or yellow or less frequently white. A green pigment may be secreted into the agar, especially or PDA. Some species produce a characteristic sweet or “coconut’ odor. Due to variability and overlapping morphological characteristics, misidentification can occur. However, morphological identification is still widely used, even though the characteristics could not be used to accurately identify species to species level but enough to give indication or clues of sections or species complex (Balajee et al., 2008). The survey by American Society for Microbiology (Seema and James, 2005) reported that 89 % of laboratories in the United States are still using morphological methods as the isolation and cultural identification are easy, quick and more affordable compared to molecular methods. In a few studies, identification solely based on morphological characteristics has been applied (Gupta et al., 2005; Chekireb et al., 2009)

1.5.3.2 Molecular Identification and Characterization: Molecular characterization means characterizing  at  the molecular level  without  any  effect  of environment  or development  or physiological state of the organism. That is why DNA based markers are called molecular makers and characterization using these molecular characterization. Molecular identification and characterization using DNA sequencing is widely used. DNA sequencing used for identification very much depends on the target locus. The locus should be ortologous, have a high level of interspecies  variation  with  low  intraspecific  variation,  easy  to  amplify  and  a  standardized

“universal” primer set, and the locus should not undergo recombination (White et al., 1990; Balajee et al., 2007).

The internal transcribed spacer (ITS) region has been proposed as the prime fungal barcode as it satisfies the requirement of a “universal” marker including highly variable within taxonomically distinct fungal species and also easy to be amplified (Samson et al., 2010). The region is part of nuclear ribosomal with two segments: ITS1 and ITS2 divided by 5.8S rDNA and located between nuclear small- and large- subunit rRNA genes. The region can be used as the initial step of identification including an unknown fungal isolates to be categorized into appropriate genus, subgenus and section up to species level (Balajee et al., 2009)

1.6 Protease Production

Proteases may be derived from a wide variety of plants, animals and microorganisms. However, most proteases are obtained from microbial sources especially in fungi. In many cases, isoenzyme forms of the enzyme have been identified (Manjunah and Rao, 1979). Many microorganism possess proteolytic activity and some of the extracellular proteases isolated are serine proteases which enable the degradation of proteins and peptides in the natural environment (Oren, 2002; DeCastro et al., 2006). Enzymes which exhibit optimal activities at various ranges of salt concentration, pH and temperature are of tremendous importance for industrial processes (Rohban et al., 2009).

The total cost of enzyme production and downstream processing play a major role in the successful application of technology in enzyme production. Optimization of process parameters assesses the effect of media components on growth of microorganism and enzyme production. Beg et al. (2003), stated that the traditional one-at-a-time approach leads to increase in production, but it does not take into consideration the interaction of different physico-chemical parameters. Other strategies for increasing protease production include screening for strains which produce high amount of proteases, cloning and over-expression, optimization of fermentation media etc. (Gupta et al., 2002).

Several statistical and non-statistical methods have been used to study the effect of medium components on protease production (Montgomery, 2002; Akolkar, 2009). In order to achieve maximum enzyme production, the composition of media is different for different microorganisms, hence the media constituents and concentrations have to be optimized. Optimum production of enzymes involves efficient and economical process development (Oskouie et al., 2008). Stastical

optimization of growth medium for protease production by Haloferax lucentensis by response surface methodology was reported by Manikandan et al. (2011).

1.6.1 Submerged fermentation (SmF) for Protease production

Proteases are usually produced during the stationary phase and their production is regulated by carbon and nitrogen stress. Submerged fermentation (SmF) is used for protease production. Submerged fermentation (SmF) technique is the cultivation of microorganisms on liquid broth. SmF system for enzyme production are generally conducted in stirred reactors under aerobic conditions or fed batch systems (Bali, 2003). Here, the microbes are cultivated in substrates submerged or dissolved in aqueous phase. In this case, the different parameters can be monitored and controlled and hence, the process of scale up from laboratory scale to industry is much easier (Paul, 2005).

1.6.2 Solid State Fermentation (SSF) for Protease production

In solid state fermentation (SSF), microbes are grown on moist solid substrates without free- flowing water. Use of cheap substrates, less requirement for water and production of metabolites in concentrated form resulting in downstream processing which is less expensive and time consuming, make SSF a very economical alternative for enzyme production. Alkaline protease production using Aspergillus versicolor by solid state fermentation was described by Choudhary and Jain (2012).

1.6.3    Factors Affecting Microbial Protease Production

For commercial practice, the optimization of medium composition is done to maintain a balance between the several medium components in production media. Optimization helps minimizing the amount of unutilized components at the end of fermentation. In addition, there are no defined medium established for the best production of alkaline serine proteases from different microbial sources. Each organism or strain has its own nutritional requirement for maximum enzyme production.

1.6.3.1 Age of inoculum: The effect of age of inoculum on alkaline protease production by some bacteria such as Bacillus licheniformis (Sen and Satyanarayana, 1993) and Streptomyces rectus var. proteolyticus (Mizuzawa et al., 1969) has been studied. The age of inoculum has been reported to be not influencing the production by these bacteria.

1.6.3.2 Size of inoculum: The size of inoculum has been reported to have influence on the yield of alkaline protease in some bacteria. Sinha and Satyanarayana (1991) who studied the alkaline protease production by Bacillus licheniformis N3 have reported good yield with the use of inoculum at 0.5-8 % level. Sen and Satyanarayana (1993) have observed that an inoculum level of

2 % was favorable for enzyme production by Bacillus licheniformis S40. For the enzyme production by Bacillus coagulan PB77, the reported optimum level of inoculum was 4 % (Gaiju et al., 1996). A higher inoculum level, 5-10 % has been reported to be optimum for the enzyme production by Streptomyces rectus var. proteolyticus (Mizusawa et al., 1969)

1.6.3.3 Effect of Carbon source: Glucose is frequently used in bioprocesses for protease production. Studies have also indicated a reduction in protease production due to catabolic repression by glucose (Hanlon et al., 1982; Frankena et al., 1985; Frankena et al., 1986; Kole et al., 1988). Increased yields of alkaline proteases were also reported by several workers in the presence of different sugars such as lactose (Malathi and Chakraborty, 1991), maltose (Tsuchiya et al., 1991), sucrose (Phadatare et al., 1993) and fructose (Sen and Satyanarayana, 1993). In commercial practice, high carbohydrate concentrations repressed enzyme production. Therefore, carbohydrate was added either continuously or in aliquots throughout the fermentation to supplement the exhausted component and keep the volume limited and thereby reduce the power requirements (Aunstrup, 1980). Whey, a waste byproduct of the dairy industry containing mainly lactose and salts, has been demonstrated as a potential substrate for alkaline protease production (McKay, 1992; Donaghy and McKay, 1993). Similarly, maximum alkaline protease secretion was observed in the presence of pure cellulose (Solka-floc) as the principal carbon source (Gusek et al., 1988). Molasses used as carbon source for protease production (Mabrouk et al., 1998). Molasses contains sucrose, glucose, fructose and important minerals such as calcium, iron and magnesium.

1.6.3.4 Effect of Nitrogen source: Most microorganisms can utilize both inorganic and organic forms of nitrogen which are essential to produce amino acids, nucleic acids, proteins and other cell wall components. The alkaline protease comprises 15.6 % nitrogen (Kole et al., 1988) and its production is dependent on the availability of both carbon and nitrogen sources in the medium (Kole et al., 1988). The complex nitrogen sources are usually used for alkaline protease production. Enzyme synthesis was found to be repressed by rapidly metabolizable nitrogen sources

such as amino acids or ammonium ion concentrations in the medium (Cruegar and Cruegar, 1984; Frankena et al., 1986; Giesecke et al., 1991). Soybean meal was also reported to be a suitable nitrogen source for protease production (Chandrasekaran and Dhar, 1983; Tsai et al., 1988; Sen and Satyanarayana, 1993; Cheng et al., 1995). Urea used as a nitrogen source for protease production (Mabrouk et al., 1998).

1.6.3.5 Effect of pH on Production Medium: The important characteristic of most alkalophilic microorganisms is their strong dependence on the extracellular pH for cell growth and enzyme production. For increased protease production from these alkalophiles, the pH of the medium must be maintained above 7.5 throughout the fermentation period (Aunstrup, 1980). The advantage in the use of carbonate in the medium for an alkaline protease has been well demonstrated (Horikoshi and Akiba, 1982). In another study, Das and Prasad (2010) considered the pH of 8.0 as the best pH for protease production.

1.6.3.6 Aeration and agitation: During fermentation, the aeration rate indirectly specifies the dissolved oxygen level in the fermentation broth. Different dissolved oxygen profiles can be obtained by: (i) variations in the aeration rate; (ii) variations in the agitation speed of the bioreactor; or (iii) use of oxygen-rich or oxygen-deficient gas phase (appropriate air-oxygen or air-nitrogen mixtures) as the oxygen source (Michalik, 1985; Moon and Parulekar, 1991). The variation in the agitation speed influences the extent of mixing in the shake flasks or the bioreactor and will also affect the nutrient availability. Optimum yields of alkaline protease are produced at 200 rpm for B. subtilis (Chu et al., 1992) and B. licheniformis (Sen and Satyanarayana, 1993).

The production of an enzyme exhibits a characteristic relationship with regard to the growth phase of that organism. In general, the synthesis of protease in Bacillus species is constitutive or partially inducible and is controlled by several complex mechanisms operative during the transition state between exponential growth and the stationary phase (Priest, 1977; Strauch and Hoch, 1993). The production of extracellular proteases during the stationary phase of growth is characteristic of many bacterial species (Priest et al., 1977). The sequence as well as the rate of enzyme production is, however, variable with the specific organism. At early stationary phase, two or more proteases are secreted and the ratio of the amount of the individual proteases produced also varied with the Bacillus strains (Uehara et al., 1974; Priest, 1977). In several cases, the function of the enzyme is not very clear, but its synthesis is connected with the onset of a high rate of protein turnover and often sporulation (Power and Adams, 1986; Chu et al., 1992).

1.6.4 Purification of Protease

Purification of proteases is vital for the understanding of their properties and action. Most proteases are extracellular in nature. The culture of microorganism is separated from the fermented broth by centrifugation or filtration and the culture supernatant is subjected to different methods of purification. In case the protease is intracellular, suitable methods for cell disruption are to be adopted (Walsh, 2001). Different methods have been used for downstream processing to purify extracellular proteases from the culture broth. Commonly used methods include ammonium sulphate precipitation followed by affinity chromatography and gel filtration (Kumar et al., 1999). Ammonium sulphate is used for precipitating proteases from culture supernatant. Ammonium sulphate is inexpensive, highly soluble and does not denature the proteases, hence this is preferred by many researchers (Adinarayana et al., 2003; Anita and Rabeeth, 2010).

Gel filtration using Sephacryl (Kumar et al., 1999), Sepharose (Singh et al., 2001) and Sephadex (Adinarayana et al., 2003) is used for separation based on size. The desired protein gets diluted and this method has lower capacity for loading proteins. Gel filtration is suitable for biomolecules such as proteins sensitive to changes in pH, concentrations of metal ions or co-factors, and harsh environmental conditions (James  et al., 1997). Gel filtration  can be used for protein  DNA purification, buffer exchange and desalting. It requires mild conditions and is relatively simple to carry out (Devasena, 2010). The buffer is chosen based on the type of material to be separated. As the buffer is introduced into the column, specific volumes of the fractions are collected over time (Acharya et al., 2014). Large molecules elute faster because they do not enter the pores in the beads, this is followed by the elution of medium sized molecules and finally small sized molecules which are able to penetrate the pores of the beads in the gel before elution. The size of the pores in the gel also affects the rate at which the components of the biomolecules travel through the gel. The different fractions collected are tested for the presence of the desired compound to be separated or purified (Masuda et al., 1988; Jebor et al., 2014).

1.7 Biochemical and Kinetic Properties of Protease

1.7.1 pH optima and Stability of Protease.

Effect of pH is important in the neighborhood of the active sites, which will overall affect the activity, structural stability and solubility of the enzyme (Chaplin and Bucke, 1990). Most of the commercially available subtilisin type proteases are also active in the pH range of 8-12 (Gupta et

al., 2002). Alkaline proteases of the genus Bacillus show an optimal activity and a good stability at high alkaline pH values (Margesin et al., 1992). According to Sun et al. (2009), the enzyme, protease, was more active between pH 7.0 – 10.0 (Sun and Xu, 2009). Bajaj and Sharma (2011) also reported that B. subtilis I-2 protease showed optimum activity at pH 8.0.

The function of protein is absolutely dependent on its three-dimensional structure. Changes in pH may alter electrostatic interactions between charged amino acids; thereby inducing conformational changes in the three-dimensional structure of the enzyme leading to reduced substrate binding and/or catalytic activity. Changes in pH may not only affect the shape of an enzyme but it may also change the shape or charge properties of the substrates (Bajaj and Manhas, 2012; Bajaj and Sharma, 2011; Singh and Bajaj, 2015). According to Bajaj et al., (2014), stability at pH 7.0-10.0, suggests potential application of this enzyme in various biotechnological industries (Bajaj et al.,

2014). The enzyme also retained up to 65 % stability at pH 11 and 12. pH-stability analysis of B. cereus K-3 protease indicate that protease has maximum stability at pH 8-10 for 1-2 h. However, at acidic or high alkaline pH activity decreased. Furthermore, activity reduction was observed to be more drastic towards acidic range as compared to alkaline.

1.7.2 Temperature optima and Thermostabilty of Protease

The heat  stability  of enzymes  is  affected  by  at  least  two  factors  alone  or in  combination. Polysaccharides and divalent cations, if any, can stabilize the molecule (Öztürk, 2001). Sun and Xu (2009) reported that, optimum temperature for protease activity was observed at 40 oC with substantial activity between 30 oC and 50 oC (Sun and Xu, 2009; Sun et al., 2009). Studies reported by Bushra et al., (2010) stated that, the optimum temperature of the purified protease from A. niger was 50 oC. The result revealed that the protease retained a considerable amount of its activity at low temperatures. The protease retained up to 94 % of its original activity at 50 °C. It reduced to about 28 % of the original activity at 80 °C, This indicates that the protease was considerably stable at low temperatures. The optimum temperature of the protease is consistent with the protease of A. terreus 50 °C (Bushra et al., 2010); and A. oryzae 50 °C (Sumantha et al., 2005). Thermostablity refers to prolonged stability of enzyme at high temperatures. It is also the ability of enzyme to resist against thermal unfolding in the absence of substrate, while thermophilicity is the capability of enzymes to work at elevated temperatures in the presence of substrate (Sarath- Babu et al., 2004, Bhatti et al., 2006). The thermostablity mechanisms for thermozymes are varied and depend upon molecular interactions such as hydrogen bonds, electrostatic and hydrophobic

interactions, disulfide bonds, and metal binding which can promote a superior conformational structure for the enzyme (Bajaj and Sharma, 2011). Thermal denaturation may occur in two steps; N—U—D

Where, N is native enzyme, U is unfolded enzyme that could be reversibly refolded upon cooling and D is the denatured enzyme formed after prolonged exposure to heat and therefore cannot be recovered on cooling. In recent years, interest in thermostable enzymes has increased dramatically as resistance to thermal inactivation has become a desirable property of the enzymes used in many industrial applications. Thermostable enzymes are generally defined as those with an optimum temperature above that of the maximum growth of an organism or with exceptional stability above

50 ºC over an extended period of time (Singh et al., 2000). One of the ways to identify enzymes, which are thermally stable, is to exploit natural sources. Different agents like temperature and chemicals promote enzyme inactivation. Temperature produces opposed effects on enzyme activity and stability and it is therefore a key variable in any biocatalytic process (Wasserman,

1984). Biocatalyst stability which is the capacity to retain activity through time is undoubtedly the limiting factor in most bioprocesses, biocatalyst stabilization being the central issue of biotechnology (Illanes, 1999). Biocatalyst thermostablity allow a higher operation temperature, which is clearly advantageous because of higher reactivity, higher process yield, lower viscosity and fewer contamination problem (Mozhaev, 1993). Enzyme thermal inactivation is the consequence of weakening the intermolecular forces responsible for the preservation of its 3D- structure, leading to a reduction in its catalytic capacity (Misset, 1993). Inactivation may involve covalent or non-covalent bond disruption with subsequent molecular aggregation or improper folding (Bommarius and Broering, 2005).  Even though there is no firm evidence to suggest that thermostable enzymes are necessarily derived from thermophilic organisms, however there is a greater chance of finding thermostable proteins from thermophilic bacteria (Rahman et al., 1994). A wide range of microbial proteases from thermophilic species has been extensively purified and characterized. These include Thermus sp., Desulfurococcus strain Tok12S1 and Bacillus sp. Among them alkaline proteases derived from alkaliphilic bacilli, are known to be active and stable in highly alkaline conditions (Rahman et al., 1994). The earliest thermophilic and alkaliphilic Bacillus sp. was B. stearothermophilus strain F1 isolated by Salleh and friends in 1977, which was stable at 60 ºC (Rahman et al., 1994; Haki and Rakshit, 2003).  Bajaj et al. (2014) reported, that thermostablity of B. cereus K-3 protease was thoroughly stable at 50-70 °C for 40 min, however,

there was abrupt decrease in activity at higher temperature (>80 °C). However, 80 min pre- incubation of enzyme lead to gradual activity reduction at 60°C and above. There are several reports of thermostable protease production from Bacillus spp. like B. pumilus KS12 (Rajput and Gupta, 2013), B. licheniformis KBDL4 (Pathak and Deshmukh, 2012) and B. cereus SIU1 (Singh et al., 2012). In addition, Sun and Xu (2009) also observed that thermo stability was measured to be stable at 40 oC – 60 oC (Sun et al., 2009). Various thermostablity parameters, such as half-life of the enzyme preparation (t1/2), D-value, Z-value, the activation energy of thermal inactivation (Ea), enthalpy of activation of the thermal inactivation (ΔH o), entropy of activation of thermal inactivation (ΔS o) and the Gibbs free energy of activation of thermal inactivation (ΔG o) were used to evaluate the thermostablity of protease.

1.7.3 Metal Ions

Alkaline proteases require a divalent cation like Ca2+, Mg2+ and Mn2+ or a combination of these cations, for maximum activity. These cations were also found to enhance the thermal stability of a Bacillus alkaline protease. It is believed that these cations protect the enzyme against thermal denaturation and play a vital role in maintaining the active conformation of the enzyme at high temperatures (Kumar and Takagi, 1999). In particular metal ions like Ca2+, Mg2+ and Mn2+ ions either individually or in combination have been found to give maximal activity to the enzyme. Especially the metal ion Ca2+  is reported to increase activity and thermal stability of alkaline protease at increased temperatures (Kumar, 2002). Other metal ions that are used for stabilizing proteases include Ba2+, Co2+, Fe3+ and Zn2+ (Johnvesly and Naik, 2001). B. cereus NS-2 fibrinolytic protease activity was increased in the presence of Fe2+, Ca2+, Mn2+, Zn2+, Cu2+  and Mg2+. However, Pb2+ and Hg2+ strongly inhibited protease activity (Bajaj et al., 2014).

1.8 Biotechnological Applications of Proteases

Alkaline proteases are robust enzymes with considerable industrial potential in detergents, leather processing, pharmaceuticals, food and feed processing and silver recovery. The different applications currently using alkaline proteases are:

1.8.1 Detergent Industry

Proteases find application in laundry detergent and dish washing detergents and cleaning detergents (Godfrey and West, 1996; Showell, 1999). Enzymes have been added to laundry detergents since last 50 years to facilitate the release of proteinaceous material in stains such as

those of milk and blood. The proteinaceous dirt coagulates on the fabric in the absence of proteinases as a result of washing condition. The enzyme removes not only the stain, such as blood, but also other materials including proteins from body secretion and food such as milk, egg, fish and meat. An ideal detergent enzyme should be stable and active in the detergent solution and should have adequate temperature stability to be effective in a wide range of washing temperature (Aurachalam et al., 2009). Burnus the first enzymatic detergent produced in 1913, consisted of crude pancreatic extract and sodium carbonate. BIO-40, the first detergent containing bacterial enzyme was produced in 1956.

The protease is most suitable for detergent application if its isoelectric pH (pI) coincides with the pH of the detergent solution. More recently a variety of bacterial proteases active and stable at alkaline pH also stable in oxidizing agents, bleach and SDS are found to be suitable for detergent applications (Kumar and Takagi, 1999; Gupta et al., 2002). Some of the fungal proteases are also reported to be suitable for detergent application (Phadatare et al., 1993; Tanksale et al., 2001; Hajji et al., 2007). Recently, alkaline proteases from Bacillus cereus, Bacillus pumilus strain CBS, Streptomyces sp. strain AB1, Bacillus licheniformis, Aspergillus flavus, Aspergillus niger, Bacillus brevis, Bacillus subtilis AG-1 have exhibited excellent deteregent compatibily in the presence of certain stabilizers such CaCl2 and glycine (Abou-Elela et al., 2011; Bezawada et al., 2011; Jaouadi et al., 2011).

1.8.2 Leather Industry

Alkaline proteases are used in three important steps of leather tanning, example;

• Soaking: In 1987, Taylor et.al, reported that Monsheimer and Pfleider used alkaline proteases in soaking from Bacterial and fungal sources and they claimed that this reduced the need for the liming chemicals by 30 to 60 %. Proteases such as alcalase from novo industry and Milenzyme from Miles laboratories are used in soaking as they are compatible with the surfactants and sodium chloride which are used to prevent microbial spoilage of the hide. The advantages of enzyme soaking includes the shortening of the wetting back time, loosening of the scud and initiating of opening of the fibre structure (Taylor et al., 1987)

• Dehairing: Dehairing removes hair from the skin. Alkaline proteases from the obligate alkalophilic Bacillus spp perform the dehairing of hides by attacking the various proteins of hide, especially  the  matrix  proteins,  elastin  and  keratin.  Alkaline  proteolytic  keratinases  from

Chrysosporium keratinophilum can be important in leather tanning industries in preference to traditional methods involving sodium sulphide (Dozie et al., 1994). Proteases reduce the use of lime and sulphide up to 50 % required in non-enzymatic process (Kalisz, 1988). Alkaline proteases carry out the process at pH 8.0-10.0 and 35-40 oC for 6 hr. The hair is removed at the root rather than broken off the skin surface as in the case with the lime sulphide method.

• Bating: Several species of bacteria including Bacillus subtilis produce neutral and alkaline protease which is suitable for bating (Pvanakrishnan Dhar, 1986).

1.8.3 Textile Industry

Proteases find application in processing of wool and degumming of silk. Sericin which constitutes about 25 % of weight of raw silk fibres confers a rough texture to raw silk fibres. The thermo stability and their activity at high pH and the mitigation of pollution characteristic have made proteolytic enzymes an ideal candidate for laundry applications. Conventional methods used to remove sericin from inner core of fibroin is by conducting shrink-proofing and twist-setting for the silk yarns using starch (Kanehisa, 2000). This is an expensive process, and the alternative method is the use of proteases for degumming of silk prior to dyeing (Freddi et al., 2003). Proteases are used to wash printing screens to remove proteinaceous gums used for thickening of printing pastes. Bio-polishing and bio-stoning are the current trends in the area of enzyme processing (Ramachandran and Karthik, 2004).

1.8.4 Pharmaceutical Industry

Proteases execute a large variety of functions, ranging from the cellular level to the organ and organism level, to produce cascade systems such as haemostasis and inflammation, which are responsible for the complex processes involved in the normal physiology of the cell as well as in abnormal pathophysiological conditions. Their involvement in the life cycle of disease causing organisms has led them to become a prospective target for developing therapeutic agents against fatal diseases such as cancer and AIDS (Mala et al., 1998). Microbial proteases are increasingly used in treatment of various disorders namely cancer, inflammation, cardiovascular disorders, and necrotic  wounds  (Chanalia  et  al.,  2011).  Proteases  are used  an  immune stimulatory  agents (Biziulenvicius, 2006). Increased antibiotic concentration at a target site when protease was concomitantly used with an antibiotic (Okumura et al., 1997). Proteases are used extensively in the pharmaceutical industry for preparation of medicines such as ointments for debridement of

wounds. It is also used in denture cleaners and as contact-lens enzyme cleaners (Gupta et al., 2002).

The immobilized alkaline protease from Bacillus subtilis possessing therapeutic properties has been used for development of ointment compositions, soft gel-based medicinal formulas, gauze, non-woven tissues and new bandage materials (Davidenko, 1999; Mukherjee et al., 2011). Rao et al. (1998) have reported the oral administration of proteases from Aspergillus oryzae to correct certain lytic enzyme deficiency syndromes. Broad spectrum antibiotics in combination with clostridia collagenase or subtilisin, is used to treat burns and wounds. Aqua-Biotechnology has launched a skin care product Zonase XTM, which removes the dead cells in the outer layers of the human skin and accelerates the renewal and healing process of the skin.

1.8.5 Food and Feed Industry

Proteases are used for preparation of fish sauce and soy sauce as they are prepared in high salt (20-

30%) containing brines (Yongsawatdigul et al., 2007). Proteases are used for the manufacture of protein rich therapeutic diets, hypoallergenic infant food formulations and also fortification of fruit juices and soft drinks. Alkaline protease from Bacillus licheniformis is used for the production of protein hydrolysate with angiotensin I converting enzyme inhibitor activity from sardine muscles; used in blood pressure regulation (Matsui et al., 1993). The brewing industry uses Neutrase (neutral protease) insensitive to natural plant proteinase inhibitors (Rao et al., 1998). The major application of proteases in the dairy industry is in the manufacture of cheese. The milk-coagulating enzymes fall into three main categories: animal rennets, microbial milk coagulants, and genetically engineered chymosin.

Both animal and microbial milk coagulating proteases belong to a class of acid aspartate proteases and have molecular weights between 30,000 kDa and 40,000 kDa (Yongsawatdigul et al., 2007). Rennet extracted from the fourth stomach of unweaned calves contains the highest ratio of chymosin to pepsin activity. A world shortage of calf rennet due to the increased demand for cheese  production  has  intensified  the search  for alternative microbial  milk  coagulants.  The microbial enzymes exhibited two major drawbacks, i.e. the presence of high levels of nonspecific and heat-stable proteases, which led to the development of bitterness in cheese after storage; and a poor yield.

Extensive research in this area has resulted in the production of enzymes that are completely inactivated at normal pasteurization temperatures and contain very low levels of nonspecific

proteases. In cheese making, the primary function of proteases is to hydrolyze the particular peptide bond to generate para-k-casein and macropeptides. Chymosin is preferred due to its high specificity for casein, which is responsible for its excellent performance in cheese making (Allen et al., 1986).

Wheat flour is a major component of baking processes. It contains an insoluble protein called gluten, which determines the properties of the bakery dough. Endo and exo-proteinases from Aspergillus oryzae have been used to modify wheat gluten by limited proteolysis. Enzymatic treatment of the dough facilitates its handling and machining and permits the production of a wider range of products. The addition of proteases reduces the mixing time and results in increased loaf volumes. Bacterial proteases are used to improve the extensibility and strength of the dough (Argos, 1987).

Alkaline elastase (Takagi et al., 1992) and alkaline protease (Wilson et al., 1992) are meat tenderizing enzymes possessing the ability to hydrolyze muscle fibre proteins as well as connective tissue proteins. Alkaline protease (B72) from Bacillus subtilis and B. licheniformis PWD-1 were used for production of proteinaceous fodder from feather keratin (Dalev, 1990, 1994; Cheng et al.,1995).

1.8.6 Silver Recovery

Silver is used in photographic industry in vast quantities. A photographic film is made up of a support layer (glass, plastic sheet, or paper) coated with an emulsion layer consisting of silver halide crystals in gelatin (Moore et al., 1996). Proteases can be used for recovery of silver from used X-ray films which contain about 1.5 -2% (w/w) silver in its gelatin layers (Nakiboglu et al., 2001). Silver can be recovered by using chemical solutions to strip gelatin silver layer (Syed et al.,

2002) and also by oxidation of silver following electrolysis (Ajiwe and Anyadiegwn, 2000). Stripping of gelatin using chemicals is hazardous, not economical and time consuming (Sankar et al., 2010). An increase in temperature was reported to cause increase in gelatin hydrolysis by Sankar et al. (2010). Nakiboglu et al. (2001) reported that 50ºC was optimum for stripping gelatin at the optimum pH 8 by enzyme of Bacillus subtilis ATCC 6633. Sankar et al. (2010) found that pH 10 is effective. Recovery of silver from used X- ray films by burning causes environmental pollution which can be overcome by the use of microbial enzymes, which also enables the polyester film base to be recycled (Kumar and Takagi, 1999).

1.9 Aim and Objectives of the Study

1.9.1 Aim of the study

This study was aimed at the isolation, molecular identification, production, partial purification, and characterization of protease from fungi obtained from laundry waste water disposing site at Odim gate, University of Nigeria Nsukka.

1.9.2 Specific Objectives of the Study

This research was designed to;

✓   Isolate protease producing fungi from laundry waste water disposal site

✓   Screen the fungal isolates for protease production

✓   Carry out morphological and molecular characterization of the fungi isolate

✓   Determine the incubation period for maximum protease production

✓   Optimize protease production based on the effect of pH

✓   Mass produce protease from the fungal isolate at the optimum condition

✓   Partially purify protease by ammonium sulphate precipitation, dialysis and gel filtration

✓   Characterize the partially purified protease ✓   Determine the pH and thermal stability assessment of the enzyme

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