1. Introduction

1.1 Enzymes

Enzymes are biomolecular machines of exquisite catalytic efficiency.

The term ‘Enzyme’ was first coined by Kuhne in 1878. Alike chemical

catalysts, enzymes too reduce the free energy of activation and speed-up

the reaction without altering the equilibrium. The database BRENDA

(BRaunschweig ENzyme DAtabase) has literature on enzymes and data

for all enzyme classes (~4800 entries in six main classes in 2008) that

have been classified according to the EC scheme of the IUBMB

[International Union of Biochemistry and Molecular Biology, (Chang et al,

2009) irrespectively of the enzyme's source.

Enzymes efficiently conduct reactions in biological systems where

prevailing physiological conditions such as pH and temperature are not

sufficient enough to support life processes like generation of nerve

impulses and intense muscular activity (Delvin, 1986). Enzymes remain

unchanged during the course of their action on a substrate. Although

most enzymes are proteins, there are few exceptions like Ribozymes.

Enzymes in some cases require certain additional elements like co-

factors and co-enzymes for their activity. They are either monomeric

(made of single polypeptide chain) or oligomeric (made of 2 or more

polypeptide chains). The important characteristic features of enzymes

include: Substrate specificity, Operation under mild conditions,

Sterospecificity, Remain unaltered by reaction, Reusability and Natural

and eco-friendly.

Recent times have witnessed an increased environmental

consciousness among the global community. The unsurpassed selectivity

and operation under mild physiological conditions conjoined with their

eco-friendly nature are the prime factors that are leading to replacement

of time-honored chemical processes with enzymes. In the context of

present times it may not be inappropriate to say that the base of

industrial biotechnology is perched on biocatalysis. Application of

enzymes reduces expulsion of wastes and contaminants from processes

and aids in development of sustainable technologies with less energy

requirements. Advances achieved in the fields of recombinant technology,

mutagenesis, proteomics and genomics have revolutionized the field of

enzymology. These technicalities facilitate in development of stable and

effective enzymes with high catalytic potential. Further they also help in

enhancing the production of enzymes in host systems of choice.

Figure 1.1: Enzyme development in State of art and Classical modes

1.1.1 Classification, Applications and Sources of Enzymes

Enzymes are ubiquitous in nature and are produced by almost all life

forms. Enzymes are either intracellular or extracellular. Some enzymes

are bound to cell membranes and few others are compartmentalized

inside cell organelles depending on their catalytic role. Extracellular

enzymes mostly catabolize nutrients and facilitate their entry into cell

where as intracellular enzymes are essentially involved in synthesis,

catabolic and metabolic activities of the cell.

Enzymes are divided into 6 classes depending on the type of reaction

they catalyze (Table 1.1):

1. Oxidoreductases,

2. Transferases

3. Hydrolases

4. Lyases

5. Isomerases

6. Ligases.

The classification of enzymes with specific examples for each class of

enzymes is presented in Table 1.1.

Despite the contending reservations such as limited substrate

specificity, limited availability, requirement of co-factors, limited stability

of protein catalyst, voiced against enzymes, still biocatalysts hold a huge

share in the catalyst market. According to a recent market research held

by BCC research group, global enzyme market is expected to hit $2.7b

mark in 2012 (www.bccresearch.com).

A-la-carte of applications are offered by enzymes and they are used in

various fields like food and beverages, feed, detergents, paper and pulp,

leather industries, organic synthesis, diagnostics, clinical analysis,

pharmaceuticals, etc (Table 1.2). There is an increase in the demand for

specialty enzymes with applications in analytics, personal care products

and DNA technology.

Table 1.1 Enzyme Classification with reaction catalyzed and

examples

Enzyme class Reaction catalyzed Examples

Oxidoreductases

Oxidation Reduction AH2 + B A + BH2

Alcohol dehydrogenase Cytochrome oxidase

Transferases

Group transfer A-X + B A + B-X

Hexokinase Transaminases Transmethylases Phosphorylase

Hydrolases

Hydrolysis A-B + H2O AH + BOH

Protease Lipase Choline esterase Alkaline phosphatase Urease

Lyases Addition Elimination A-B + X-Y AX-BY

Aldolase Fumarase Histidase

Isomerases

Interconversion of isomers A A’

Triose phosphate isomerase Retinine isomerase Glucose phosphate isomerase

Ligases

Condensation

(usually dependent on ATP)

Glutamine synthase Acetyl coA carboxylase Succinate thiokinase

Table:1.2 Enzymes used in industrial applications

Table 1.3 A list of enzymes of each class with usage in industrial

processes

Class Industrial enzymes

1: Oxidoreductases Catalases

Glucose oxidases

Laccases

2: Transferases Fructosyltransferases

Glucosyltransferases

3: Hydrolases Amylases

Cellulases

Lipases

Mannanases

Pectinases

Phytases

Protease

Pullulanases

Xylanases

4: Lyases Pectate

Lyases

Alpha acetolactate decarboxylases

5: Isomerases Glucose isomerases

6: Ligases Not used at present

Enzymes are ubiquitous in nature and are produced by almost all

living forms. Enzymes of plant and animal origin are well studied and

extensively investigated for applications. But when it comes to

production of enzymes on a commercial scale, microorganisms remain as

ideal sources. Microbes are protein factories with ability to produce

diverse enzymes. They are harnessed to produce high quantities of

enzymes for commercial purposes. Microorganisms due to their short

doubling times, moderate nutritional requirements, ease of genetic

manipulation and highly adaptable nature offer several advantages over

other sources of enzymes.

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1.1.2 Hydrolases

Hydrolases (EC 3) are placed in class of enzyme classification. They

catalyze the cleavage of bonds with involvement of water molecules.

These enzymes catalyze the cleavage of C–O, C–N, C–C bonds with either

removal or addition of water molecules. Hydrolases are involved in

reactions such as hydrolysis, condensations and alchoholysis.

Hydrolases is one class with a large group of industrially important

enzymes. One predominant class of enzymes among hydrolases is

proteases.

1.2 Proteases EC 3.4

Proteases are a complex group of ubiquitous enzymes in nature with

immense physiological and commercial significance. The term ‘Protease’

refers to a group of enzymes that catalyze the cleavage of peptide bonds

in protein substrates. According to EC nomenclature of enzymes,

proteases are placed in class 3 i.e., Hydrolases and subclass EC 3.4,

i.e. Peptidases (peptide bond hydrolases). As portrayed by few

researchers, Proteases are ‘Mother Nature’s Swiss army Knives’ which

cleave long sequences of amino acids into shorter fragments and

component amino acids with utmost substrate and site specificity (Hong-

Bin and Kuo-Chen, 2009). Proteases essentially influence the vital

metabolic regulations and ultimately survival of a cell at various levels

via peptide hydrolysis (Charles, 1997). The process of peptide hydrolysis

is a substrate specific and site –directed action that directly affects the

synthesis, composition, size, shape, turnover and finally destruction of

proteins (Hong-Bin and Kuo-Chen, 2009).

1.2.1 Physiological Significance of Proteases

Regardless of the taxonomic kingdom they belong to, virtually all

biological systems produce proteases. Although proteases catalyze a

single reaction i.e., hydrolysis of peptide bond, the various ways with

which they carry out this reaction and multiple positioning of these

enzymes in cells allows them to play a crucial role in numerous

physiological and pathophysiological processes due to. Several studies

conducted till date have proven the regulatory role played by proteases in

almost all the decisive aspects of life like conception, birth, growth,

maturation, aging and death of all organisms (Hong-Bin and Kuo-Chen,

2009). These enzymes play many vital physiological roles and remain as

essential homeostatic controlling factors in both prokaryotes and

eukaryotes. The homeostatic processes of significant protease association

are digestion of food, lysosomal degradation, transport of secretory

proteins across membranes, cell growth and migration, blood clotting,

matrix remodeling, immunological defenses like inflammation and Cell

death. Proteases have a key role to play in cancer, Zymogen activation

and hormone release. In case of plants, proteases play important role in

their life cycle (Andreas, 2004). In addition from photosynthesis to

photomorphogenesis, programmed cell death, circadian rhythm, and

defense response in plants also point involvement of proteolysis (Mark,

2001).

Proteases also serve important functions in Fungi and Bacteria.

Formation of spores in bacteria (Kornberg, et al. 1968), ascospores in

yeasts (Esposito and Klapholz 1981), fruiting bodies in slime molds and

conidial discharge in fungi (Phadatare, et al., 1989) involve an intense

protein turnover by proteases. Replication of viruses and their assembly

into capsids, Multiplication of various parasitic organisms, their spread

and transmission of infectious diseases in insects, animals and human

hosts are governed by proteases at various levels. Now it is known 50

human genes code for proteases (Puente, et al., 2003). Hence, proteases

are considered as biomarkers in diagnostics. Owing to their significant

role in the physiological and metabolic regulations, proteases have

become prime tools in medical research and drug development. Further

proteases behold 5-10% of the current market as pharmaceutical targets

(Salisbury and Ellman, 2006).

1.2.2 Commercial potential of Proteases

Proteases form a large group of industrial enzymes with multitude of

applications in various fields like food, pharmaceutical, detergent, leather

and dairy industries. Proteases account for nearly 60% of total enzyme

sales (Haard 1992). The market research conducted by an international

industry market research company, The Freedonia Group, Inc., predicts

that enzyme market will recover from difficult 2009 and will head to

reach $7 billion in 2013. They also foresee world enzyme demand to rise

6.3% yearly through 2013 (World Enzymes, 2009). Further fastest growth

is anticipated from the rapidly developing economies of the Asia and the

Africa/Mideast regions, Latin America and Eastern Europe. In particular,

the demand for proteases in US market as per the recent market

research report of The Freedonia Group (Enzymes, 2008) is presented in

Table 1.1.

Table 1.4 Estimated and predicted Protease Demand in US by

Product & Market (million dollars)

Item 1997 2002 2007 2012 2017

Nondurable Goods Shipments

(bil $)

1603 1701 2378 2655 3060

$ protease/mil $nondurables 220 249 234 257 260

Protease Demand 353 423 556 681 796

By Product:

Microbial Protease 155 240 346 460 570

Fibrinolytic Protease 90 60 72 65 56

Rennet/Chymosin 39 45 50 54 58

Other Protease 69 78 88 102 112

By Market:

Pharmaceutical 125 168 267 344 409

Research &

Biotechnology

11 15 19 25 33

Food & Beverage

Processing

64 76 88 100 113

Cleaning Product 117 125 134 149 162

Other Markets 36 39 48 63 79

% protease 34.0 33.0 30.0 27.5 23.8

Total Enzyme Demand 1037 1283 1855 2480 3340

According to the market research conducted by the same Freedonia

group, demand for enzymes in India is mainly concentrated in industrial

enzymes, more specifically in cleansing products (detergents), food and

beverage and textile and leather markets. But even within these

industries the usage rates of enzymes in comparison to developed

economies is very low. Although this is mainly attributed to the

government policies, recent moves of the government are quiet

encouraging. The details of sector –wise enzyme demand in India are

presented in the table*** below (Enzymes , 2007).

Table 1.5 INDIA -- Enzyme Demand By Type & Market (million

dollars) (Enzymes, 2007)

1.2.3 Sources of Proteases

Proteases are ubiquitous in nature due to their physiological

significance. A wide diversity of living organisms produce proteases such

as plants, animals and microorganisms (Rao, et al. 1998). Approximately

human genome of 2.0% and genome percentage of ~5% of disease

causing agents is accountable to protease production (Puente, et al.,

2003). Fortunately, these biocatalysts can be separated from the

respective cell source without losing their vitality. Besides, these

macromolecules retain and exhibit the same catalytic efficiency even

under in vitro conditions. Where papain, bromelain and ficin represent

Item 1996 2001 2006 2011 2016

Enzyme Demand 20 31 65 135 250

By Type

Carbohydrase 10 13 26 61 105

Protease 6 11 22 38 71

Other Enzymes 4 7 17 36 74

By Market

Industrial 17 5 47 96 166

Food &

Beverage

3 5 11 25 52

Other Industrial 14 20 36 71 114

Specialty 3 6 18 39 84

% India 6.3 7.6 8.6 10.5 11.9

Asia/Pacific Enzyme

Demand

320 410 760 1285 2100

some of the well-known proteases of plant origin (Rao, et al. 1998),

trypsin, chymotrypsin, pepsin and rennin account for commonly known

animal proteases. These proteases have found extensive application in

the food and dairy industries.

However, commercial production of plant and animal proteases is

subject to several complexities. As far as the production of plant

proteases is concerned, several factors apart from time constraint govern

the issue like availability of cultivable land, suitable climatic conditions,

quantity of plant material processed, etc. Further animal protease

production also encompasses problems like availability of live stock and

slaughter related political and agricultural policies (Rao, et al. 1998).

1.2.3.1 Microbial proteases

Microbial proteases are probably the most extensively studied

enzymes since the dawn of Enzymology. Even though proteases of

commercial use are derived from animals, plants and microorganisms,

microbial proteases hold a high share of approximately 40% of total

world wide enzyme sales due to their undue advantages. The consistent

interest in microbial proteases is not only for commercial reasons but

their study enables deciphering the role of these enzymes in various

cellular and metabolic processes. Apart from aiding in nutritional uptake

proteases also play a key role in sporulation and differentiation,

maintenance of cellular protein pool and overall protein turnover.

Intracellular proteases take part in cellular processes and extracellular

proteases enable absorption of hydrolyzed products by cell (Kalisz 1988).

The role of microbial proteases in the development and manifestations

of various diseases can not be underrated. These biocatalysts play a

crucial role in several chronic and systemic infections and also in severe

diseases with high death toll like AIDS and cancer. Characterization of

proteases along side the virulence factors facilitates the design of

effective and novel therapeutic strategies. One such case is of HIV

protease where synthetic HIV protease inhibitors have found successful

application in the treatment of AIDS. Similarly development of synthetic

inhibitors/2nd generation antibiotics against bacterial proteases is

undertaken to tackle different diseases.

However microorganisms represent the most attractive and an

unexhausted source of proteases. The relative ease of handling these tiny

creatures enables them to continue as sources of choice for commercial

production of proteases. Few inherent advantages of microorganisms

over plants and animals include their ease of large scale cultivation,

relatively simplified down stream processes, weather unaffected

production, ease of genetic manipulation etc (Gupta, et al. 2002).

Additional advantage of microbial enzymes is that most of them are

extracellular in nature. This increases their ease of isolation in pure

form. These incentives offered by microbes aid in orchestration of an

eco-friendly and cost-effective protease production scheme.

1.2.4 Classification of Proteases

According to the EC recommendations of the Nomenclature

Committee of IUBMB (International Union of Biochemistry and Molecular

Biology), Proteases have been placed in the subgroup 4 of group 3

(Hydrolases) (IUBMB 1992). However the huge diversity of structure and

action exhibited by this unique class of enzymes complicates their

classification. A detailed classification proteases in presented in the

Table *****. Presently, Proteases are classified based on 3 major criterion

(Barrett 1994):

1. catalysis

2. catalytic site

3. structure

Proteases have been further classified (Merops, 1999) into families

based on the amino acid sequence homology (Argos 1987) and into clans

based on their evolutionary origin (Rawlings and Barrett 1993). A Family

comprises of a set of homologous peptidases. In order to be a member of

a family, the significant score for sequence similarity with at least one of

the catalytic domains of a sequence family should be less than 0.0001

for BLAST (BLAST, 1999), or greater than 6 for RDF (RDF, 1999) and

ProfilSearch (ProfilSearch 1999). Proteases having a common ancestor

belong to the same clan.

Based on the site of action, proteases are classified as Exopeptidases

and Endopeptidases.

Table 1.6 Classification of proteases (Rao et al., 1998)

http://genome.ukm.my/prolyses/ECproteasenomenclature.html

Type of Protease EC Number

EXOPEPTIDASES

Aminopeptidases 3.4.11

Dipeptidases 3.4.13

Dipeptidyl peptidases 3.4.14

Tripeptidyl peptidases 3.4.14

Peptidyl dipeptidase 3.4.15

Carboxypeptidases 3.4.16-3.4.18

Serine- type carboxypeptidases 3.4.16

Metallocarboxypeptidases 3.4.17

Cysteine- type carboxypeptidases 3.4.18

Omega peptidases 3.4.19

ENDOPEPTIDASES 3.4.21-3.4.34

Serine protease 3.4.21

Cysteine protease 3.4.22

Aspartic protease 3.4.23

Metallo protease 3.4.24

Endopeptidases of unknown catalytic mechanism 3.4.99

1.2.4.1 Exopeptidases

Exopeptidases attack the peptide bonds located near the termini of

the polypeptide chains. Exopeptidases that cleave peptide bond at the

amino terminus are called as aminopeptidases and those that cleave at

the carboxyl terminus are called as carboxypeptidases.

1.2.4.1.1 Aminopeptidases (EC 3.4.11)

Aminopeptidases (EC 3.4.11-19) act at free ’N’ or amino terminus of

the polypeptide chain and releases one amino acid or a chain of 2 or 3 aa

containing peptides. Aminopeptidases are known to remove the N

terminal ‘Met’ (Methionine) from heterologously expressed proteins.

Based on their preference for either neutral (uncharged) or acidic side

chains they are classified as Aminopeptidase N and aminopeptidase A

respectively. They are found in a variety of bacteria and fungi.

Aminopeptidases from bacteria and fungi exhibit a clear distinction in

substrate specificity and are evident from their respective hydrolysis

product profiles. Although most of the aminopeptidases are intracellular

in nature, an extracellular aminopeptidase has been reported from

Aspergillus niger (Rao, et al. 1998).

1.2.4.1.2 Carboxypeptidases (EC 3.4.16-19)

Carboxypeptidases (EC 3.4.16-19) refer to the group of enzymes that

act at the ‘C’ or carboxyl terminus of the polypeptide chain to remove L-

amino acids either as single amino acid residue or as a dipeptide unit.

Depending on the active site mechanism carboxypeptidases are

categorized into families namely, Carboxypeptidases that use amino acid

residues serine or cysteine in active site are termed as serine-

carboxypeptidases (EC 3.4.16) and cysteine-carboxypeptidases (EC

3.4.18) respectively. Carboxypeptidases that make use of metal ion in

active site are termed as metallo-carboxypeptidases (EC 3.4.17).

Carboxypeptidases have been isolated from humans, animals and plants.

Carboxypeptidases are also categorized based on their substrate

specificity. Those carboxypeptidases that prefer aromatic amino acids or

those amino acids with branched chain hydrocarbons are called as

Carboxypeptidase A (A stands for aromatic/ aliphatic).

Carboxypeptidases that cleave amino acids bearing positive charges like

arginine or lysine are called as carboxypeptidases B (B stands for Basic).

Glutamate carboxypeptidase is a metallo carboxypeptidase that

specifically cleaves a glutamate residue from the C terminus of a peptide

namely N-acetyl-L-aspartyl-L-glutamate. Prolyl carboxypeptidase V is

another special serine carboxypeptidase that specifically catalyzes the

cleavage of C terminal residue in peptides of sequence –Pro-Xaa (pro –

proline; Xaa- amino acid at C terminus of peptide).

Dipetidases (EC 3.4.13) and Omega peptidases (EC 3.4.19) are other

important categories of exopeptidases. Enzymes that specifically cleave

dipeptide unit from either N (EC 3.4.14) terminus or C terminus (EC

3.4.15) are referred to as Dipeptidases. However, Omega peptidases

catalyze the hydrolysis of substituted amino acid residue at either N

terminus (EC 3.4.14) or C terminus (EC 3.4.15).

1.2.4.2 Endopeptidases

On the other hand endopeptidases specifically target and cleave the

peptide bonds that are located internally away from the termini. Further

based on the functional group present at the catalytic site, proteases are

classified into 5 groups namely 1) Seine proteases (EC 3.4.21) 2) Aspartic

proteases 3) Cysteine proteases. 4) Metallo proteases 5) Unknown

Proteases. A code letter has been assigned to each family of peptidases

for denoting type of catalysis: S - Serine, C - Cysteine, A- Aspartic, M-

Metallo, or U for Unknown protease type, respectively.

1.2.4.2.1 Serine Proteases

Serine proteases which are characterized by the occurrence of a

serine residue at their active site are classified into 20 families and sub-

grouped into 6 clans (Barrett, 1994). This widespread group of proteases

has been reported from viruses, bacteria, fungi and other eukaryotes

(Rao, et al. 1998). Some of the thiol reagents like PCMB are also known

to inhibit few serine proteases with residue of cysteine at active site.

Serine proteases are usually active at 7 and above pH. The pH optima of

these enzymes range between pH 7 and 11 and they record an isoelectric

point amid of pH 4 and 6. Serine proteases generally demonstrate broad

substrate specificity. Serine proteases are usually of low molecular mass

ranging between 18-35KDa although very few large serine proteases like

90KDa serine protease of B. subtilisK (nattom) have reported (Yamagata,

et al. 1995; Kato, et al. 1992). Interestingly serine proteases conserve

glycine residues present near the catalytic serine residue to form Gly-

Xaa-Ser-Yaa-Gly.

1.2.4.2.1.1 Serine alkaline proteases

Serine proteases that are active at highly alkaline pH are serine

alkaline proteases. The pH optima and isoelectric point of these enzymes

is around pH 10 and pH 9 respectively. Serine alkaline protease is one of

the largest subgroups of serine proteases (Rao, et al. 1998). They

hydrolyze a peptide bond, which has tyrosine, phenylalanine, or leucine

at the carboxyl site of the splitting bond. They have a molecular mass in

the range of 15-30 KDa.

Subtilisins comprise of another interesting group of alkaline

proteases. Mainly subtilisins produced by Bacillus sp. stand as the

second largest family of serine proteases. Subtilisin Carlberg and

Subtilisin Novo or bacterial proteases Nagase (BPN’) are two well studied

subtilisins. Although subtilisin Carlberg produced by B.

amyloliquefaciens has extensive application in detergents, Subtilisin

Novo produced by B. amyloliquefaciens is of less commercial importance

(Rao et al., 1998).

1.2.4.2.2 Cysteine Proteases

This unique group of proteases produced by both prokaryotes and

eukaryotes is dependent on reducing agents like HCN or cysteine for

their activity. Cysteine proteases are divided into four groups based on

their specificity for the side chain namely 1) papain like 2) trypsin-like

with preference for cleavage at the arginine residue, 3) specific for

glutamic acid, and 4) others. Although pH optima of cysteine proteases

lies in the neutral range, however few of them have been reported with

pH optima in the acidic range. Catalytic mechanism of these proteases

proceeds via a catalytic dyad comprising of cysteine and histidine. Thiol

group of a cysteine residue has a crucial role in the catalytic mechanism.

Sulfhydryl agents like PCMB show an inhibitory affect over cysteine

proteases while metalchelating agents and DFP remain ineffective.

Further this group of enzymes is susceptible to oxidation and can react

with a variety of reagents; heavy metals, iodoacetate, N-ethyl-maleimide

etc. (Kenny, 1999).

1.2.4.2.3 Aspartic Proteases

Aspartic acid proteases are commonly called as acidic proteases. The

catalytic activity of this group of endopeptidases depends on aspartic

acid residue. Aspartic acid proteases are classified into three families,

namely, pepsin, retropepsin and enzymes from pararetroviruses. Most of

the aspartic proteases have their pH optima at lower pH range (pH 3-4)

and their isoelectric points range between pH 3 and 4.5. Molecular

masses of these enzymes are reported to be in the range of 30 to 45 kDa.

1.2.4.2.4 Metalloproteases

Metalloproteases comprises of a varied group of proteases that require

metal ion for action. Most of the enzymes have a catalytically active Zinc

ion. Due to their metal ion dependency, Metalloproteases are inhibited

either by dialysis or chelating agents like EDTA. However sulfyhydryl

agents have no inhibitory affect on them. Most of the Metalloproteases

contain His-Glu-Xaa-Xaa-His (HEXXH). It is involved in providing a site

for binding metal ion. These enzymes are produced by bacteria, fungi

and higher organisms (Barrett, 1995). Collagenases, hemorrhagic

poisons of snake and thermolysins are few examples of Metalloproteases.

Physiological significance of these enzymes is evident from the role of

matrix Metalloproteases in the extracellular matrix degradation during

morphogenesis of tissue, differentiation, and healing of wounds.

Meatalloproteases are divided into families which are in turn sub

grouped into clans. Based on their specificity of action, Metalloproteases

are divided into 4 groups. Where alkaline metalloproteases demonstrate

broad substrate specificity there, Neutral proteases are specific towards

hydrophobic amino acids.

1.2.4.2.5 Unknown type proteases

Certain miscellaneous proteases do not fit into any of the standard

classes of classification and hence are termed as Unknown group of

proteases e.g., ATP-dependent proteases which require ATP for their

activity (Menon and Goldberg 1987).

Apart from the above mentioned strategies, proteases are also

classified based on their pH optima into 3 classes namely Acid, Neutral

and alkaline proteases (Rao, et al. 1998).

1.2.5 Mechanism of Action of Proteases

A clear understanding of the mechanism of action of proteases is

crucial in research viewpoint. This will enable exploration of ways for

modifying and increasing the versatility of these biocatalysts. It has been

revealed from studies that different types of mechanisms depend on the

configuration of active site. The protease catalytic site is flanked by

specific subsites, to accommodate side chain of one aa from the

substrate.

Protease active site

The catalytic site and scissile bonds are indicated by and --¦--

respectively.

1.2.5.1 Mechanism of action of serine proteases

Serine proteases carry out hydrolysis of a peptide bond via two-step

reaction. Schematic representation of the mechanism of action of serine

proteases is presented in the Figure****. Hydrolysis of the peptide bond

involves catalytic triad usually composed of His- Asp- Ser. The residue at P1

position decides the primary specificity and the site of cleavage of peptide

PPrrootteeaassee:: NN SSnn ---------- SS33 –– SS22 –– SS11 SS11’’ –– SS22’’ –– SS33’’ ---------- SSnn’’ CC

SSuubbssttrraattee:: NN PPnn ---------- PP33 –– PP22 –– PP11 ----¦¦---- PP11’’ –– PP22’’ –– PP33’’ ----------PPnn’’ CC

bond whereas residues at other positions influence the rate of cleavage.

The 1st step is formation of an acyl enzyme intermediate amid the

substrate and serine. This step is followed by a transition state that is

tetrahedral and –vely charged followed by peptide bond cleavage. A water

molecule is released which attacks the nucleophile in place of serine

residue. During this process some of the glycine residues located near

the catalytic serine moieties are conserved.

Figure 1.3 http://genome.ukm.my/prolyses/serinemech.html

1.2.5.2 Mechanism of action of metalloproteases

Metalloproteases engross a metal ion that is divalent for activity. The

His-Glu-Xaa-Xaa-His (HEXXH) motif provides site for metal ion to bind.

The catalytic method directs the construction of a non-covalent

tetrahedral intermediate later to the action of zinc bind water above the

carbonyl group. Further this intermediate is decomposed by shift of

proton from glutamic acid to leaving group.

Figure 1.4 http://genome.ukm.my/prolyses/metallomech.html

1.3 Alkaline Proteases

Alkaline proteases (EC.3.4.21-24, 99) are a distinct class of

physiologically and commercially important group of proteases that are

active at neutral to alkaline pH range. These enzymes either possess a

serine centre (serine protease) or a metal ion based catalytic mechanism

(metalloprotease). Alkaline proteases are a robust group of enzymes with

broad substrate specificities. These enzymes remain functional under

harsh working conditions such as temperatures up till 70ºC, pH of 11

and higher concentrations of detergents, polyphosphates, chelating

agents like EDTA and oxidizing agents such as sodium perborate

(Cowan, 1994). Alkaline proteases are inhibited by agents like DFP but

not by TLCK or TPCK.

Alkaline proteases are produced by diverse group of organisms from

higher organisms and insects to microorganisms (Singh et al., 2001a).

Several groups of microorganisms including fungi, molds, yeasts and

bacteria are reported of alkaline protease production. However microbial

alkaline proteases have gained huge commercial importance and they

play a dominating role in world enzyme market with a two thirds share in

the detergent industry since their introduction in 1914. Literature survey

indicates bacteria as the most sources for alkaline protease production.

Bacillus species among bacteria are more extensively exploited in this

regard. Among the other potential producers are Pseudomonas species

(Ogino et. al., 1999; Bayoudh et. al., 2000), Streptomyces strains among

actinomycetes (Petinate et. al., 1999) and Aspergilli in fungi (Chakrabarti

et. al., 2000; Rajamani and Hilda 1987). Conidiobolus species (Bhosale et.

al., 1995), Rhizopus species (Banerjee and Bhattacharya 1993) Candida

species among yeasts (Poza et. al., 2001) are also reported as potent

alkaline protease producers.

Alkaline proteases are among the most commercially exploited group

of enzymes (Gupta et al., 2002b) with applications in various industries.

Primarily these enzymes have been used as detergent additives. Alkaline

proteases have an immense application in food processing and feed

preparation. Tannery is another major niche where these enzymes are

used for dehairing and refining of hides and skins to get quality leather.

Extraction of silver from used X-ray films and degumming of silk to

improve its luster (Kanehisa 2000; Puri 2001) are other areas of

appliance. Alkaline proteases are also used for waste treatment,

delignification of hemp (Dorado et al. 2001), pest control (Kim et al. 1999)

and synthesis of peptides (Clapes et al. 1997; Isono and Nakajima 2000;

Kise et al. 1990; Morihara 1987). Further these enzymes also have

several medical and therapeutic applications. An elaborate survey of

literature with respect to the applications of alkaline proteases is

presented in later sections. A list of commercial bacterial alkaline

protease producers and their applications is provided in the table.

Table 1.7 Commercial bacterial alkaline proteases, sources, applications and

their industrial suppliers n.s. Not specified

Supplier Product trade name Microbial source Application

Novo Nordisk, Denmark Alcalase Savinase Esperase Biofeed pro Durazym Novozyme 471MP Novozyme 243 Nue

Bacillus licheniformis Bacillus sp. B. lentus B. licheniformis Bacillus sp. n.s. B. licheniformis Bacillus sp.

Detergent, silk degumming Detergent, textile Detergent, food, silk degumming Feed Detergent Photographic gelatin hydrolysis Denture cleaners Leather

Genencor International, USA Purafact Primatan

B. lentus Bacterial source

Detergent Leather

Gist-Brocades, The Netherlands Subtilisin Maxacal Maxatase

B. alcalophilus Bacillus sp. Bacillus sp.

Detergent Detergent Detergent

Solvay Enzymes, Germany Opticlean Optimase Maxapem

HT-proteolytic

Protease

B. alcalophilus B. licheniformis Protein engineered variant of Bacillus sp. B. subtilis

B. licheniformis

Detergent Detergent Detergent

Alcohol, baking, brewing, feed, food, leather, photographic waste Food, waste

Amano Pharmaceticals, Japan Proleather Collagenase Amano protease S

Bacillus sp. Clostridium sp. Bacillus sp.

Food Technical Food

Enzyme Development, USA Enzeco alkaline protease Enzeco alkaline protease-L FG Enzeco high alkaline protease

B. licheniformis B. licheniformis Bacillus sp.

Industrial Food Industrial

Nagase Biochemicals, Japan Bioprase concentrate Ps. protease Ps. elastase Cryst. protease Cryst. protease Bioprase Bioprase SP-10

B. subtilis Pseudomonas aeruginosa Pseudomonas aeruginosa B. subtilis (K2) B. subtilis (bioteus) B. subtilis B. subtilis

Cosmetic, pharmaceuticals Research Research Research Research Detergent, cleaning Food

Godo Shusei, Japan Godo-Bap B. licheniformis Detergent, food Rohm, Germany Corolase 7089 B. subtilis Food Wuxi Synder Bioproducts, China Wuxi Bacillus sp. Detergent Advance Biochemicals, India Protosol Bacillus sp. Detergent

1.3.1 Gordian knots in the way to commercial success of

proteases

Alkaline proteases have held a fiscal share in global enzyme market

for decades from now. Despite their strong industrial and research

potential, alkaline proteases are not harnessed to the fullest. Regardless

of the fact that these enzymes have reined the cleansing product market

for quiet some time, the changing needs of the customers with changing

times have set very high standards for these enzymes to sustain and

raise their position in global market. The most significant among the

characteristic features of an enzyme that jostle its way to commercial

success are thermostability, operational pH range and longer shelf-life.

Although alkaline proteases are amongst the most extensively studied

group of enzymes, still there are several lapses in our understanding of

these biocatalysts. As quoted earlier commercial viability of these

enzymes is hooked to their stability and operation under robust

industrial conditions. Several researchers have oriented their studies to

delineate the structure-function relationship for improving the stability of

these enzymes. The three major problems that have been addressed

include 1) alteration of pH optimum 2) Enhancement of thermostability

and 3) prevention of autoproteolytic inactivation.

Subtilisisns which are extensively used in detergents pose a problem

of autolytic digestion. A correlation has been deduced between the

autoplysis and conformation stability of the enzyme. Computer modeling

studies conjugated with SDS have enabled in achieving at a logical

inference that autoproteolysis can be prevented by introducing mutations

at the site of proteolysis.

Moreover alteration of substrate specificity is also being attempted to

enhance industrial potential of these enzymes. Strain improvement by

strategies like mutagenesis and recombinant DNA technology are being

attempted to produce proteases with desired characteristic features to

suit multitude of applications.

Regardless of the sophistication and technical advancements achieved

in the fields of protein and genetic engineering, isolation of novel

microorganisms from native environments with substantial production of

alkaline protease with desired enzymatic properties will always remain

an open option. High-throughput microbial screening and selective

enrichment techniques can be employed to instigate the unexplored

microbial consortia of the nature for most versatile and coveted catalytic

properties.

1.4 Response surface methodology (RSM)

Response Surface Methodology (RSM) is a collection of statistical and

mathematical techniques useful for developing, improving, and

optimizing processes (Myers et al, 2009). This is a branch of experimental

design with critical significance in developing and/or optimizing the

performance of processes. RSM has extensive applications in different

industrial fields and sciences such as Biological, Clinical and Food

Sciences, Chemical, Physical, Design and Engineering sciences and

Social sciences.

1.4.1 RSM: A sequential approach

RSM has a sequential nature. Usually the experiments are conducted

in 3 phases which include Phase 0, phase 1 and phase2. Phase 0 is also

called as screening experiment where in the independent variables which

play a critical role in generating the response are identified. Phase 1

comprises of adjusting the current experimental settings to attain a near

optimum response. This process is carried out by using first order model

and a technique called method of steepest ascent (descent). This

phase is followed by phase 2 where in the experimenter usually utilizes a

second or higher order polynomial to obtain a suitable model that will

fairly accurate the true response function. This orderly experimental

process is usually conducted inside an area of independent variable

known as operability region or experimentation region or region of

interest. The response surface is represented graphically for easy

analysis. The response surface can be plotted either in 3 – dimensional

space as Response surface plots or as contour plots.

1.5 Scope and aim of the present Investigation

Despite the fact that several microbial species have been reported

with alkaline protease production, only those microbes with substantial

production holding a GRAS (Generally Regarded As Safe) status can be

exploited commercially. Keeping in view the global and Indigenous

demand for alkaline proteases (Ref table 1&2), the present investigation

has been initiated.

The present thesis work has been taken up on identifying the undue

significance of alkaline proteases in research and industry. The purpose

of the proposed project is to isolate an alkaline protease organism by

appropriate screening techniques. Identification of the selected isolate

with desired enzymatic properties will be carried out. Cultural conditions

for alkaline protease production will be studied and optimized.

Purification and characterization of the enzyme will be carried out and

finally the applications of the purified enzyme will be investigated. The

specific objectives of the present research work are presented below.

1.6. Specific Research Objectives

Isolation and screening of alkaline protease producing microorganisms

Identification of the isolate

Purification of the enzyme

Characterization of the purified enzymes

Optimization of the process variables for maximal production of the

enzyme

Evaluation of the industrial application of enzyme