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CHAPTER 2
REVIEW OF LITERATURE 2.1 Proteinaceous waste 2.1.1 Keratinaceous waste- feather waste
Vast quantity of chickens are being utilised every day in the society that produces a large
amount of feathers waste in poultry industries. So far, feathers are known to have been
chemically and physically prepared to be used as feather meal as well as digestible nutritional
protein for animal feed. Keratin is a major constituent of feathers, possessing almost 90% of
feather weight [54]. Keratin-consisting materials have always been plentiful in the nature but
restricted in practical usages, mainly because of their insolubility and non-degradability by the
ordinary proteolytic enzymes. New developments of keratinase production have attracted many
attentions to apply keratinase in poultry industry. Feathers waste in poultry industries present a
high-quality supply of keratins. This valuable source of keratin could be used either as a source
of fertilizer glues and films, or many selected amino acids, and proteins which are applied in
animal feed industry [55].
Obviously, large amounts of keratinase for industrial scale processes are essentially needed
which is not cheap. Many researches show bacteria able to produce keratinase. However, best
host cell for overproduction of keratinase will remain unknown. Several efforts have been done
to overproduce keratinase as demands increasing. It is now certain that many different species
such as bacteria, actinomycetes, and fungi are able to produce keratinase. However, the level of
production and providing conditions are still remaining and are yet to be discovered [56].
2.1.2 Chicken Feather Waste
Poultry industry is continuously producing increasing amount of poultry meat and
noticeable quantities of organic residues such as feather, bone meal, blood, offal and so on.
6
Chicken feathers, making up about 5% of the body weight of poultry, is a considerable waste
product of the poultry industry being produced about 4 million tons per year world-wide [57,
58]. Disposal of waste feathers is a major concern for poultry industry and accumulation of this
huge volume of the waste feathers results in environmental pollution and protein wastage.
Figure 2.1: Chicken feathers image [82] Currently a minor quantity of waste feathers is used in other industrial applications such as
clothing, insulation and bedding [59], producing biodegradable polymers [60] and enzymes [61]
and also as a medium for culturing microbes. A higher quantity of pretreated feather is utilized to
produce a digestible dietary protein feedstuff for poultry and livestock [62-66]. However, to
decrease the risk of disease transmission via feed and food chain legislation on the recovery of
organic materials for animal feed is becoming tighter (Commission of the European
Communities, 2000), [67, 68].
2.2 Pretreatment methods for hydrolysis of poultry feathers Because of the complex, rigid and fibrous structure of keratin, poultry feather is a challenge to
anaerobic digestion. It’s poorly degradable under anaerobic conditions. [60] However,
application of appropriate pretreatments methods hydrolyzes feather and breaks down its tough
structure to corresponding amino acids and small peptides [62, 69].
For more than half a century many studies have been performed and various pretreatment
methods have been applied to improve the digestibility of feather meal, dietary animal protein
7
feedstuff and feather biogas potential [70, 71]. Feather meal treatment methods are usually
categorized into two groups: hydrothermal treatments and microbial keratinolysis [62, 72].
2.2.1 Hydrothermal pretreatment
Hydrothermal pre-treatment includes thermo-chemical treatment methods (such as acidic
hydrolysis and alkali hydrolysis), and also steam pressure cooking [62, 73]. These methods
usually need high temperatures [71] or high pressure [74, 75] with addition of diluted acids such
as hydrochloric acid [21] or alkali such as sodium hydroxide [62, 74]. “Acidic solutions promote
the loss of some amino acids such as tryptophan. [75]. Alkaline reactions are slow and
degradation of some amino acids with hydroxide is less. Hence the use of bases is recommended.
A stepwise diagram for the hydrolysis of protein rich material under alkaline condition is
indicated in Fig. 2.2 [76].
Figure 2.2: Protein hydrolysis during thermo-chemical treatment [76]
As a whole, hydrothermal hydrolysis usually consumes high amount of energy and employs
expensive equipment during lengthy processes (8 to 12 hrs), [77, and 64]. Thus, optimization of
8
the treatment conditions is an important issue from technological and economical points of view
when applying this method.
2.2.2 Biological pretreatment
Biodegradation of feathers is another alternative method. Some bacterial strains can produce
keratinase proteases which have keratinolytic activity and are capable to keratinolyse feather α-
keratin. These enzymes help the bacteria to obtain carbon, sulfur and energy for their growth and
maintenance from the degradation of α -keratin [78]. Various keratinases from different
microorganisms such as Bacillus sp. Bacillus licheniformis [79-81] Burkholderia,
Chryseobacterium, Pseudomonas, Microbacterium sp., Chryseobacterium sp., Streptomyces sp.
has been isolated and studied to date [62, 78].
Microbial proteases are classified into acidic, neutral, or alkaline groups, depends on the required
conditions for their activity and on the characteristics of the active site group of the enzyme, i.e.
metallo-, aspartic- , cysteine- or sulphydryl- or serine-type. Alkaline proteases which are active
in a neutral to alkaline pH, especially serine-types, are the most important group of enzymes
used in protein hydrolysis, waste treatment and many other industrial applications. Alkaline
protease from Bacillus subtilis was used for the keratinolysis of waste feathers [82].
Subtilisins are extracellular alkaline serine proteases, which catalyse the hydrolysis of proteins
and peptide amides. Savinase is one of these enzymes; Alcalase, Esperase and Maxatase are
others. These enzymes are all produced using species of Bacillus. Maxatase and Alcalase come
from B. licheniformis, Esperase from an alkalophilic strain of a B. licheniformis, and Savinase
from an alkalophilic strain of B. amyloliquefaciens [82]. An important advantage of enzyme
treatment method is fully biodegradability of enzymes by themselves as proteins. Hence, unlike
other remediation methods, there is no buildup of unrecovered enzymes or chemicals that must
be removed from the system at the end of degradation process. Although enzymatic treatment is
a promising technology; it has some limitations and disadvantages, as well. Currently, the main
disadvantage of using alkaline proteases is the high cost of the enzymes production. Much of the
cost of producing enzymes is related to high purification of enzymes solutions to avoid the side
effects and side activities of the crude enzyme solution which is cheaper. Furthermore, in
contrast with microbes which can reproduce themselves and increase their population to be able
to consume a large quantity of substrate and survive in harsh environments, extracellular
9
enzymes like alkaline protease do not have reproducibility. Namely, increasing the enzyme
population must be done through adding new enzymes from outside into the system. On the other
hand, these alkaline proteases lose some reactivity after they interact with pollutants and could
eventually become completely inactive. Hence they do not have the adaptability to the harsh
environment even though they can survive in a wide range of environmental conditions. This
means that the enzyme concentrations must be monitored and controlled during the process in
order to optimize enzyme kinetics for site-specific conditions [82].
2.2.3 Chemical-Biological pretreatment
Keratins are insoluble macromolecule comprises super coiled long polypeptide chains with high
degree of cross linked disulphide bonds between contiguous chains. According to the literatures
disulfide bonds in keratin significantly decrease protein digestibility [83]. And “for complete
easy degradation of feather all enzymatic keratinolysis from any organism essentially needs to be
assisted by a suitable redox [84]”. Therefore, it has been suggested that some reductants, such as
thioglycollate, copper sulphate, ammonia and sodium sulphite [85] and others, might cleavage
the disulfide bonds in keratin and allows the proteases to have access to their peptide bond
substrates [86], and consequently improve the degradability of feathers. For instance Ramnani et
al., 2007 found that savinase is capable of near complete feather degradation (up to 96%) in the
presence of sodium sulfite [84].
2.3 Keratin Structure Chicken feathers are composed of over 90% of keratin protein, small amounts of lipids and
water. Feathers keratin consists of high quantities of small and essential amino acid residues such
as glycyl, alanyl and seryl as well as cysteinyl and valyl. Keratin is also the main protein
components of hair, wool, nails, horn, and hoofs. Animal hair, hoofs, horns and wool contain β -
keratin, and bird’s feather contains α-keratin. The polypeptides in α-keratin are closely
associated pairs of β helices, whereas α-keratin has high proportion of β pleated sheets. “This
conformation confers an axial distance between adjacent residues of 0.35 nm in β -sheets,
compared to 0.15 nm in α-helices. The β sheets have a far more extended conformation than the
α–helices”.
10
Keratins are insoluble macromolecule comprises a tight packing of supercoiled long polypeptide
chains with a molecular weight of approximately 10 kDa. High degree of cross linked cystin
disulphide bonds between contiguous chains in keratinous material imparts high stability and
resistance to degradation [60, 62-64]. Hence, a keratinous material is a tough, fibrous matrix
being mechanically firm, chemically unreactive, waterinsoluble and protease-resistant [78]. Such
a molecular structure makes feathers poorly degradable under anaerobic digestion condition. Fig.
2.3 shows keratin molecular structure:
Figure 2.3: Keratin molecular structure [87] 2.4 Enzymes According to a new technical market research report, WORLD MARKETS FOR
FERMENTATION INGREDIENTS (FOD020C), the global market for fermentation products
was nearly $16 billion in 2008, and is expected to increase to $22.4 billion by the end of 2013,
for a compound annual growth rate (CAGR) of 7%. The amino acids segment has the largest
share of the market at $5.4 billion in 2008, and an expected increase by 2013 to more than $7.8
billion, for a CAGR of 7.6%. The market for industrial enzymes was the second-largest segment
at $3.8 billion in 2008, with an expected rise to $4.9 billion in 2013, for a CAGR of 8.9%. This
report is an update of World Markets for Fermentation Ingredients (FOD020B), published in
11
2005. During the past four years, substantial changes of the industry setup have taken place. The
most important products manufactured by fermentation are still the same crude antibiotics,
organic acids, amino acids, enzymes, vitamins, polysaccharides, and carotenoids. For virtually
all of these categories, markets expanded strongly, but the production landscape changed
massively. The market value of crude-fermentation-derived antibiotics is estimated at $1.8
billion for the year 2008. This value is much less than the estimates of many analysts as it refers
to the actual trade value of crude products and is supposed to reflect reality more
accurately. Consumption increased by more than 10% per year during the past five years, but
prices, after some erratic movements in 2006 and 2007, are back to the standard low levels. In
contrast to expectations, expansion of consumption took place primarily in the veterinary and in-
feed sectors. The report reviews the global fermentation industry with emphasis on major
fermentation-derived products used in food, feed, pharmaceutical, and technical applications. It
provides the most up-to-date information on quantities manufactured, prices and market value
developments, and on industry structures. It enables the reader to understand the industry in
general, provides in particular insight into the inter-relationship between the ethanol and other
carbohydrate-using industries [88].
Source: BCC Research [88]
Figure 2.4: Global Market for Fermentation Product, 2008 and 2013
12
Enzyme demand worldwide to reach $7 billion in 2013, the world market for enzymes will
recover from a difficult, 2009 to reach $7 billion in 2013 continued strong demand for specialty
enzymes. With the environment and cost issues surrounding conventional chemical processes
being subjected to considerable scrutiny, biotechnology rapidly is gaining ground due to the
various advantages it offers over conventional technologies. Industrial enzymes represent the
heart of biotechnology processes. The field of industrial enzymes now is experiencing major
R&D initiatives, resulting both in the development of a number of new products and in
improvement in the process and performance of several existing products.
Currently, new and emerging applications are driving demand and the industry is responding
with a continuous stream of innovative products. Significant future growth will require
investments by all the participants in research and applications development. This BCC study
examines current commercial applications worldwide, their markets and growth opportunities. It
also looks into market penetrations of newer grades of enzymes and their applications and sales,
as well as new developments and potential applications on the horizon. This report gives a clear,
quantitative picture of the supply and demand scenario and highlights technological and
investment opportunities in the field [89].
•
Figure 2.5: Global enzyme markets by application sectors, through 2009 ($ Millions), according to BCC (2008)
According to a new technical market research report, Enzymes for Industrial Applications
(BIO030E) from BCC Research (www.bccresearch.com), the global market for industrial
13
enzymes will be worth $2.3 billion in 2007. This is expected to increase to over $2.7 billion by
2012, a compound average annual growth rate (CAGR) of 4%.
The greatest growth rate is expected in the animal enzymes sector, with a CAGR of 6% between
2007 and 2012, helped in large part by the increased use of phytase enzymes to fight phosphate
pollution.
The market is broken down into applications of technical, food and animal feed enzymes. Of
these, technical enzymes have over 50% of the market. Valued at nearly $1.1 billion in 2007, this
segment is expected to be worth $1.4 billion by 2012, a CAGR of 3.5%. The animal feed enzyme
segment is currently worth $280 million and will be worth $375 million in 2012, a CAGR of 4%.
The higher growth in this sector will be helped in part by the increased use of phytase enzymes
to fight phosphate pollution. New and emerging applications have helped drive demand for
enzymes, and the industry is responding with a continuous stream of innovative products.
Table 2.1: Global enzymes market based on application sectors ($millions)
Global enzymes market based on application sectors ($millions)
Application sector
2005 2006 2007 2012 CAGR%
2007-2012Technical Enzymes
1,075 1,105 1,140 1,355 3.5
Food Enzymes
775 800 830 1,010 4.0
Animal Feed Enzymes
240 260 280 375 6.0
Total 2,090 2,165 2,250 2,740 4.0
14
2.4.1 Proteases
Proteases refer to a group of enzymes whose catalytic function is to hydrolyze peptide bonds of
proteins. They are also called proteolytic enzymes or proteinases. Protease forms a large group
of enzymes belonging to the class of hydrolases [90], ubiquitous in nature and performs a major
role with respect to their applications in both physiological and commercial fields.These enzymes
are widely distributed nearly in all plants, animals and microorganisms. In higher organisms
about 2% of the genes codes are formed by these enzymes [91]. Traditionally the proteinases
have been regarded as degradative enzymes which are capable of cleaving protein foods. They
liberate small peptides and amino acids needed by the body. Also they participate in the turnover
of cellular protein. Indeed, this is one of the best characteristic of the proteinases, such as the
mammalian digestive enzymes trypsin, chymotrypsin, and pepsin and the lysosome enzymes
cathepsin B and cathepsin D. Proteolytic enzymes have the ability to carry out selective
modification of proteins by limited cleavage such as activation of zymogenic forms of
enzymes, blood clotting and lysis of fibrin clots [92], and processing and transport of secretory
proteins across the membranes. These properties add considerable interest to an already
important group of enzymes. Additionally proteolytic enzymes have been used for a long time
in various forms of therapy [93]. Their use in medicine is gaining more and more attention
because several clinical studies are indicating their applications in oncology, inflammatory
conditions, blood rheology control and immune regulation. These are also used in crucial
biological processes such as regulation of metabolism, enzyme modification, photogenecity,
complement system, apoptosis pathways, invertebrate prophenoloxidase activating cascade
etc [94]. Furthermore, a study of proteolytic enzymes is valued because of their importance as
reagents in laboratory, clinical, and industrial processes. Proteinases from both microbial and non-
microbial sources, are extensively used in the food industry (baking, brewing, cheese anufacturing,
meat tenderizing) [95], in the tanning industry, and in the manufacture of biological detergents
[96]. Thus, there is an increasing interest in the proteinases and peptidases of both eukaryotic
and prokaryotic microorganisms. Proteases execute a large variety of pharmaceutical
functions; particularly their involvement in the life cycle of disease causing organisms has led
them to become a potential target for developing therapeutic agents against fatal diseases such as
cancer and AIDS [97]. The vast diversity of proteases, in contrast to the specificity of their
15
action, has attracted worldwide attention in an attempt to exploit their physiological and
biotechnological applications [98, 99]. Though proteases are enzymes of meta-bolic as well as
commercial importance, there is not much literature available on their biochemical and
biotechnological aspects [99-103]. However, the earlier reviews are lack of satisfactory
information, the biologicalconcept of proteases, which offers new possibilities and potentials for
their biotechnological functions.
2.4.1.1 Types of Proteases
Proteinases may be classified in a number of ways, for example, on the basis of the pH
range over which they are active (acid, neutral, or alkaline), or their ability to hydrolyze
specific proteins (keratinase, elastase, collagenase etc.), or their similarity to well
characterized proteinases such as pepsin, trypsin, chymotrypsin, or the mammalian
cathepsins. Hartley,1960 described the most satisfactory system of classification based on
the presence of main catalytic amino acid residue in their active site:(1) Serine
proteinases , with a serine and histidine; (2) Cysteine proteinases, with a cysteine; (3)
Aspartic proteinases , with an aspartate group and (4) Metalloproteases , with a metallic
ion (Zn++, Ca++ or Mn++) in their active site. However, currently proteases are classified on
the basis of three major criteria: (a) depending upon site of action; (b) depending upon
optimum pH and (c) miscellaneous. Proteases are grossly subdivided into two major
groups, i.e., exopeptidases and endopeptidases, depending on their site of action.
Exopeptidases cleave the peptide bond proximal to the amino or carboxy termini of the
substrate, whereas endopeptidases cleave peptide bonds distant from the termini of the
substrate .In general, depending upon the optimum pH proteases are named as: (i) Acid
proteases, active in the pH range 2 - 3.5; (ii) Neutral Proteases, active in the pH between 6.5
and 7.5 and (iii) Alkaline proteases, active in the pH between 7.5 and 10.5. A few
miscellaneous proteases which do not precisely fit into the standard classification, e.g., TP-
dependent proteases which require ATP for activity [104]. Based on their amino acid
sequences, proteases are classified into different families [105] and further subdivided into
“clans” to accommodate sets of peptidases that have diverged from a common ancestor.
Each family of ptidases has been assigned a code letter denoting the type of catalysis, i.e., S,
16
C, A, M, or U for serine, cysteine, aspartic, metallo, or unknown type respectively.
2.4.1.1.1 Exopeptidases
The exopeptidases act only near the ends of polypeptide chains. Based on their site of
action at the N or C terminus (Table 2.2), they are classified as amino- and
Carboxypeptidase, respectively.
Table 2.2: Classes and active site of exopeptidases
*Open circles represent the amino acid residues in the polypeptide chain. Solid
circles indicate the terminal amino acids. Solid triangle indicates site of peptide
cleavage
2.4.1.1.1.1 Aminopeptidases
Aminopeptidases act at a free N terminus of the polypeptide chain and liberate a single amino
acid residue, a dipeptide, or a tripeptide (Table 2.2). They are known for removing the N-
terminal Met that may be found in heterologously expressed proteins but not in many naturally
occurring mature proteins. Aminopeptidases occur in a wide variety of microbial species
including bacteria and fungi [106]. In general, aminopeptidases are intracellular enzymes, but
there has been a single report on an extracellular aminopeptidase produced by Aspergillus
oryzae [107]. The substrate specificities of the enzymes from bacteria and fungi are distinctly
different in that the organisms can be differentiated on the basis of the profiles of the products of
17
hydrolysis [108]. Aminopeptidase I from Escherichia coli is a large protease (400 kDa). It has a
broad pH optimum of 7.5 to 10.5 and requires Mg+2 or Mn+2 for optimal activity [109]. A leucine
aminopeptidase was purified about 670 fold from germinated grains of barley (Hordeum vulgare).
This leucine aminopeptidase is remarkably similar to mammalian leucine aminopeptidase (EC
3.4.1.1). The Bacillus licheniformis aminopeptidase has a molecular weight of 34.00 kDa. Its
activity is enhanced by Co+2 ions. Similarly, another species of Bacillus genus i.e. B.
stearothermophilus produces aminopeptidase. Structurally it is a made up of two subunits whose
molecular mass is about 80 to 100 kDa [110] and is activated by Zn+2, Mn+2, or Co +2 ions.
2.4.1.1.1.2 Carboxypeptidases
The Carboxypeptidase act at C terminals of the polypeptide chain and liberate a single amino
acid or a dipeptide. Carboxypeptidases are divided into three major groups, serine
carboxypeptidases, metallocarboxypeptidases, and cysteine carboxypeptidases, based on the
nature of the amino acid residues at the active site of the enzymes. The serine carboxypeptidases
isolated from Penicillium sp., Saccharomyces sp., and Aspergillus sp. are similar in their
substrate specificities but differ slightly in other properties such as pH optimum, stability,
molecular weight, and effect of inhibitors. Metallocarboxypeptidases from Saccharomyces sp.
[111] and Pseudomonas sp. [112] require Zn+2 or Co +2 for their activity. The enzymes are also
hydrolyze the peptides in which the peptidyl group is replaced by a pteroyl moiety or by
acyl groups.
2.4.1.1.2 Endopeptidases
Endopeptidases are characterized by their preferential action at the peptide bonds in the inner
regions of the polypeptide chain away from the N and C termini. The presence of the free amino
or carboxyl group has a negative influence on enzyme activity. The endopeptidases are divided
into four subgroups based on their catalytic mechanism, (i) serine proteases, (ii) aspartic
proteases, (iii) cysteine proteases, and (iv) metalloprotease. Table 2.3 summarizes general
properties of four classes of endopeptidases [261]. To facilitate quick and unambiguous reference
to a particular family of peptidases, have been assigned a code letter denoting the catalytic type,
i.e., S, C, A, M, or U (Unknown Protease) followed by an arbitrarily assigned number.
18
Table 2.3: Classes and some general properties of endopeptidases
Property Serine Cysteine Aspartic Metallo
Old Name Serine Thiol Carboxyl Metallo
Enzyme Nomenclature 3.4.21 3.4.22 3.4.23 3.4.24
Active site component Serine Cysteine Aspartic Zn++
acid
pH range 7-9 3-7 2-6 5-9
Temperature range (0C) 20-80 25-70 40-70 40-60
Molecular mass (kDa) 20-135 20-65 30-60 20-60
Inhibitors PMSF, DIFP E-64, Iodoacetate Pepstatin EDTA
Location Intra- and extra-cellular Lysosomes Lysosomes Intra-and extracellular
Examples Elastin, Plasmin Cathepsins B & L CathepsinD Gelatinase
2.4.1.1.2.1 Aspartic proteases (EC 3.4.23)
Aspartic proteases, commonly known as acidic proteases, depend on aspartic acid residues for
their catalytic function. Acidic proteases have been grouped into three families, namely, pepsin
(AP1), retropepsin (AP2), and enzymes from Para retroviruses (AP3) [113]. The members of
families AP1 and AP2 are known to be related to each other, while those of family AP3 show
some relatedness to AP1 and AP2. Most aspartic proteases show maximal activity at low pH (pH
3 to 4) and have isoelectric points in the range of pH 3 to 4.5. Their molecular masses are in the
range of 30 to 45 kDa, the exception being larger enzymes in Podospora anserine [114]. The
members of the AP1 family have a bilobal structure with the active-site cleft located between
the lobes [115]. The active-site aspartic acid residue is situated within the motif Asp-Xaa-Gly, in
which Xaa can be Ser or Thr. The aspartic proteases are generally inhibited by pepstatin [265].
They are also sensitive to diazoketone compounds such as diazoacetyl-DL-norleucine methyl
ester (DAN) and 1, 2-epoxy-3-(p -nitrophenoxy) propane (EPNP) in the presence of copper
19
ions. Microbial acid proteases exhibit specificity against aromatic or bulky amino acid residues on
both sides of the peptide bond, which is similar to pepsin, but their action is less stringent than
that of pepsin. Microbial aspartic proteases can be broadly divided into two groups, (i) pepsin-
like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and (ii) rennin-
like enzymes produced by Endothia and Mucor sp. An interesting property of many of the fungal
acid proteases i.e. protease of unidentified species of Penicillium is able to activate bovine
trypsinogen [116]. Morihara and Oka, 1973 [117] reported a relationship between this
trypsinogen kinase activity and the ability of proteinases to hydrolyze specific oligopeptides at
bond involving the carboxyl group of lysine, although lysine and arginine containing bonds in
the insulin B chain are not cleaved. It is interesting to note that proteinases from the
protozoan Tetrahymena pyriformis [118] and Dictyostelium discoideum [119] have
trypsinogen kinase activity. Amino acid sequence analysis [120] and X-ray crys-tallographic
analysis [121, 122, 123] of the proteinases of Rhizopus chinensis, Penicillium janthinellum,
Penicillium roqueforti and Endothia parasitica have revealed a considerable degree of homology
between the fungal proteinases and mammalian aspartic proteinases including pepsin and rennin,
suggesting that they all evolved from a common ancestral gene [124].
2.4.1.1.2.2 Serine proteases (EC 3.4.21)
Serine proteases are characterized by the presence of a serine group in their active site. These are
numerous and widespread among viruses, bacteria, and eukaryotes, suggesting that they are vital
to the organisms. Serine proteases are found in the exopeptidases, endopeptidase, oligopeptidase,
and omega peptidase groups. Based on their structural similarities, serine proteases have been
grouped into 20 families, which have been further, subdivided into about six clans with common
ancestors [125]. The primary structures of the members of four clans, chymotrypsin (SA),
subtilisin (SB), carboxypeptidase C (SC), and Escherichia D-Ala-D-Ala peptidase A (SE) are
totally unrelated, suggesting that at least four separate evolutionary origins for serine proteases.
Clans SA, SB, and SC have a common reaction mechanism consisting of a common catalytic
triad of the three amino acids, serine (nucleophile), aspartate (electrophile), and histidine
(base). Although the geometric orientations of these residues are similar, the protein folds are
quite different, forming a typical example of a convergent evolution. The catalytic
20
mechanisms of clans SE and SF (repressor LexA) are distinctly different from those of clans
SA, SB, and SC, since they lack the classical Ser-His-Asp triad. Another interesting feature of
the serine proteases is the conservation of glycine residues in the vicinity of the catalytic serine
residue to form the motif Gly-Xaa-Ser-Yaa-Gly. Serine proteases are recognized by their
irreversible inhibition by 3, 4-dichloroisocoumarin (3, 4-DCI), diisopropylfluorophosphate
(DFP), phenylmethylsulfonyl fluoride (PMSF) and tosyl-L-lysine chloromethyl ketone
(TLCK). Some of the serine proteases are inhibited by thiol reagents such as p-
chloromercuribenzoate (PCMB) due to the presence of a cysteine residue near the active site.
Serine proteases are generally active at neutral and alkaline pH, with an optimum between pH 7
and 11. They have broad substrate specificities including esterolytic and amidase activity. Their
molecular masses range between 18 and 35 kDa, for the serine protease from Blakeslea trispora,
which has a molecular mass of 126 kDa [126]. The isoelectric points of serine proteases are
generally between pH 4 and 6. Serine alkaline proteases that are active at highly alkaline pH
represent the largest subgroup of serine proteases.
Serine proteases are inhibited by DFP or a potato protease inhibitor but not by
tosyl-L-phenylalanine chloromethyl ketone (TPCK) or TLCK. Their substrate specificity is
similar to but less stringent than that of chymotrypsin. These enzymes hydrolyze a peptide
bond which has tyrosine, phenylalanine, or leucine at the carboxyl side of the splitting bond. The
optimal pH of alkaline proteases is around pH 10, and their isoelectric point is around pH 9. Their
molecular masses are in the range of 15 to 30 kDa. Although alkaline serine proteases are
produced by several bacteria such as Arthrobacter, Streptomyces, and Flavobacterium sp.
[127], subtilisins produced by Bacillus sp. are the best known. Alkaline proteases are also
produced by S. cerevisiae [128] and filamentous fungi such as Conidiobolus sp. [129] and
Aspergillus and Neurospora sp. [130]. Subtilisin of Bacillus origin represents the second largest
family of serine proteases. Two different types of alkaline proteases, subtilisin Carlsberg and
subtilisin Novo or bacterial protease Nagase (BPN9), have been identified. Subtilisin Carlsberg
produced by Bacillus licheniformis was discovered in 1947 by Linderstrom, Lang, and Ottesen at
the Carlsberg laboratory [131]. Subtilisin Novo or BPN9 is produced by Bacillus
amyloliquefaciens. Subtilisin Carlsberg is widely used in detergents. Both subtilisins have a
molecular mass of 27.5 kDa but differ from each other by 58 aminoacids. These have similar
21
properties such as an optimal temperature of 60°C and an optimal pH of 10. Both enzymes
exhibit broad substrate specificity and have an active-site triad made up of Ser 221, His 64
and Asp 32. The active-site conformation of subtilisin is similar to that of trypsin and
chymotrypsin despite the dissimilarity in their overall molecular arrangements. The serine
alkaline protease from the fungus Conidiobolus coronatus has a distinct different struc-
ture from subtilisin Carlsberg in spite of their functional similarities [132].
2.4.1.1.2.3 Cysteine proteases (EC 3.4.22)
Cysteine proteases occur in both prokaryotes and eukaryotes. About 20 families
of cysteine proteases have been recognized. The activity of all cysteine proteases depends on a
catalytic dyad consisting of cysteine and histidine. The order of Cys and His (Cys-His or His-Cys)
residues differ among the families [127]. Generally, cysteine proteases are active only in the
presence of reducing agents such as HCN or cysteine. Based on their side chain specificity, they
are broadly divided into four groups: (i) papain-like, (ii) trypsin- like with preference for
cleavage at the arginine residue, (iii) specific to glutamic acid, and (iv) others. Papain is the best
known example of cysteine protease. Cysteine proteases have neutral pH optima,
although a few of them, e.g., lysosome proteases, are maximally active at acidic pH. These are
susceptible to sulfhydryl agents such as PCMB but are unaffected by DFP and metal-chelating
agents. Clostripain, produced by the anaerobic bacterium Clostridium histolyticum, exhibits a
stringent specificity for arginyl residues at the carboxyl side of the splitting bond and differs
from papain in its obligate requirement for calcium. Streptopain, the cysteine protease produced
by Streptococcus sp., shows a broad specificity, including oxidized insulin B chain and other
synthetic substrates. Clostripain has an isoelectric point of pH 4.9 and a molecular mass of 50 kDa,
whereas the isoelectric point and molecular mass of streptopain are pH 8.4 and 32 kDa,
respectively.
2.4.1.1.2.4 Metalloproteases (EC 3.4.24)
Metalloproteases are the most diverse types of the catalytic proteases [114]. This type of enzymes
characterized by the requirement for a divalent metal ion for their activity. These include
22
enzymes from a variety of origins such as collagenases from higher organisms, hemorrhagic
toxins from snake venoms, and thermolysin from bacteria [135, 136]. About 30 families of
metalloprotease have been recognized, of which 17 (M1) contain only endopeptidases, 12 (M2)
contain only exopeptidases, and 1 (M3) contains both endo and exopeptidases. Based on the
specificity of their action, metalloproteases are divided into four groups, (i) neutral, (ii) alkaline,
(iii) Myxobacter I, and (iv) Myxobacter II. The neutral proteases show specificity for
hydrophobic amino acids, while the alkaline pro-teases possess a very broad specificity.
Myxobacter protease I is specific for small amino acid residues on either side of the cleavage
bond, whereas protease II is specific for lysine residue on the amino side of the peptide bond.
All of them are inhibited by chelating agents such as EDTA but not by sulfhydryl agents or DFP. A
few examples of metalloproteinases have been reported in fungi, and most have been shown to
be zinc-containing enzymes. Gripon et al., [135] have suggested that the enzymes of Penicillium
caseicolum and Penicillium roqueforti and the neutral proteinase of Aspergillus oryzae and
Aspergillus sojae represent a distinct group of enzymes for which they suggest the name acid
metalloproteinase. These have lower pH optima and molecular weights i.e. pH 5 and 19,000
respectively, and a different specificity with the oxidized insulin B chain from the thermolysin
like neutral metalloproteinases. The Penicillium proteinases are also insensitive to
phosphoramidon, a specific neutral metalloproteinase inhibitor. The basidiomycete Tricholoma
columbetta produces a low molecular weight neutral proteinase [136] which has some
resemblance to the metalloproteinase of another basidiomycete, Armillaria mellea [137].
2.4.1.2 Distribution of proteases
Proteases are widely distributed in each part of biological source. Due to this, it belongs to
one of the subtype of digestive enzyme. Plant kingdom occupies the topmost rank (43.85 %)
for finding proteases, followed by bacteria (18.09 %), fungi (15.08 %), animals (11.15 %), algae
(7.42 %) and viruses (4.41 %). Isolated proteases only contribute 27 to 67 % of biological origin
irrespective of either animal, microbial or plant proteases while remaining proteases are not well
studied. Cysteine protease abundantly occurs (34.92 %) in plants. Microbes have ability to
secrete large quantities of serine (13.21 %) and aspartic (8.81%) proteases. Recently, glutamic
acid protease of microbial origin has been recorded. Serine, cysteine and aspartic proteases are
23
commonly found in animals (Fig. 2.2). Plant proteases are virtually present in every part of plants
viz., root, stem, leaf, flower, fruit, seed, gum and latex. Plant latex is the richest source of
protease. About 43.91 % of plant proteases have not been fully characterized. Very rare finding
is recorded on asparaginyl protease. On the other hand, usually cysteine and serine
endoproteases occurred in plants. Aspartic protease and aminopeptidases are rarely found in
plants. Figure 2.6 summarizes the distribution of protease enzymes in biological sources.
2.4.1.2.1 Plant Proteases Crude preparation of the enzyme has a wide specificity due to the presence of various proteinase
and peptidase isozymes. The performance of the enzyme depends on the plant source, the
climatic conditions for growth and the methods used in its extraction and purification; for
example, if the fruit is healthy, then enzyme found is more active. Milk clotting, a property of
proteolytic enzyme was recorded in the latex of Carica papaya [138]. Carica papaya latex is rich
in proteolytic enzymes, commercially called papain [139]. Papain is a traditional plant protease
and has a long history of use [140]. It is extracted from the latex of unripe papaya fruits, which
are grown in subtropical areas of west and central Africa and Asia (Tanzania, Uganda, Zaire, Sri
Lanka, Thailand and India). The papain is active between pH 5.0 and 9.0 and is stable up to 80 or
90ºC. It is widely used in industry as a meat tenderizer and has also other uses in the
pharmaceutical, detergent, veterinary and food industry. Method of crystallization of papain was
established by Monti et al., 2000 [141]. Milk clotting enzyme is present in the fruit of Withania
coagulans [142]. Proteolytic enzyme was successfully separated from the lattices of Ficus carica
and Ficus glabrata [143]. Kramer and Whitaker, 1964 determined the properties of proteolytic
enzyme, purified from the latex of Ficus carica [144]. Proteolytic activity was found in some
plant latex including Calotropis procera, Calotropis gigantea, Cryptostcgia grandiflora, Carica
papaya and Ficus carica [139]. Ginger rhizome is reported for new source of proteolytic enzyme
[145] and the extracted proteolytic enzyme named, Zingibain, showed more activity at its
optimum pH 5.0 and optimum temperature at 60°C. The amino acid sequence was evaluted for
tryptic peptides of the thiol proteinase i.e. actidin which is found in the fruit of Actinidia
chinensis [146]. Proteolytic activity was reported in green asparagus, kiwi fruits and miut;
optimum temperature for activities of Green Asparagus and Miut were 40-45°C and that of kiwi
24
fruit was 60°C [147]. Germinated finger millet (Eleusine coracana) seed-lings showed
proteolytic activity against hemoglobin and albumin [148]. A Benzyloxycarbonylalanylcitruline -
p-nitroanilide is a powerful substrate for papain, bromelain, ficin and many other plant cysteine
proteinases [149]. A latex serine protease i.e. Euphorbian P of Euphorbia pulcherrima had
molecular mass about 74kDa, pI 4.7 and optimum pH 7.0 [150]. Two serine centered proteolytic
enzymes, namely euphorbian d 1 and d 2 are separated from Elaeophorbia drupifera latex on the
basis of their Mr of 117 K and 65 K respectively [151]. A new protease is well characterized in
developing maize endosperm and high lysine opaque-2 maize endosperm which plays an
important role in modification of storage protein [152]. Ficin E is well characterized serine
protease of Ficus elastica latex having molecular mass about 50 kDa, pI 3.7 and optimum pH 6.0
[153]. An important and popular milk product i.e. cheese was prepared by incorporating
vegetable rennet i.e. protease of Calotropis procera [154]. Curcain is a new protease of Jatropha
curcas latex whose molecular mass about 22 kDa and isoelectric point is 5.8 [155]. This isolated
enzyme of J. curcas plays an important role in the healing process of skin injury i.e. wound
[156]. Bromelain is a proteolytic enzyme derived from stem and the juice of pineapples (Ananas
comosus). This enzyme is an alternative to papain and is used to tenderize meat. In modern
therapy, bromelain is consumed as a digestive and demonstrates anti-edematous, anti-
inflammatory, anti-thrombotic and fibrinolytic activities [157]. Total 17 proteases were purified
from the lattices of 8 different species of Euphorbiaceae family having molecular mass in
between 33-117 kDa [158]. Isolated aminopeptidase of oat leaf shows optimum activity at pH
8.4 by using azocasein and rubisco as substrate [159]. Three protease fractions were obtained by
purification of a wild thistle (Cynara cardunculus) extract with ammonium sulphate precipitation.
Optimum pH and temperature on the proteolytic activity of the crude extract was found to be 5.7
and 37°C respectively [160]. Milk clotting activity of some plants found in Pakistan including
Opuntia phylloclades, Cereus triangularis, Euphorbia caducifolia, Calotropis procera, Carica
papaya, Ficus bengalensis, Ficus elastica and Euphorbia hista was determined by using
skimmed milk powder as a substrate [161]. Immunochemical properties of Protease A of
dormant cotton seed was established using double immunodiffusion technique through
immunological affinity in between trypsin and protease A [162]. Multiple forms of the cysteine
proteinases ananain and comosain were derived from pineapple stem [159]. An attempt has been
25
made by Terp et al., 2000 [162] and Fontanini and Jones, 2002 [163] to characterize hordolisin
and subtilisin -like serine protease called SEP - 1 from barley. Amino acid sequences of these
proteases are matched with other plant subtilases. Thus, both enzymes belong to the cucumisin -
like group. A type of serine protease of tomato flesh of Lycopersicon esculentum, showed
optimum activity at pH 7 and 45°C temperature with Km value of 0.48 per cent by using casein
as a substrate [164]. The ovules of flowering plants are difficult to obtain in the amounts
sufficient for extraction and purification of proteolytic enzymes. For this reason studies have
been focused primarily on the characteristics of proteases of the ovary pistil style and stigma. A
proteolytic enzyme was extracted from the latex of Ficus hispida having optimum temperature
and pH 40°C and 7.0 respectively with the isoelectric point of 4.4 to 4.7 [165]. A high molecular
weight (80kDa) cucumisin like serine protease, isolated from the latex of Euphorbia supine by
two steps chromatography [166]. Tropical squash seeds of Cucurbita ficifolia possess serine
protease, which hydrolyses casein and Suc-Ala-Ala-Pro-PhepNA at pH 10.5; oxidized insulin B-
chain is also cleaved by this enzyme on several of its peptide bonds. This enzyme is optimally
active at 9.2 pH and 55°C temperature [167]. A new subtilisin like protease called plantagolisin
identified from leaves extract of Plantago maior by using affinity chromatography on bacitracin
sepharose and ion exchange chromatography on Mono Q in FPLC. This was obtained at pH 11
and 70°C temperature [168]. A high molecular weight (60 kDa) proteolytic enzyme occurred in
leaf extract of Calotropis procera which had optimum temperature 70°C [169]. Roots of higher
plants (Allium porrum, Zea mays and Helianthus indicus) could secrete protease and maintain
the proteolytic activity for a long time. The culture medium of aseptically cultivated seedlings of
these plants showed highest proteolytic activity at pH 7 [170]. An acidic serine protease occurred
in the root extract of gramineae member i .e Zea mays, whose molecular mass is about 54 kDa
[171]. The seeds of Albizzia lebbeck and Helianthus indicus show milk clotting activity. This
milk clotting protease is extracted by using ammonium sulphate precipitation [172]. Two
examples of novel cysteine proteases are well characterized from germinating cotyledons of
soybean. Molecular masses and isoelectric point of both enzymes are 26.178 kDa, 26.429 kDa,
pI 4.4 and pI 4.7 respectively [112]. A usual thermostable aspartic protease of Ficus racemosa
latex exhibits a broad spectrum pH range between pH 4.5 - 6.5 and maximum activity at 60 ±
0.50C. This enzyme has 44.50 ± 0.50 kDa molecular weight [173]. Pedilanthin, a novel protease
26
identified to homogeneity of Pedilanthus tithymaloids latex, which had 63.1 kDa molecular
mass. This enzyme had optimum pH 8.5 and temperature optima at 68°C [174]. A low
molecular weight (25kda) alkaline serine protease of Holarrhena antidysentrica seeds had
optimum activity at pH 7.5 which exhibited its highest activity at 350C using 1% casein as a
substrate. The Km and Vmax values are 1.1mg ml-1 and 38971 Units min -1 mg-1 respectively
[175]. A high molecular weight serine protease (134.3kDa) named as indicain has been identified
to homogeneity from the latex of Morus indica using ammonium sulphate precipitation,
hydrophobic interaction and size exclusion chromatography. The pH and temperature optimum
of this enzyme is 8.5 and 80°C respectively. The extinction coefficient and isoelctric pH of this
enzyme was 41.24 and pI 4.8 respectively. The molecular structure consists of 52 tryptophan,
198 tyrosine and 42 cysteine residue [176]. A good potential of industrial application of
bromelain was recorded in textile, brewing and fermentation industry [177]. This enzyme has
been clinically fully analyzed by Taussin and Batkin, 1988 [178]. A new cysteine protease as a
vegetable source for milk clotting enzyme was puri -fied from the root latex of Jacaratia
corumbensis and enzyme showed pH and temperature optima in between pH 6.5 - 7.0 and 55°C
respectively with 33 kDa molecular mass [179]. A new papain like endopeptidase i.e. asclepain c
-II has been isolated and well characterized from the petiole latex of Asclepias curassavica. This
enzyme displayed molecular mass of 23.59 kDa, pI > 9.3, maximum proteolytic activity at pH
9.4 - 10.2 and showed poor thermostability [180]. An alkaline chymotrypsin like serine protease
i.e. dubiumin of Solanum dubium seeds showed optimum enzyme activity at 70°C temperature.
This enzyme had 66 kDa molecular mass and pI is 6 [181]. Benghalensin, a serine protease is
identified to homogeneity from the latex of Ficus benghalensis by a single step procedure using
anion exchange chromatography. This enzyme had 47 kDa molecular mass, pI is 4.4, optimum
pH is 8 and optimum temperature is 55°C. The molecular structure of enzyme consists of 17
tryptophan, 31 tyrosine and 09 cysteine residue [182]. A comparative study in between
proteolytic activity, milk clotting activity and gelatinolytic activity is given in latex of twenty
one laticiferous plants, belonging to seven different latex bearing families of Khandesh region
of Maharashtra, India. Highest milk clotting activity was reported in the latex of
Euphorbia nivulia. The decreasing order of potential proteolytic activity are in the
order of Euphorbianivulia > Carica papaya > Calotropis procera > Ficus carrica
27
based on Tyrosine Unit [183]. Shivaprasad et al., 2009 reported the thrombin like activity of
cysteine protease, Pergularain eI of Pergularia extensa latex having molecular mass
23.35 kDa and the N-terminal amino acid sequence as L -P -H -D-V [184]. The latex of
Aslepiadaceae member i.e. Calotropis gigantea possess fibrinogenolytic, fibrinolytic and
proteolytic activity. Inhibition pattern shows that the isolated protease enzyme
belongs to cysteine protease family [185]. Procerain B is a novel cysteine protease of the
latex of Calotropis procera, enzyme shows broad optimum pH and temperature range i
.e pH 6.5 to 8.5 and 40 to 600C temperature respectively. This enzyme had 25.70 kDa
molecular weight and pI is 9.52 [186]. Crinumin is a chymotrypsin like serine protease
isolated from the latex of Crinum asiaticum. This enzyme is characterized for its
physiological and chemical properties. It has a wide range of pH stability (4.5 to 11.5 and
optimum at pH 8.5), optimum temperature at 700C, 67.7 kDa molecular mass, 6.9 extinction
coefficient, and number of 13 tryptophan, 24 tyrosine and 15 cysteine residues with 7
isulphide bridges [187]. A number of subtilases have been isolated from various
cucurbitaceous plants. Cucumisin a protease enzyme of sarcocarp of melon fruit, Cucumis
melo) has been characterized completely [188] and supported by Uchikoba et al., 1995
[189]. However, protease D, though it is present in same part of this plant need to be
characterized more [190]. A high molecular weight serine protease (78 kDa) called
kiwano protease is present in similar part i.e. sarcocarp of Cucumis metuliferus. It had
maximum activity at pH 8.0 and 300C temperature [206]. A protease obtained from the
sarcocarp of wax gourd (Benincasa hispida). The first 33 residues amino acids in the N -
terminus were sequenced, and it was shown that first 25 residues of amino acids
matched with cucumisin. Its molecular weight is 67.00 kDa. It ad optimum pH and
temperature is 9.0 and 600C respectively [191]. Interestingly cucumisin like serine
protease is present in the sarcocarp of snake gourd (Tricosanthes bracteata) [192].
28
Figure 2.6: Per cent wise distribution of proteases (A) Distribution of proteases; (B) Plant protease; (C) Microbial proteases; (D) Animal proteases and (E) Plants part used for isolation and characterization of
proteases
2.4.1.2.2 Animal Proteases
The most common and known proteases of animal origin are pancreatic trypsin, chymotrypsin,
pepsin and rennin [193, 194]. These are prepared in pure form in bulk quantities. However, their
production depends on the availability of livestock for slaughter, which is governed by political
and agricultural policies. Trypsin is one of the three principal digestive proteinases. In the
digestive process, trypsin acts with the other proteinases to break down dietary protein molecules
29
to their component peptides and amino acids. Trypsin continues the process of digestion (that
begins in the stomach) in the small intestine where a slightly alkaline environment (about pH 8)
promotes its maximal enzymatic activity. This enzyme is active only against the peptide bonds in
protein molecules that have carboxyl groups donated by arginine and lysine [195]. Trypsin is the
most discriminating of all the proteolytic enzymes in terms of the restricted
number of chemical bonds that it will attack [196]. Chymotrypsin is a proteolytic, or protein
digesting enzyme, active in the mammalian intestinal tract. It catalyzes the hydrolysis of peptide
bonds in which the carboxyl groups are provided by one of the three aromatic amino acids i.e.
phenylalanine, tyrosine or tryptophan. Pure chymotrypsin is an expensive enzyme and is used
only for diagnostic and analytical applications [197]. Now a days chymotrypsin tablets are
available, that are used as safe painkillers. Pepsin is an enzyme produced in the mucosal lining of
the stomach that acts to degrade protein. In the laboratory studies, pepsin is most efficient in
cleaving bonds involving the aromatic amino acids, phenylalanine, tryptophan, and tyrosine.
Pepsin is an aspartyl protease and resembles human immunodeficiency virus type 1 (HIV-1)
protease, responsible for the maturation of HIV-1 [198]. Rennet is a pepsin -like protease ,
having the property of clotting , or curdling of milk . Rennet is obtained from the inner
lining of fourth or true stomachs (abomasum) of milk-fed calves [199]. It is used extensively in
the making of cheese and junket. The specialized nature of the enzyme is due to its specificity in
cleaving a single peptide bond in casein to generate insoluble para casein and C-terminal
glycopeptide [14]. Acid proteinase activity has been detected in both African (Trypanosoma
brucei) and Latin American (Trypanosoma cruzi) species of trypanosomes. Although it was
suggested initially that a T. brucei rhodesiense proteinase isolated from trypomastigotes was like
cathepsin D, i.e. an aspartic proteinase [200], it has now been shown that the major proteinase
from T. brucei bloodstream forms must be a cysteine proteinase, since activity is stimulated by
both ethylene glycolbis - (P -aminoethyl ether) N, N -tetraacetic acid and cysteine, inhibited by p
-chloromercuribenzoate, and unaffected by pepstatin [201]. The enzyme had an optimum pH
around 4 for acid denatured hemoglobin hydrolysis [202]. Proteinase activity has also been
reported in Trypanosoma rangeli [203], The partially purified protease of flagellate parasite
obtained from genitourinary tract of cattle i.e. Tritrichomonas foetus [204] which is responsible
for the hydrolysis of denatured hemoglobin, azocasein, and a -N -benzoyl -L -argininamide. It
30
has a molecular weight of between 17,500 and 20,000, having optimum pH between 6.5 and 7.0.
Its activity was blocked by a number of cysteine proteinase inhibitors but not by pepstatin
suggesting that the isolated enzyme belongs to cysteine type protease. Proteinases are also
present in phytoflagellates. Proteolytic activity in malarial parasites was first reported by
Moulder and Evans, 1946 [209] for Plasmodium gallinaceum, a chicken parasite. The first
attempt was made for characterization of protease in Plasmodium berghei and Plasmodium
knowlesi species [210]. They also reported soluble proteolytic activity with pH optima in
between 4 and 8 for hemoglobin hydrolysis. Peptidase activity has been detected in
Tetrahymena, particularly in the cytoplasm and on the outer cell surface of Tetrahymena
thermophila [211]. Proteolytic activity in the rabbit brain homogenate was determined by using
haemoglobin and casein as a substrate [212]. Cathepsin D, a specific aspartic protease occurs in
brain of diabetic rat induced by alloxan monohydrate injection. Also proteolytic activators are
found in cerebral extract of the same rat [159]. Cathepsin L- like proteinase is characterized from
goat brain [213]. Purified enzyme shows the 5.0 optimum pH and 50°C optimum temperature.
The effect of guar gum, lignin and pectin on proteolytic enzyme was investigated in
gastrointestinal tract of rat [214]. A novel protease Cathepsin P of mouse placenta has been well
characterized [215]. Chymosin, the major component of rennet (milk clotting enzyme) is an acid
protease, which is isolated from abomasal tissue of goat kid (Capra hircus). The purified
chymosin had a molecular mass of 36 kDa with maximal activity at 30°C at pH 5.5 [216].
Plasmin and plasminogen derived activities were measured in bovine and human milk with a
chromogenic tripeptide H-D-valyl-L-lysine-p-nitroanilide substrate [217]. A thrombin like serine
proteinase occurred in the snake venom i.e. Bothrops asper. The purified enzyme has a molecular
mass about 27 kDa [218]. Various kinds of proteolytic enzymes were purified from animal
source [219]. Astrup (1951) investigates the activation of proteolytic enzyme in blood (plasmin
and fibrinolysin) by an interaction between its proenzyme and the insoluble tissue activator
(fibrinokinase) [220]. Blackwood et al., 1962 [221] reported the changes occurred in the
proteolytic enzyme systems of rat tissues in response to heterogeneous growth of human ovarian
tumors. A digestive protease enzyme occurred in larval guts of fifth instar stage of Spilosoma
obliqua (Lepidoptera: Arctiidae). This protease was purified using ammonium sulfate
fractionation, ion-exchange chromatography, and hemoglobin-sepharose affinity
31
chromatography. The purified protease had a molecular mass of 90 kDa and a pH optimum of
11. The purified protease optimally hydrolyzed casein at 50°C [173]. Two cysteine peptidases
coined “Metamorphosis Associated Cysteine Peptidase” (MACP-I and MACP-II) are identified
to homogeneity from 40-46 h after puparium formation stage of insect (Ceratitis capitata) which
is a key moment of metamorphosis. Both enzymes showed sensitivity against a specific
inhibitor of papain-like cysteine peptidases i.e. Ep-475. MACP -I is a single chain protein
with 80 kDa molecular mass and it includes several isoforms with pI values of pH 6.25-
6.35, 6.7, and 7.2. This enzyme has an optimum pH of 5 and its pH stability ranges from
pH 4 to 6. The molecular weight and N-terminal sequence (VNIESDTADQ) suggest that
MACP-I is a novel enzyme. On the other hand cathepsin B -like cysteine protease i.e.
MACP -II) belongs to acidic protease family. The molecular weight of this enzyme is 30
kDa and pI is 5.85. N-terminal sequence of this enzyme is LPEQFE -P -QF [174]. A bacterium
species i.e. Bacillus sp. PN 51 is isolated from bat feces. An alkaline serine protease is
characterized from this Bacillus sp. This enzyme has highest protease activity at 600C at
pH 10. Enzyme activity of this enzyme is strongly inhibited by PMSF and chymostatin,
suggesting that the purified enzyme belongs to serine family of protease [175].
2.4.1.2.3 Microbial Proteases
The inability of the plant and animal proteases to meet current world demands has
led to an increased interest in microbial proteases. Microorganisms represent an
excellent source of enzymes owing to their broad biochemical diversity and their
susceptibility to genetic manipulation. Microbial proteases account for approximately
40% of the total worldwide enzyme sales. Proteases from microbial sources are preferred
to the enzymes from plant and animal sources since they possess almost all the
characteristics desired for their biotechnological applications.
2.4.1.2.3.1 Bacterial Protease
Milk clotting enzyme (MCE) is produced by Egyptian Bacillus sphaericus NRC 24. MCE
is obtained by fractional precipitation with acetone, followed by ion exchange
chromatography by using DEAE Sephadex A 25 and finally by Sephadex G 100 column.
32
The pH 6.5 is the optimum pH of MCE at 550C [176]. Protease enzyme of mutated strain
of Bacillus subtilis shows proteolysis phenomenon in the manufacturing of Canadian
cheddar cheese. This is advantageous in the formation of softer curd [177]. A microbial
rennet i.e. milk clotting protease enzyme is partially purified from cell free supernatant
of Bacillus cereus [178] Extracellular alkaline protease isolated from Gram positive
Bacillus firmus MTCC 7728, showed maximum activity t pH 9 and 400C temperature.
Maximum proteolytic activity was observed after 48 h growth, when growth is reached
stationary phase [179]. A new acidic protease is obtained from the culture medium of
thermotolerant Gram positive Bacillus badius MTCC 7727. This enzyme exhibits optimum
activity at pH 5 and 400C temperature [180]. A neutral protease is partially purified from
Bacillus subtilis. The optimal conditions for protease production was an optimum
substrate concentration 0.5%, optimum incubation period 30 h, optimum temperature
and pH is 400C and 7 respectively. This protease enzyme is partially purified by
ammonium sulphate precipitation and Sephadex G 200 filtration. Purified protease enzyme
had a maximum activity at pH 7 [181]. A halotolerant strain of Bacillus subtilis FP -133 is
isolated from fish paste. This strain is identified for its intracellular protease production.
The molecular mass of this intracellular protease is about 59 kDa and the enzyme
(protein) consists of four subunits, each with a molecular mass of 14 kDa [182]. A solid
state fermentation is employed for the production of alkaline protease by a thermophilic
strain of Bacillus subtilis DM-04 using agro industrial waste product and kitchen waste
material viz., mustard oil cake, wheat bran, rice bran, Imperata cylindrical grass, banana
leaves, potato peels and used tea leaves. The crude protease enzyme shows optimum
activity at 400C under alkaline condition [183]. A newly isolated halotolerant Bacillus
aquimaris VITP 4 is used for the production of extracellular protease. The optimum pH
and temperature for production of enzyme is pH 7.5 and 37°C respectively [184]. A
thermophillic neutral protease is characterized from thermophilic Bacillus strain HS 08.
Molecular mass and optimum pH and temperature of this enzyme is 30.9 kDa, pH 7.5 and
650C temperature respectively. Azocasein is the best substrate for enzyme activity [185].
The growth and protease production by Bacillus sp. (SBP - 29) was examined and maximum
protease activity achieved by using soybean meal as substrate; enzyme has optimum
33
temperature and pH optima is 60°C and 9.5 respectively [186]. Surfactants, laundry detergent
and organic solvent resistant alkaline protease occurred in cell free supernatant of
Bacillus sp. HR - 08. The Zymogram analysis of the crude extract revealed the presence
of five extracellular proteases. One of the protease enzymes is partially purified by
three step procedure including ammonium sulphate precipitation, DEAE sepharose ion
exchange chromatography and Sephacryl S 200 gel filtration. The purified protease has
an optimum temperature and pH of 600C and 10 respectively and molecular mass of this
enzyme is 29 kDa [187]. An alkaline protease occurred in cell free broth of Bacillus
circulans BM 15 strain. This enzyme showed optimum activity at pH 7 and 400C; having
molecular mass about 30 kDa [188]. An alkaline serine protease from Bacillus circulans has
been characterized in detail for its robustness and ecofriendly application potential in
leather processing and detergent industries. Molecular mass of the purified enzyme is around
to 39.5 kDa and it exhibits optimum activity at 70°C under alkaline pH environment [189]. A
bacterial strain Bacillus licheniformis is isolated from Tihamet Aseer, Saudi Arabia. Protease of
this candidate displays proteolytic property at optimal pH and temperature 9.0 and 55°C
respectively [190]. Thermostable alkaline protease was purified from Bacillus licheniformis MIR
2 9 [191]. Another strain of Bacillus i.e. Bacillus licheniformis NH 1 is able to produce detergent
stable and thermostable alkaline serine protease. The protease had optimal activity at 68°C
temperature and pH is 10.5 [192]. A novel organic solvent stable alkaline protease is identified in
cell free supernatant of a new strain i .e Bacillus licheniformis YP 1 A, which is isolated from
crude oil contaminant soil. This enzyme retained more than 95% of its initial activity after
preincubating at 40°C for 1 h in presence of 50% (V/V) organic solvents such as DMSO, DMF
and cyclohexane. This protease is active in a broad range of pH from 8 to 12 with the
optimum pH 9.5 and optimum temperature as 600C [193]. Twelve strains of Bacillus
licheniformis were isolated from traditionally fermented African locust bean (iru) for
the production of protease. One of the strain of Bacillus licheniformis LBBL-11 exhibits
highest proteolytic activity. Maximum protease production by using this strain occurred
after 48 h of growth which is the end period of exponential phase of growth. The
protease from this Bacillus sp. had optimum pH is 8 at 600C [194]. Highly thermostable
protease was purified and characterized from broth culture of Bacillus
34
steareothermophillus TLS33 [195] and Bacillus pumilus [196]. An alkalophilic strain of
Bacillus pumilus MK 6-5 is able to produce thermostable alkaline protease. This enzyme is
purified by using ammonium sulphate precipitation, ion exchange and gel filtration
chromatography. Inhibition profile of this enzyme exhibited by PMSF suggested that
the protease belonged to serine protease [196]. A low molecular weight (34.60 kDa) alkaline
serine protease is identified to homogeneity using ammonium sulphate precipitation and
gel filtration from Bacillus pumilus CBS. The N-terminal sequence of first 21 amino
acids (aa) of the purified enzyme is AQTVPYGIPQIKAPAVHAQGY. The highest
homology of 98.1% is observed with BPP-A protease of Bacillus pumilus MS-1. This
protease is strongly inhibited by PMSF and DFP, showing that it belongs to the serine
proteases superfamily. The optimum pH is 10.6 while the optimum temperature is 650C
[198]. An alkalophilic strain of bacterium i.e. Bacillus cereus is able to produce an
extracellular alkaline protease, which is suitable for commercial laundry detergent. This
enzyme shows maximum activity against casein at pH 10.5 and 500C temperature and 28
kDa is the molecular mass of the enzyme [199]. The Bacillus cereus MCM B-326 was
isolated from buffalo hide and characterized for production of extracellular protease.
Maximum protease production occurred at pH 9 and 300C under shake culture condition
by using starch soybean meal medium. This enzyme is used in leather processing unit
due to its dehairing principle [200]. An extracellular bleach stable protease producing
strain was isolated from marine water sample and identified as Bacillus mojavensis A21. The
A21 alkaline protease is purified from the culture supernatant to homogeneity using
acetone precipitation, Sephadex G-75 gel filtration and CM-Sepharose ion exchange
chromatography, with a 6.43 -fold increase in specific activity and 16.56 % recovery. The
molecular weight of the purified protease is 20 kDa. The enzyme is highly active over a
wide range of pH from 7 to 13, with an optimum pH at 8.5. The N-terminal amino acid
sequence of the first 20 amino acids of this purified protease is
DINGGGATLPQKLYQTSGVL. B. mojavensis A21 protease showed low homology with
bacterial peptidases, suggesting that the isolated enzyme is a new and novel protease
[201]. A comparative account on hydrolytic activities of exogenous protease in four
strains of Pseudomonas sp. viz., Pseudomonas sp. C 61, Pseudomonas sp. C 20, Pseudomonas
35
fragi (ATCC 4973) and Pseudomonas fluorescens (NRRL-B-1244) is given . Molecular
mass of isolated protease from C 61, C 20 and P. fluorescens is 46.80, 49.20 and 46.10 kDa
respectively. While P. fragi produces two proteases whose molecular mass is about 49.20 and
34.20 kDa [202]. Gram negative bacteria i.e. Pseudomonas aeruginosa exhibits the
proteolytic activity [203]. Temperature and nutrient factors affect the proteinase
production by P. fluorescens and P. aeruginosa in broth and milk [204]. A protease enzyme
is identified in cell free supernatant of Pseudomonas aeruginosa PD 100. Ammonium
sulphate precipitation, Sephadex G 50 filtration and CM -sephadex chromatography is
employed for the purification of protease. This protease has molecular mass about 36 kDa,
pI is 6.2 and optimum pH 8 at 600C. It finds potential application for waste treatment, used in
detergent and leather industry [205]. A potent bacterium for extracellular protease
production was identified as Pseudomonas sp. RAJR 044. Mutant of this strain JNGR 242
shows highest yield of protease productivity i.e. 2.5 times higher than parent one. The
purified protease enzyme of both strains i.e. parent and mutant a single homogenous band
on SDS-PAGE gel corresponding to 14.4 kDa. Inhibition study demonstrates that the
isolated protease belongs to serine protease family [206]. A solvent tolerant, thermostable
and alkaline metalloprotease occurs in cell free broth culture of alkalophilic Pseudomonas
aeruginosa MTCC 7926. The purified protease has an optimum temperature and pH of 25-
650C and 6-11 respectively and molecular mass is 35 kDa. Purified metalloprotease
showed industrial interest in dehairing of animal skin, anti-staphylococcal activity and
processing of X -ray film for recover of silver [207]. Forty three bacterial strains are
isolated from crude oil contaminated samples by using toluene and cyclohexane
enrichment medium. Out of these, ten bacterial species show highest protease activity.
Among them one of the isolate i.e. PT 121 is identified as Pseudomonas aeruginosa.
Protease of PT 121 shows highest enzyme activity and it acts as a catalyst for
aspartame precursor Cbz-Asp-Phe-NH2 synthesis in presence of 50%dimethylsulphoxuide
[208]. Watanabe, 1988 characterizes the proteolytic properties in more than 100 strains of
oral isolates of Mycoplasma salivarium with respect to aminopeptidase and
carboxypeptidase activity [209]. Petit and Guespin, 1992 [210] describe extracellular
proteolytic activity of protease which occurs in submerged growth of a Gram negative soil
36
bacterium Myxococcus xanthus DK 1622. This enzyme shows the milk clotting activity
and it belongs to aspartic protease family on the basis of inhibition by pepstatin [210].
Protease activity is first time detected in Streptomyces rimosus TM-55 after 12 h of
growth in submerged culture. The optimum pH of this enzyme is 6.5 and temperature is
400C [211]. Alkaline protease is identified from Streptomyces gulbergensis. Purified
enzyme shows optimum pH is 9.0 and 450C temperature. This enzyme is applied in
washing surgical instruments [212]. A comparative account is given in total 157 stock
cultures of lactic acid bacteria for their ability to produce extracellular proteinase with
milk clotting activity. One of the cultures is identified as Enterococcus faecalis TUA
2495 L, which has highest milk clotting activity. The estimated molecular mass of the
purified enzyme of this strain is 35 kDa and pI is 5.4 [213]. A bacterial strain i.e. Listeria
monocytogenes was isolated from degraded cow meat. It exhibits proteolytic activity
against casein, gelatin and egg albumin at pH 9 and 30°C temperature [214]. A novel
extracellular alkaline protease enzyme is identified in cell free supernatant of marine
bacterium eredinobacter turnirae. Optimum temperature and pH for maximum enzyme
activity of this protease is 500C and 9 respectively and molecular mass is 40 kDa [215].
Protease activity was detected in total 317 isolated mesophilic Streptomycetes sp., were
obtained from several areas around Egypt [216]. A low molecular weight (18.4 kDa)
glutamic acid specific protease is identified from Streptomyces griseus [217]. A novel
thermostable and detergent stable subtilisin like serine protease called as aqualysin I is
identified from the culture medium of Thermus aquaticus YT - 1. This enzyme is purified
by applying FPLC system with a mono-S-column. It maintains the stability in presence of
urea and Tween 20 [218]. A novel aspartic protease is present in lactic acid bacteria
(Oenococcus oeni), which is isolated from Argentinean wine by using ammonium
sulphate precipitation and sephadex G-100 filtration. Structurally this enzyme has two
identical subunits whose molecular mass is about 33.10 and 17.00 kDa [219]. Keratinase is
a type of protease enzyme, which is identified from Chrysobacterium sp. Kr6 growing on
poultry feathers [220]. Keratinolytic metalloprotease is purified from bacterium
Microbacterium sp. strain kr 10 by sequential liquid chromatography on Sephadex G-100
37
and Q-Sepharose column. Estimated molecular mass of this enzyme is 42 kDa. The enzyme
had pH and temperature optima of 7.5 and 500C respectively [221].
2.4.1.2.3.2 Fungal Protease
The fungal proteases are active over wide pH range (pH 4 to 11) and exhibit broad
substrate specificity. Aspergillus oryzae produces acid, neutral, and alkaline proteases.
However, these have a lower reaction rate and worse heat tolerance than the bacterial
enzymes do. Fungal enzymes can be conveniently produced in a solid-state fermentation
process. Milk clotting enzyme i.e. microbial rennet is identified from Mucor pussilus [222]. A
solid state fermentation [223] and submerged fermentation [224] are employed for the
production fungal rennet by thermophillic strain of Mucor miehei. This enzyme is
used in cheese production which satisfies the characteristics of organoleptic quality [223].
The solid state fermentation is an appropriate system for rennin production. A report
includes ability of protease production in twelve species of genus Mucor. All the strains
could produce protease enzyme after 120h period of incubation at 280C. Highest
proteolytic activity is recorded in Mucor racemosus Fres. F. chibinensis [225]. The
production of a rennin like milk clotting enzyme is possible by Penicillium citrinum 805
using corn-steep water as a medium. The enzyme has maximum activity at 600C and at pH
6 [226]. Protease enzyme of Candida albicans shows proteolytic activity against
proteins isolated from human saliva [227]. A good protease produced by Aspergillus
fumigatus when grown on glucose-peptone-gelatin medium, pH 5 and 300C for four days
[228]. The fungus Acremonium typhinum produces a novel endoprotease during
symbiotic endophytic infection of the grass, Poa ampla. This enzyme belongs to thiol
containing serine protease family [229]. The wheat bran is most suitable substrate for the
production of alkaline protease produced by Trichoderma koningii [230]. Penicillium
expansum produced an alkaline protease in culture broth which had maximum activity at pH
10.5 and 350C temperature, whose molecular mass is 20.5 kDa. Purification of this
enzyme is achieved by acetone precipitation and column chromatography on Sephadex G-
100 and DEAE Sephadex A-50 [231]. The structure of aspartic proteinase is estimated from
zygomycetes fungus Rhizomucor pusillus. Structurally this enzyme contains two
38
asparagine linked high mannose type oligosaccharide chains at Asn 79 and Asn188 [232]. A
milk clotting enzyme was identified from Penicillium oxalicum through fractional
precipitation with ethanol. This enzyme showed optimum activity at pH 4.3 and 650C
[233]. New extracellular subtilisin like, high molecular weight (75 kDa) alkaline protease
(PoSI) is isolated from white rot fungus Pleurotus ostereatus culture broth. Isoelectric
point of this enzyme is 4.5 [234]. A rice pathogenic fungus i.e. Sarocladium oryzae
secretes proteinase enzyme in the culture medium. This enzyme showed the proteolytic
activity against azocasein as a substrate at pH 9.0 [235]. Acetone precipitation, ion
exchange chromatography and gel filtration methods are used for the purification
protease enzyme from this fungal strain. Estimated molecular mass of the enzyme is 23
kDa and it shows maximum activity at pH 8 and 400C temperature [236]. Similarly a new
milk clotting protease produced by solid state fermentation by Aspergillus oryzae LS1 by
using wheat bran as a substrate [237]. Five different kinds of agricultural wastes viz., corn
cob, oat husk, sugar cane bagasses, corn husk and cassava peel used in submerged
fermentation for protease production by using Penicillium janthinellum [238]. The rice
bran is used as a substrate for the production of neutral metalloprotease by solid state
fermentation using Rhizopus microsporus NRRL 3671 [239] and a local strain of
Aspergillus oryzae (Ozykat-1) [240]. This enzyme showed maximum activity at a temperature
600C and pH 7. Maximum yield protease could achieve by using wheat bran as a
substrate by a highly potent, local strain of Aspergillus awamori: Nakazawa MTCC 6652
[242] by using a modified form of solid state fermentation. Extracellular bleach stable
an alkaline serine protease produced by Aspergillus clavatus ES 1. This enzyme showed
maximum activity at pH 8.5 and 50°C temperature [243].
2.4.1.2.3.3 Viral proteases
Viral proteases have gained importance due to their functional involvement in the
processing of proteins of viruses that cause certain fatal diseases such as AIDS and
cancer. Serine, aspartic, and cysteine peptidases are found in various viruses [97]. All of
the virus-encoded peptidases are endopeptidases; there are no metallopeptidases.
Retroviral aspartyl proteases that are required for viral assembly and replication are
39
homodimers and are expressed as a part of the polyprotein precursor. The mature
protease is released by autolysis of the precursor. An extensive literature is available on
the expression, purification, and enzymatic analysis of retroviral aspartic protease and
its mutants [319]. Recent research is focused on the three-dimensional structure of viral
proteases and their interaction with synthetic inhibitors with a view to designing potent
inhibitors that could combat the relentlessly spreading and devastating epidemic of
AIDS. Crystal and three dimensional structure of aspartyl protease of the HIV-I, has
been determined to 3 A0 resolution; the structure suggested a mechanism for the
autoproteolytic release of protease and a role in the control of virus maturation [320].
Three dimensional structure of the protease has been elucidated from human
cytomegalovirus (hCMV) at 2.27A0 resolution [321].
2.4.1.2.3.4 Algal Protease
Higher level of proteolytic activity occurred in multicellular algae than in unicellular
algae [322]. Matrix metallo protease inhibitor has been identified as a potential therapeutic
candidate from brown algae Ecklonia cava [323]. A comparative account on proteolytic
activity is given in total 47 species of macroalgae including 9 species of chlorophyta, 22
species of rhodophyta and 16 species of phaeophyta [324]. A fibrinolytic enzyme, serine
protease family member is identified from a marine green alga, Codium divaricatum
and it is abbreviated as C. divaricatum protease (CDP). Its molecular is 31 kDa and it
showed maximum activity at pH 9 [325]. Similarly another fibrinolytic trypsin like serine
protease enzyme is identified from Codium latum, a member of marine green alga, and it
is abbreviated as C. latum protease (CLP) [326].
2.4.1.3 Mechanism of action of protease and Physicochemical properties of proteases
The mechanism of the action of proteases has been an interesting subject to researchers.
Purification of proteases to homogeneity is a prerequisite for studying their mechanism of action.
Vast numbers of purification procedures for proteases, involving affinity chromatography, ion-
exchange chromatography, and gel filtration techniques, have been well documented in the
literature. Primary methods of purification of plant proteases often include precipitation
40
(either by using solvent i.e. acetone or salt i.e. ammonium sulfate), ion exchange chromatography
and gel filtration; secondary, yet more specific, techniques include affinity chromatography,
chromatofocusing and hydrophobic interaction chromatography. After the purification enzymes
are subjected to characterization by considering these tools, some physicochemical and
biochemical properties of few earlier reported plant proteases listed in Table 2.1, mostly include
plant proteases studied during the past four to five decades.
2.4.1.3.1 Serine Proteases
Peptide bond hydrolysis is a very common process. A wide variety of enzymes can perform
proteolytic reactions. Members of one large family of protease are called “serine proteases”
because of the important serine group at the active site. All of the serine proteases contain three
residues at their active site: a serine, a histidine, and an aspartate. These comprise the
characteristic catalytic triad. In the numbering scheme for chymotrypsin (a numbering scheme
which is typically used in studies of any of the mammalian serine proteases), the residues are
Ser 195, His 57, and Asp102. Serine proteases increase the rate of peptide bond hydrolysis by
~1010 times compared to the uncatalyzed reaction. Performing this feat requires a specific
structure. As mentioned above, the serine proteases all have three residues that are critical for
catalysis: a serine, a histidine, and an aspartic acid. These are conserved in all of the serine
proteases, and are superimposable in the crystal structures of these proteins.
The side-chain of the amino acid residue of substrate peptide can bind to the recognition site
on the enzyme. Serine195 performs a nucleophilic attack on the substrate. Histidine 57 abstracts
a proton from Ser 195 during the process. The result of the nucleophilic attack is a covalent bond
between the Ser 195 side-chain oxygen and the substrate. The negative charge that develops
on the peptide carbonyl oxygen is stabilized by hydrogen bonds formed from two protease
backbone amide protons. This region of the protein is called the “oxyanion hole”, because it
stabilizes the negative charge on the oxygen. The oxyanion hole is critical for catalysis [327].
Histidine 57 donates a proton to the substrate amide nitrogen, allowing release of the C-terminal
part of the substrate as a free peptide (peptide 1). The final step is an attack by water on the
ester bond between the peptide and the Ser195 oxygen. This forms the second product of
peptide with a normal carboxyl group, and regenerates the serine hydroxyl. The second peptide
41
then dissociates from the enzyme to allow another catalytic cycle to begin [Fig. 2.7]. As it is
apparent in the series of drawings below, serine 195 is the residue that performs the actual
catalysis, while the other residues seem to be important for positioning of the serine and for
stabilizing the intermediate states. Aspartate102 is relatively far from the substrate; it helps
position histidine 57, and raises the pKa of histidine 57 to allow the histidine to act as a base.
This is important to the catalytic process: mutation of aspartate102 to asparagine decreases the
kcat by ~10,000-fold [328]. Serine proteases are very useful as model enzymes for studying
catalytic processes, because they use a variety of techniques for accelerating the reaction rate.
They use acid-base catalysis; the histidine abstraction of the serine proton, and the histidine
donation of the proton to allow release of the first peptide. Serine proteases also use charge
stabilization (the oxyanion hole) to lower the energy of the transition state and use covalent
catalysis to assist in the reaction.
Figure 2.7: Mechanism of action of Serine Protease
Serine proteases are very useful as model enzymes for studying catalytic processes, because
they use a variety of techniques for accelerating the reaction rate. They use acid-base catalysis;
the histidine abstraction of the serine proton, and the histidine donation of the proton to allow
release of the first peptide. Serine proteases also use charge stabilization (the oxyanion hole) to
lower the energy of the transition state and use covalent catalysis to assist in the reaction.
42
2.4.1.3.2 Aspartic Proteases
Aspartic proteases are a family of eukaryotic protease enzyme that utilizes an
aspartate residue for catalysis of their peptide substrates. In general they have two
highly conserved aspartates in the active site and are active at acidic pH. Suguna
et al., 1987 proposed a general mechanism for peptide cleavage by aspartyl protease [Fig.
2.8]. While a number of different mechanisms for aspartyl proteases have been proposed, the
most widely accepted is a general acid base mechanism involving coordination of water
molecule between the two highly conserved aspartate residues.
One aspartate activates water by abstracting a proton, enabling the water to attack the
carbonyl carbon of the substrate scissile bond, generating a tetrahedral oxyanion intermediate.
Rearrangement of this intermediate leads to protonation of the scissile amide.
Figure 2.8: Mechanism of action of Peptide cleavage by Aspartic Protease
2.4.1.3.3 Metalloproteases
Metalloproteases constitute a family of enzymes from the group of proteinases,
classified by the nature of the most prominent functional group in their active
site. The mechanism of the action of metalloproteases is slightly different from
that of the above described proteases. These enzymes depend on the presence of
bound divalent cations. Kester and Matthews, 1977 suggested an acid base catalysis for
metalloproteases [Fig. 2.9], by taking interaction between water molecule and the Zn+2 ions
[329].
43
Figure 2.9: Mechanism of action of Metallo Protease
Manzetti et al., 2003 provided evidence that a coordination between water and the zinc, where
in a histidine from the HExxHxxGxxH-motif participates in catalysis by allowing the Zn+2
ions to assume a quasi-penta coordinated state, via its dissociation from it [330]. In this state, the
Zn+2 ion is coordinated with two oxygen atoms from the catalytic glutamic acid, the substrate’s
carbonyl oxygen atom and the two histidine residues and can polarize the glutamic acid’s oxygen
atom, proximate the scissile bond, and induce it to act as reversible electron donor. This forms an
oxyanion transition state. At this state water molecules act on the dissociated scissile bond and
complete the hydrolyzation of the substrate.
2.4.1.3.4 Cysteine proteases
Cysteine proteases catalyze the hydrolysis of carboxylic acid derivatives through a double-
displacement pathway involving general acid-base formation and hydrolysis of an acyl-thiol
intermediate. The mechanism of action of cysteine proteases is thus very similar to that of
serine proteases.
Figure 2.10 : Mechanism of action of Cysteine Protease
44
A striking similarity is also observed in the reaction mechanism for several peptidases of different
evolutionary origins. The plant peptidase papain can be considered the archetype of cysteine
peptidases and constitutes a good model for this family of enzymes. They catalyze the
hydrolysis of peptide, amide ester, thiol ester, and thiono ester bonds [331]. The initial step
in the catalytic process (Figure, where Im and + Him refer to the imidazole and protonated
imidazole respectively) involves the noncovalent binding of the free enzyme and the substrate to
form the complex. This is followed by the acylation of the enzyme, with the formation and
release of the first product, the amine R’-NH2. In the next deacylation step, the acyl-enzyme
reacts with a water molecule to release the second product, with the regeneration of free
enzyme [Fig. 2.10].
The enzyme papain consists of a single protein chain folded to form two domains containing a
cleft for the substrate to bind. The crystal structure of papain confirmed the Cys25- His159
pairing [332]. The presence of a conserved asparagine residue (Asn175) in the proximity of
catalytic histidine (His159) creating a Cys-His-Asn triad in cysteine peptidases is considered
analogous to the Ser-His-Asp arrangement found in serine proteases. Studies on the mechanism of
action of proteases have revealed that they exhibit different types of mechanisms based on
their active-site configuration. The serine proteases contain a Ser-His-Asp catalytic triad, and
the hydrolysis of the peptide bond involves an acylation step followed by a deacylation step.
Aspartic proteases are characterized by an Asp-Thr-Gly motif in their active site and by acid-
base catalysis as their mechanisms of action. The activity of metalloproteases depends on the
binding of a divalent metal ion to a HExxHxxGxxH-motif. Cysteine proteases adopt a
hydrolysis mechanism involving a general acid-base formation followed by hydrolysis of an
acyl-thiol intermediate.
2.4.1.4 Physiological Functions of Proteases
Proteases execute a large variety of complex physiological functions. Their importance in
conducting the essential metabolic and regulatory functions is evident from their occurrence
in all forms of living organisms. Proteases play a critical role in many physiological and
pathological processes such as protein catabolism, blood coagulation, cell growth and
migration, tissue arrangement, morphogenesis in development, inflammation, tumor growth
45
and metastasis, activation of zymogens, release of hormones and pharmacologically active pep-
tides from precursor proteins, and transport of secretory proteins across mem-
branes. In general, extracellular proteases catalyze the hydrolysis of large proteins to smaller
molecules for subsequent absorption by the cell, where as intracellular proteases play a critical
role in the regulation of metabolism. Some of the major activities in which the proteases
participate are described below.
2.4.1.5 Protein Turnover
All living cells maintain a particular rate of protein turnover by continuous, albeit balanced,
degradation and synthesis of proteins. Catabolism of proteins provides a ready pool of amino
acids as precursors of the synthesis of proteins. Intracellular proteases are known to participate in
executing the proper protein turnover for the cell. In E. coli, ATP-dependent protease La, the
lon gene product, is responsible for hydrolysis of abnormal proteins [333]. The turnover of
intracellular proteins in eukaryotes is also affected by a pathway involving ATP-dependent
proteases [334]. Evidence for the participation of proteolytic activity in controlling the protein
turnover was demonstrated by the lack of proper turnover in protease-deficient mutants.
2.4.1.6 Sale of Proteases and other enzymes
The present estimated value of the worldwide sales of industrial enzymes is $1 billion [335].
Hydrolytic enzymes contribute 75 % of total enzyme sale including protease, carbohydrase and
lipase. Proteases represent one of the three largest groups of industrial enzymes and account
for about 59% [Fig. 2.11] of the total worldwide sales of enzymes.
46
Amylase, 18%
Other Protease , 21% Carbohydrase, 10%
Rennin, 10% Lipase, 3%
Trypsin, 3% Pharmaceutical
Enzyme, 10%
Alkaline Protease, 25%
Figure 2.11: Distribution of enzyme sales. Yellow portion indicates the total sales of proteases.
2.4.2 Keratinase
Keratinase is a protease capable of digesting keratins in chicken feathers as well as animal wool
and hair. Proteases are by and large classified into two major groups based on their cleavage
habits. First group is called “Endopeptidases” which cleaves non-terminal peptide bonds inside
polypeptide chains. Second group so-called “Exoproteases” breaks down peptide bond at the
amino termini (aminopeptidases) or at the carboxy termini (carboxypeptidases) of their
substrates. Proteases are further categorized based on functional groups of their active sites. Four
major groups are: serine proteases, cysteine proteases, aspartic proteases, and metalloproteases.
Keratinases are mostly known to be endopeptidase which is a member of serine protease family
[103].
Keratins are less likely to be digested by enzyme such as trypsin, pepsin, and papain [104]
because the stiff packing of the protein chain in α-helix and β-sheet structures resists
andmechanically stabilizes the keratin to microbial degradation. However, keratin can be
degraded by a number of species of saprophytic and parasitic fungi, a few actinomyces and
Bacillus species [105].
Keratins proteolysis like the other proteins is effectively directed by proteases. Nevertheless,
keratinases are known to have an effect on their hydrolysis [94]. Keratinases have already been
47
purified from several microorganisms such as fungi, a few bacteria, and some Streptomyces
species [105]. Keratinase belongs to a group of proteinase enzymes that have high level of
activity on insoluble keratin, playing a crucial role in hydrolyzing feather, hair, wool, collagen
and casein in removing barriers in waste water treatment systems. Not only have these enzymes
been applied in sewage systems but have also recently emerged in many applications including
food, textile, medicine, and cosmetics industries. In fact, using of keratinases in skin medications
to get rid of acne and psoriasis as well as removing of human callus in medical applications is
well known. It is also utilized for the erection of a vaccine for dermatophytosis therapy [94, 103].
More interestingly, keratinases are well identified in leather industry to have been employed in
dehairing process of animal skins instead of treating them with sodium sulfide [327].
The majority of known keratinases are endopeptidases belonging to the serine protease family.
Amino acid sequences of several Bacillus keratinases are known to show striking sequence
homology to Carlsberg subtilisin (E.C. 3.4.21.62), a well-described member of the serine
protease family. All three catalytic active sites (Asp32, His64, Ser221) characteristic to
subtilisins can be identified in the primary sequence of keratinases. Subtilisins and related
extracellular proteases bear a triad of 'pre', 'pro' and 'mature' regions. The N-terminal 'pre' part
serves as a signal sequence directing the translocation of the newly synthesized precursor
molecules through the cell membrane. The adjacent 'pro' region acts as an intramolecular
chaperone that promotes the correct fold of the protease domain and is a prerequisite for the
protease maturation. In the last step of maturation, the enzyme is activated via an autocatalytic
removal of the 'pro' region. Kinetic parameters of Bacillus licheniformis KK1 keratinase and
Carlsberg subtilisin were determined and compared using a set of para-nitroaniline (pNA)
conjugated oligopeptides as substrates [210]. Both enzymes showed similar kinetics with most of
the oligopeptide substrates, preferentially cleaving next to hydrophobic and aromatic residues.
The nearly identical protein sequence and the similar biochemical characteristics suggest a tight
relationship between keratinases and subtilisins isolated from Bacillus strains.
2.4.2.1 Sources of microbial keratinases: Diversity among keratinase-producing
microorganisms
Keratinases are very widespread in the microbial world and they can be identified from
48
microorganisms of the three domains: Eucarya, Bacteria, and Archaea. These microorganisms
have been isolated from the most distinct sites, from Antarctic soils to hot springs, including
aerobic and anaerobic environments. Therefore, microbial keratinases present a great diversity in
their biochemical and biophysical properties. The characteristics of some microbial keratinases are
summarized in Table 2.4.
In natural environments, keratinolytic fungi are involved in recycling the carbon, nitrogen, and
sulfur of the keratins. Their presence and distribution seem to depend on keratin
availability, especially where humans and animals exert strong selective pressure on the
environment. A number of studies focused on the keratinolytic potential of dermathophytic
fungi such as Trichophyton and Microsporum, mainly due to their medical and veterinary
implications. Although some studies on the biotechnological potential of such genera are
available [26], little commercial interest was attracted by this group because of their potential
pathogenicity. Among nondermatophytic fungi, keratinases showing attractive biochemical
properties were reported to be produced by Aspergillus, Trichoderma, Doratomyces,
Myrothecium, Paecilomyces, Scopulariopsis, and also Acremonium, Alternaria, Beauveria,
Curvularia, and Penicillium. Besides the biotechnological interest, these investigations may
help in understanding the role of fungi in the degradation of complex keratinous substrates in
thenature.
Several keratinases have been isolated from a diversity of bacteria. Bacillus spp. appears as the
prominent keratinase producer. Diverse strains of Bacillus licheniformis and Bacillus subtilis are
described as keratinolytic [350, 70, 351, 73, 352], but other species such as Bacillus pumilus and
Bacillus cereus also produce keratinases [324, 296, 198]. Furthermore, B. licheniformis [350] is the
source of Versazyme™, the first thermo-resistant commercial keratinase developed by Shih and
coworkers at Bioresource International, Inc. Some thermophilic and alkaliphilic strains of
Bacillus have also been described to show keratin-degrading activity, such as Bacillus halodurans
AH-101, Bacillis pseudofirmus AL-89, and B. pseudofirmus FA30-01 [353]. Besides,
microorganisms belonging to the same genus (Bacillus) can produce different keratinases (Table
2.4). In this regard, the exploitation of microbial diversity might provide keratinases with suitable
properties for biotechnological uses. For instance, the keratinolytic potential and
eratinolytic enzymes from novel mesophilic Bacillus species isolated from the Amazon basin
49
have been recently characterized in our laboratory [354], presenting interesting features for
diverse potential applications.
Keratinase producers have been also described among actinomycetes, mainly from the
Streptomyces genus. These microorganisms, isolated from several different soil sites, are
associated with the hydrolysis of a wide range of keratinous substrates like hair, wool,
and feathers. For example, two highly keratinolytic actinomycete strains, Streptomyces flavis
2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic), were isolated from
Antarctic soil. Thethermophilic species Streptomyces gulbarguensis [30], Streptomyces
thermoviolaceus, and Streptomyces thermonitrificans have also been isolated from soils. Besides
these thermophilic strains, some mesophilic Streptomyces have also been characterized like
Streptomyces pactum DSM 40530 [33], Streptomyces graminofaciens [31] and Streptomyces
albidoflavus K1-02 [54].
In addition to these Bacillus sp. and actinomycetes, keratinase production has been associated
to an increasing number of bacteria. Since keratin degradation is facilitated at high temperatures
and pH, and thermostable hydrolases are employed in various industrial processes, the
thermophilic and alkaliphilic microorganisms are of great interest. Fervidobacterium
pennavorans, Fervidobacterium islandicum, Meiothermus ruber H328, Clostridium sporogenes,
and strains of Thermoanaerobacter sp. were isolated from extreme environments like hot
springs, geothermal vents, solfataric muds, and volcanic areas. Some alkaliphilic strains such as
Nesternkonia sp. and Nocardiopsis sp. TOA-1 have been also characterized, showing
keratinase activity in strongly alkaline pH. The investigation of keratinolytic bacteria isolated
from soils has revealed a high and undescribed diversity. For example, a single soil site revealed
several keratinolytic isolates related to Bacillus, Cytophagales, Actinomycetales, and
Proteobacteria. Several feather-degrading bacterial strains have been isolated from soil,
poultry wastes, and other sources, and characterized as mesophilic keratinase producers. These
include some Gram-positive, such as Lysobacter NCIMB 9497, Kocuria rosea, and Micro-
bacterium sp. kr10 [271], and a few Gram-negative, such as Vibrio sp. [60], Xanthomonas
maltophilia, Stenotrophomonas sp., Chryseobacterium sp. [362, 70], and Serratia sp. Many
Archaea grow in environments usually lethal to most cells, including extremes in temperature,
pH, salt content, and pressure. Thus, Archaea are valuable resource of proteases for fundamental
50
microbiology and enzymology studies, also possessing the potential for biotechnological
applications. Archaea displaying keratinolytic activities were recently revealed through the in
situ enrichment of thermophilic prokaryotes with hydrolytic activities in hot springs (68-87°C
and pH 4.1-7.0). One isolate, identified as 1507-2, grew on α-keratin at 70° C and pH 6.0, and
was found to be an archaeon of the Crenarchaeota phylum, representing a cluster of the so-
called unknown Desulfurococcales. In the same investigation, a 220-kDa thermostable keratinase
showing broad pH (6.0 to 10.0) and temperature (30 to 80°C) ranges of activity, with an
optimum at pH 7.0 and 66°C, was found in the culture supernatant of strain 1523-1 growing on
keratin.
Table 2.4: Diversity of keratinolytic microorganisms and some biochemical properties of their keratinases
Microorganism Catalytic type Molecular Optimal Optimal Reference mass (kDa) pH T (°C) Bacteria Bacillus sp. SCB-3 Metallo 134 7 40 Lee et al. 2002 B. cereus DCUW Serine 80 8.5 50 Ghosh et al. 2008 B. licheniformis FK14 Serine 35 8.5 60 Suntornsuk et al. 2005 B. licheniformis K-508 Thiol 42 8.5 52 Rozs et al. 2001 B. licheniformis MSK103 Serine 26 9-10 60-70 Yoshioka et al. 2007 B. licheniformis PWD-1 Serine 33 7.5 50 Lin et al. 1992 B. licheniformis RPk Serine 32 9.0 60 Fakhfakh et al. 2009 B. pumilis Serine 65 8.0 65 Kumar et al. 2008 B. subtilis KD-N2 Serine 30.5 8.5 55 Cai et al. 2008b B. subtilis KS-1 Serine 25.4 7.5 - Suh and Lee 2001 B. subtilis MTCC (9102) Metallo 69 6 40 Balaji et al. 2008 B. subtilis RM-01 Serine 20.1 9 45 Rai et al. 2009 Clostridium sporogenes - 28.7 8 55 Ionata et al. 2008 Chryseobacterium sp. kr6 Metallo 64 8.5 50 Riffel et al. 2007 Chryseobacterium indologenes TKU014 Metallo P1: 56 P1: 10 P1: 30-50 Wang et al. 2008a Metallo P2: 40 P2: 7-8 P2: 40 Metallo P3: 40 P3: 8-9 P3: 40-50 Fervidobacterium islandicum AW-1 Serine >200 9 100 Nam et al. 2002 Fervidobacterium pennavorans Serine 130 10 80 Friedrich and Antranikian 1996
Kocuria rosea Serine 240 10 40 Bernal et al. 2006a Kytococcus sedentarius Serine 30-50 7-7.5 40-50 Longshaw et al. 2002 Lysobacter sp. NCIMB 9497 Metallo 148 - 50 Allpress et al. 2002 Microbacterium sp. kr10 Metallo 42 7.5 50 Thys et al 2006 Nesternkonia sp. AL-20 Serine 23 10 70 Gessesse et al. 2003 Nocardiopsis sp. TOA-1 Serine 20 >12.5 60 Mitsuiki et al. 2004 Stenotrophomonas maltophilia Serine 35.2 7.8 40 Cao et al. 2009
51
Streptomyces sp. S7 Serine-metallo 44 11 45 Tatineni et al. 2008 Streptomyces sp. strain 16 Serine KI: 203.2 KI: 9 KI: 50 Xie et al. 2010 Serine KII: 100.8 KII: 9 KII: 50 Serine KIII: 31.8 KIII: 9 KIII: 50 Serine KIV: 19.2 KIV: 9 KIV: 60 Streptomyces albidoflavus Serine 18 6-9.5 40-70 Bressollier et al. 1999 Streptomyces pactum Serine 30 7-10 40-75 Böckle et al. 1995 Streptomyces gulbagensis DAS 131 - 46 9 45 Syed et al. 2009 Streptomyces thermoviolaceus - 40 8 55 Chitte et al. 1999 Thermoanaerobacter sp. 1004-09 Serine 150 9.3 60 Kublanov et al. 2009a Thermoanaerobacter keratinophilus Serine 135 8 85 Riessen and Antranikian 2001 Xanthomonas maltophilia Serine 36 8 60 De Toni et al. 2002 Fungi Aspergillus fumigatus Serine - 6.5-9 45 Santos et al. 1996 Aspergillus oryzae Metallo 60 8 50 Farag and Hassan 2004 Doratomyces microsporum Serine 30-33 8-9 50 Gradisar et al. 2005 Myrothecium verrucaria Serine 22 8.3 37 Moreira-Gasparin et al. 2009 Paecilomyces marquandii Serine 33 8.0 60-65 Gradisar et al. 2005 Scopulariopsis brevicaulis Serine 36-39 8.0 40 Anbu et al. 2005 Trichoderma atrvoviride F6 Serine 21 8-9 50-60 Cao et al. 2008 Trichophyton mentagrophytes Serine 38-41 4.5 - Tsuboi et al. 1989 Trichophyton schoenleinii - 38 5.5 50 Qin et al. 1992 Trichophyton sp. HÁ-2 Serine 34 7.8 40 Anbu et al. 2008 Trichophyton vanbreuseghemii Serine 37 8.0 - Moallaei et al. 2006 2.4.2.2 Biochemistry of keratinases
2.4.2.2.1 General biochemical characteristics
The properties of microbial keratinases may be diverse depending on the producer
microorganism. These enzymes are predominantly extracellular, although cell-bound and
intracellular enzymes have been described [371]. Some general biochemical characteristics of
selected keratinases are presented in Table 2.5. Most of the microbial keratinases are alkaline or
neutral proteases showing optima pH ranging 7.5-9.0. However, some enzymes are optimally
active outside this range, even at extreme alkalophilic pH or at slightly acidic pH [325]. A
feature showed by several keratinases is the stability over a wide pH range [371]. This property
is remarkable for the keratinase of Nocardiopsis TOA-1, which is stable over a pH range of
1.5 to 12.0 for 24 h at 30°C. Increased stability has been recently achieved by recombinant
keratinases, such as the B. licheniformis MKU3 keratinase expressed in Pichia pastoris X33
[43].
52
The temperature optima of keratinases may also be very variable, often depending on the
source and origin of the isolate (Table 2.5). The enzyme of the thermophilic F. pennavorans has
optimum temperature at 80°C while the mesophilic Stenotrophomonas maltophila DHHJ showed
maximum activity at 40°C. In some exceptional cases, as for F. islandicum AW-1, the optimum
of 100°C has been reported Nam et al. 2002.
The molecular masses of several keratinases have been determined. Despite they range
from 18 to 240 kDa for S. albidoflavus and K. rosea [54], respectively, most keratinases have
less than 50 kDa (Table 2.5). The majority of keratinases are monomeric enzymes; however,
multimeric keratinases are also reported Nam et al. 2002; Xie et al. 2010. Higher molecular
masses are often associated to keratinases with metalloprotease character or those from
thermophilic organisms (Table 2.5).
The effect of metal ions and inhibitors on keratinolytic activity has been also
investigated. The results of several studies have been recently compiled [331], showing as a
general trend that the presence of divalent metal ions like Ca2+, Mg2+, and Mn2+ often
stimulate the keratinases. This positive effect might be related to the maintenance of the active
enzyme conformation, and to the stabilization of the enzyme-substrate complex [361].
Additionally, metal ions may protect the enzyme against thermal denaturation [367, 327]. In
subtilisin-related keratinases, for instance, interaction of calcium with specific Ca2+-binding sites
or autolysis sites may explain the improved thermostability in the presence of such metal ions
[41]. On the other hand, transition and heavy metals like Cu2+, Ag+, Hg2+, and Pb2+ generally
caused inhibition of keratinolytic enzymes.
Organic solvents, detergents, and reducing agents have diverse effects on different
keratinases. However, a tenden-cy observed in several investigations is the stimulation of the
keratinolytic activity by reducing agents. Such effect is usually attributed to the reduction of
cysteine bridges in the keratinous substrate rather than direct effects on the enzyme [72].
2.4.2.2.2 Structure and catalysis of keratinase
The primary sequence of some keratinases was determined or deduced from its coding genes.
This information allows comparing keratinases with other previously characterized proteases.
The N-terminal sequence of B. licheniformis PWD-1 keratinase is identical to Carlsberg
53
subtilisin, which is a typical member of the serine-type proteases. Its coding gene kerA
presents elevated similarity and homology with the subtilisin Carlsberg and the deduced
amino acid sequence differs by only five amino acids [324]. Further investigation revealed
that the deduced sequence of kerA exactly agrees with those of substilisins Carlsberg
(NCIMB6816), NCIMB10689 and ATCC12759, except for an arginine instead of lysine at
position 144 and a valine instead of alanine at position 222 [209]. Since V222 is close to S220
at the catalytic site, this change may be related to the enhanced keratin hydrolysis. Keratinase
KerRP from B. licheniformis RPk, consisting of 274 amino acids, showed 99% homology with
kerA, and 98% with subtilisin Carlsberg, differing from kerA by 2 amino acids (K144 and A222
in KerRP and R144 and V222 in kerA) and from subtilisin Carlsberg by four amino acids
(S102, A128, K144, and N211 in KerRP and T102, P128, R144, and S211, respectively, in
subtilisin Carlsberg), but conserving the active site residues D32, H63, and S220 [41].
Likewise, a keratinase from B.licheniformis MKU3, conserving the amino acid residues that
form the catalytic triad, showed 99% similarity with both kerA and subtilisin Carlsberg.
Additionally, the amino acid sequence of a keratinase from B. licheniformis MSK103
showed homology of approximately 87% with kerA. The complete nucleotide sequencing of
the gene encoding for a high-molecular-mass extracellular feather-degrading protease (Vpr)
from B. cereus DCUW allowed the analysis of its structural domains: initial amino
acids (1-25) encoded for a putative N-terminal extracellular signal sequence for secretion,
amino acids 62-158 encoded for a subtilisin N sequence that is signature for N-terminal
processing, amino acids 172-583 encoded for a catalytic peptidase S8 domain, and amino acids
606-917 encoded a Vpr-specific protease-associated domain.
Similar to B. licheniformis, the main keratinolytic activity from B. subtilis is often
associated with serine protease activity. The gene aprA cloned from a feather-
degrading strain of B. subtilis showed significant similarity and homology with subtilisins. Despite
the limited information on purified keratinases from B. subtilis, the subtilisin-like character
appears to predominate. A keratinolytic protease purified from B. subtilis KS-1 showed an
Nterminal sequence similar to that of other serine proteases of B. subtilis [69]. A novel
keratinase from B. subtilis S14, showing no activity on collagen and an excellent dehairing
activity, presents an identical N-terminal sequence to subtilisin E [28]. This enzyme also
54
showed an internal sequence VAVIDSGLDSSHPDLNVR that is identical to that of a
bacterial alkaline subtilisin and to that of nattokinase [28].
The thermophilic feather-degrading bacterium F. penna-vorans produces the keratinase
fervidolysin. The primary sequence of fervidolysin, deduced from the coding gene fls,
showed high homology with the subtilisin-like proteases. Although the N-terminal sequence of
fervidolysin is very distinct to that of subtilisins, its active site region was similar to subtilisin-like
serine proteases. More detailed information on the configuration of fervidolysin was obtained from
the 1.7 Å resolution crystal structure [298]. Fervidolysin is composed of four domains: a catalytic
domain, two beta-sandwich domains, and the propeptide domain. The architecture of the catalytic
domain closely resembles that of a subtilisin, and a calcium-binding site was observed within the
catalytic domain, which exactly matches that of the subtilisin E-propeptide domain complex [298].
The N-terminal sequences of some fungal keratinases have been also described. These also
present significant similarity with serine proteinases. The comparison of their N-terminal
sequences indicates a closer relationship with those of subtilisins than that of keratinases from
actinomycetes (Fig. 2.12). Indeed, the keratinases from Streptomyces appear to be close to
Streptomyces griseus proteinase B (SBPG), the major component of pronase [367].
More recently, information on protein sequences of keratinolytic metalloproteases became
available. A kerati-nolytic metalloprotease purified from Chryseobacterium sp. kr6 belongs to the
M14 family of peptidases, also known as the carboxypeptidase A family [362]. This is the first
keratinolytic enzyme associated to this family. The enzyme exhibit an O-glycosylation site DS* in
the peptide 5 (KGSSADS*PNSEEK), that is usually found in proteins secreted by the related
species Chryseobacterium meningosepticum. Flavastacin, an extracellular metalloprotease from C.
meningosepticum presents a heptaglycoside linked to the DS* site, which may be associated
with protection against autoproteolysis. Peptide sequences of three keratinases from C.
indologenes were recently determined by mass spectrometry showing no significant homology
to any other reported microbial peptide [360]. The complete DNA and amino acid sequences of a
keratinolytic metalloprotease from Pseudomonas aeruginosa was also recently reported [324],
and its N-terminal sequence showed only little homology with other microbial keratinases (Fig.
2.12).
The catalytic type of many keratinases has been determined by using specific
55
substrates and inhibitors. The specificity of some keratinases is illustrated in Fig. 2. The
chymotrypsin-like keratinase from S. albidoflavus exhibited specificity for aromatic and
hydrophobic amino acid residues, as demonstrated by using synthetic peptides [367]. S. pactum
DSM 40530 produces a keratinolytic serine protease that showed substrate specificity and
stereospecificity to p-nitroanilide (pNA) derivatives of aromatic and basic amino acids lysine and
arginine (L-enantiomers), but hydrolysis of benzoyl-D-Arg-pNA was not detected. The keratinase
NAPase of Nocardiopsis sp. TOA-1, composed by 188 amino acids and conserving the catalytic
triad of the active site of serine proteases (H35, D61, and S143), showed preference for
aromatic and hydrophobic residues at the P1 position of synthetic pNa substrates; structural
similarities suggest that APase might be categorized as a chymotrypsin-type serine protease of
Streptomyces strains.
An alkaline protease from the feather-degrading Nesterenkonia sp. AL20 also exhibited higher
activity with tetrapeptides with hydrophobic residues located at P1 site. B. subtilis S14
keratinase showed preference for Arg at P1, small amino acids like Ala and Gly at P2, and
Gln or Glu at P3 [28]. Besides the well-characterized serine-type keratinases from B.
licheniformis, other keratinolytic proteases with atypical properties have been associated to this
species. B. licheniformis strain HK-1 produces a keratinolytic protease that was partially
inhibited by EDTA, 1,10-phenanthroline (Zn2+ specific chelator), PMSF, and Zn2+ [81]. The
keratinolytic B. licheniformis K-508 secreted an unusual trypsin-like thiol protease that is
strongly active towards benzoyl-Phe-Val-Arg-pNA and is not inhibited by PMSF [82]. The
purified keratinase of K. rosea was strongly inhibited by 4-(2-aminoethyl) benzene-sulfonyl
fluoride, soybean trypsin inhibitor, and chymostatin, suggesting that it belongs to the serine
protease family. The keratinolytic metalloprotease from the Gram-negative bacterium
Lysobacter sp. was strongly active towards carboxybenzoyl-Phe-pNA. The keratinase produced
by Chryseobacterium sp. Kr6 appears to belong to the metalloprotease type since it was
inhibited by EDTA and 1, 10-phenanthroline and lacks hydrolysis of the substrate benzoyl-L-Arg-
pNA [362]. The enzyme was strongly active on L-Leu-(amino-4-methylcoumarin) (AMC) and N-t-
Boc-Ile-Glu-Gly-Arg-AMC, and also active on L-Phe-AMC and L-Val-AMC, showing
preference for hydrophobic and positive amino acids. Activation by Ca2+ and inhibition by excess
Zn2+ of Gram-negative keratinases [366, 362] resembles typical bacterial metalloproteases like
56
Figure 2.12: Substrate specificity of some keratinases.
KerA from B.licheniformis PWD-1 (Evans et al. 2000), keratinase K-508 from B. licheniformis (Rozs et al. 2001), keratinase S14 from B. Subtilis (Macedo et al. 2008), SAKase from S. albidoflavus K1-02 (Bressollier et al. 1999), keratinase Sp from S. pactum (Böckle et al. 1995), SFase-2 from S. fradiae ATCC 14544 (Kitadokoro et al. 1994), keratinase AL20 from Nesterenkonia sp. (Bakhtiar et al. 2005), NAPase from Nocardiopsis sp. (Mitsuiki et al. 2004), keratinase Pm from P. marquandii, and keratinase Dm from D. microsporus (Gradisar et al. 2005), keratinase Tv from Trichophyton vanbreuseghemii (Moallaei et al. 2006), keratinase 1004-09 from Thermoanaerobacter sp. (Kublanov et al. 2009a), keratinase kr6 from Chryseobacterium sp. (Silveira et al. 2009), keratinase L from Lysobacter sp. (Allpress et al. 2002), keratinase kr10 from Microbacterium sp. kr10 (Thys and Brandelli 2006), keratinase Pa from P. aeruginosa (Lin et al. 2009), and fervidolysin from F. pennivorans (Kluskens et al. 2002). Superscript letter the enzyme showed higher affinity for Suc-Ala-Ala-Pro-Leu-pNa. Superscript letter b the enzyme specificity was also high on Suc-Ala-Ala-Pro-Phe-pNA. Superscript letter c the enzyme was also highly active on N-t-Boc-Ile-Glu-Gly-Arg-AMC. Superscript letter d obtained from the 3D modeling of the catalytic site (Kluskens et al. 2002)
thermolysin and extracellular proteases from Pseudomonas species. Keratinolytic proteases are
57
largely classified as serine or metallo proteases and, independent of their catalytic type, it seems
that hydrophobic and aromatic aminoacid residues are preferably cleaved at the P1 position
(Fig. 2.12), resembling the specificity of chymotrypsin. The presence of Arg at P1 appears to be
preferentially cleaved only by some keratinases from Bacillus sp. [82, 28] and
Chryseobacterium sp., a preference also showed by trypsin. In this sense, the specificity of
keratinases towards keratinous materials may arise from the amino acid composition of
keratins, which contains about 50-60% of hydrophobic and aromatic residues [353, 59].
Additionally, the nature of amino acids at the vicinity of the cleaved bond was showed to
influence the specificity for the P1 position, which might indicate the presence of an extended
active site [367, 28]. For instance, substitution of Ala residue of Suc-Ala-Ala-Ala-pNA by Pro at
the P2 position increased the kcat/Km value of NAPase from Nocardiopsis sp. TOA-1 by 37-
fold. The preference for longer substrates at both sides of the scissile peptide bond suggests
the suitability of eratinases for the conversion of native and complex substrates. The
cleavage of peptide bonds in the compact keratin molecules is difficult due to the restricted
enzyme-substrate interaction on the surface of keratin particles and accessibility to splitting
points. Therefore, the hydrolyzing ability of keratinolytic proteases may be due to its ability and
specificity to bind onto compact substrates, and a more exposed active site. NAPase from
Nocardiopsis sp. TOA-1 showed a strong adsorption capability (more than 70% of added
enzyme) towardskeratin, obeying a Langmuir-type adsorption isotherm, which was likely due to
the presence of an efficient binding pocket for keratin. In the Vpr protease from B. cereus
DCUW, the C-terminal domain of the enzyme has the ability to noncovalently bind
specific substrates like feather keratin via protein-protein interactions. Keratinase adsorption to
fibrous keratin was previously showed to occur through electrostatic interactions [367].
2.4.2.2.3 Hydrolysis of native proteins
Keratinases are mostly endo-proteases showing a broad spectrum of activity [270], usually
hydrolyzing soluble proteins (such as casein) more effectively than insoluble proteins (such as
keratins; Lin et al. 1992; Suh and Lee 2001; Brandelli 2005; Suntornsuk et al. 2005; Syed et al.
2009) [324, 363, 270, 361, 30]. Only few microbial keratinases show higher hydrolysis of
keratins than soluble proteins [325]. Besides some exceptions, purified keratinolytic enzymes are
58
often ineffective to hydrolyze native keratin [367, 371, 362], a behavior that is mainly attributed to
the high levels of disulfide bonds in the keratin molecules.
Keratin hydrolysis by microorganisms is reported to not simply rely on the production of
keratinolytic proteases. Release of thiol groups during microbial growth on keratinous
materials supports the essential role of the reduction of disulfide bonds for efficient keratin
degradation [276, 328]. Production of intra- and/or extracellular disulfide reductases [80, 217],
release of sulfite and thiosulfate [331], and also a cell-bound redox system [331, 86] are
reported to lead to sulfitolysis. In fungi, keratinolysis also seems to involve the mechanical attack
of the keratinous substrates through mycelial pressure and/or penetration [265]. Since the main
efforts are focused on the characterization of keratinolytic proteases, components responsible for
the reduction of disulfide bonds are probably removed during the purification procedures. Thus, the
keratinolysis process seems to involve, at least, sulfitolysis and proteolysis.
The importance of disulfide bonds to the recalcitrance of the keratin structure is
emphasized by the stimulation of keratin hydrolysis by purified keratinases through the
addition of reducing agents that promote sulfitolysis [369, 271, 72, 181]. The cut of
disulfide bonds changes the conformation of keratins and more sites for keratinase action are
exposed. Production of free thiol groups is not observed during the hydrolysis of keratinous
substrates, indicating that the breakdown of disulfide bonds is not accomplished by keratinases
themselves [324, 270, 361, 181].
Therefore, a suitable redox environment is necessary for effective keratin degradation by
keratinases, and even by other proteases [331]. pidermal ‘soft’ α-keratins such as that from
stratum corneum, which possess a low level of disulfide bonds in comparison to hair and
wool hard’ α-keratins, are generally more susceptible to hydrolysis by keratinases [270].
Another observed trend is the higher hydrolysis of feather β-keratins when compared to ‘hard’ α-
keratins from hair and wool [270, 29]. Cysteine residues, responsible by formation of disulfide
bonds, are present at higher concentrations in wool 10.5-17% [59, 330] than in feather β-keratin
4.2-7.6% [353, 31]. In α-keratin, the polypeptide chains are closely associated pairs of α-
helices, whereas β-keratin has high proportions of β-pleated sheets. These structural features
provide a more extended conformation for β-keratins in comparison to α-keratins [61], which
could result in the enhanced keratinase accessibility to the former. In this context, a keratinase
59
from Chryseobacterium sp. kr6 was reported to hydrolyze keratinous substrates in the following
order: stratum corneum from human sole > porcine skin > chicken feathers > chicken nails >
wool > hair keratin [270]. Other microbial keratinases are also reported to hydrolyze both α-
and β-keratins [72]. A keratinase from B. pseudofirmus FA30-01 was able to degrade feather, but
was poorly active towards wool and hair [327], similar to that observed for B. subtilis P13 [29]
and B. subtilis RM-01 keratinases [233]. Contrarily, keratinases from Doratomyces microsporus
and Paecilomyces marquandii hydrolyzed α-keratins from skin, nail, and hair, but not β-
keratins from chicken feathers. Therefore, other properties, such as fibril structure and
porosity [61], might dictate the differential hydrolysis of these substrates.
Although some inferences on the keratinolysis mechanism can be drawn, the elucidation of
this complex process needs to be supported by conclusive experimental data. The hydrolysis of
feather keratin by free and immobilized keratinase from B. licheniformis PWD-1 yielded a
major peak of 18 kDa in size-exclusion HPLC, which increased with the reaction time from 1 to
10 h [70]. A single peak was also observed during hydrolysis of feather keratin by
Chryseobacterium sp. kr6 keratinase [270]. Such pattern might indicate that feather keratin was
hydrolyzed at specific cleavage site(s) [70].
In summary, the multiplicity of catalytic mechanisms observed for microbial keratinases
(including serine, thiol, and metalloproteases) could have an important effect in the
natural environment, where the synergistic action of keratinolytic microorganisms and its
enzymes and metabolites may degrade the recalcitrant structure of native keratin more
easily than a pure culture. After the initial attack by keratinases and disulfide reductases, other
less specific proteases may also act on the substrate, resulting in extensive keratin degradation.
2.4.2.3 Production of keratinases
The biotechnological application of keratinolytic proteases requires the production of these enzymes
in sufficient amounts for commercial purposes. Keratinase production is usually induced by keratin
[26, 27] and, thus, a keratinous substrate (chicken feathers, feather meal, hair) is often added to the
cultivation medium. Such keratin-rich materials are produced in high amounts by agroindustrial
activities and are normally discarded as wastes. Therefore, this microbial technology connects the
production of valuable products (keratinases, microbial biomass, protein hydrolysates) from low-
60
cost substrates with an alternative and efficient way of waste management [270, 359, 328].
However, the addition of a keratinous substrate is not always required for keratinase production
[352, 298]. Other non-keratinous substrates, such as soy flour, soybean meal [29], skim milk,
shrimp shell powder [360], gelatin [271], casein, and cheese whey [60], have been reported to act
as inducers of keratinase production. Furthermore, in some cases, keratinase production appears to
be constitutive [82, 298]. Recently, peptide limitation in culture media induced the sequential
production of collagenase, elastase, and keratinase by B. cereus IZ-06b and IZ-06r. The
keratinolytic activities produced on keratinous substrates, in comparison to readily assimilable
substrates, may result from nitrogen limitation rather than keratin induction. In this case,
keratinous substrates would act only as indirect inducers.
Supplementation of keratin-containing media with different carbon and/or nitrogen sources might
result in higher levels of keratinase production. For instance, the addition of glucose [80, 26],
sucrose [27], starch [327, 30], molasses [352], and bagasses; and additional nitrogen sources,
such as urea, peptone, tryptone, yeast extract, ammonium chloride, and sodium nitrate are
reported to enhance enzyme yields [331, 27]. Conversely, the addition of supplementary
substrates carbohydrates; inorganic and/or organic nitrogen sources often decrease enzyme
production by some microorganisms, mainly due to catabolite repression mechanisms [270, 276].
Therefore, the effect of different growth substrates on keratinase production is highly variable,
depending on the microorganism, the substrate and the carbon and nitrogen concentration,
implicating that the medium composition should be determined on a case-by-case basis [270].
Besides the composition of the culture medium, incubation temperature, pH, and aeration are
among the important variables investigated in view to obtain high keratinase yields Maximum
keratinase activities are usually achieved in the late exponential or stationary growth phases [271,
371, 327, 385]. In this sense, keratinase production was bserved to be growth-associated in B.
licheniformis FK 14 [361]; similar results were observed with Chryseobacterium sp. kr6 [270]
and Streptomyces gulbargensis DAS 131 [30]. Nevertheless, Serratia sp. HPC 1383 showed the
highest proteolytic activity in the initial phase of growth (24 h) on feather meal medium, whereas
maximum biomass was achieved after 96 h. Development of mutant and recombinant microbial
strains is also investigated, representing useful techniques to enhance keratinase production and keratin
degradation [72, 251]. In the specific case of the opportunistic pathogen P. aeruginosa, cloning and
61
heterologous expression of its keratinase gene also represents a viable alternative to ensure safety
[324]. The gene kerA, which encodes a B. licheniformis keratinase, is expressed specifically for
feather hydrolysis [324]; therefore, the presence of feather keratin as the sole carbon and nitrogen
source in the culture medium may result in preferential expression of the keratinolytic protease.
This gene has been cloned and expressed in heterologous microorganisms such as Escherichia
coli and B. subtilis, but the keratinase yields are lower than the wild strain [360]. However,
increased keratinase yield was achieved by chromosomal integration of multiple copies of the
kerA gene in B. licheniformis and B. subtilis. The kerA gene was also cloned for extracellular
expression in P. pastoris, resulting in a recombinant enzyme that was glycosylated and even
though active on azokeratin. The vast majority of investigations report keratinase production
through submerged cultivations. Only recently the production of keratinolytic enzymes through
solid-state processes has been demonstrated [265]. The potential of keratinase production by
immobilized microorganisms was also reported [62].
2.4.3 Applications of Proteases and Keratinases
Keratinases from microorganisms have attracted a great deal of attention in the recent decade,
particularly due to their multitude of industrial applications such as in the feed, fertilizer,
detergent, leather and pharmaceutical industries. Currently, the most promising application of
keratinases/ keratinolytic microorganisms is the production of nutritious, cost-effective,
environmentally benign feather meal for poultry. Other applications of keratinases have yet to be
thoroughly explored before commercialization. The following section will discuss some of the
prospective applications of keratinases that are rapidly gaining importance.
All proteolytic enzymes have characteristic properties with regard to temperature, pH, ion
requirement, specificity, activity and stability. These biochemical parameters determine the
application of protease in industry apart from other factors, which include the cost of
production and development, markets and the economy of application. Proteases have a huge
variety of applications, mainly in the detergent and food industries. In view of the recent trend of
developing ecofriendly technologies, proteases are envisaged to have extensive applications in
leather treatment and in several bioremediation processes. Proteases are used extensively in the
62
pharmaceutical industry for preparation of medicines such as ointments for debridement of
wounds, etc.
2.4.3.1. Detergent industry
Proteases are one of the standard ingredients of all kinds of detergents ranging
from those used for household laundering to reagents used for cleaning contact lenses. The use of
proteases in laundry detergents accounts for approximately 25 per cent of the total worldwide
sales of enzymes. Röhm Company in Germany isolated the first enzyme for industrial use, in
1914 [360]. They used a trypsin enzyme isolated from animal’s pancreas that degrades proteins.
It proved to be so powerful compared to traditional washing powders that the original small
packages size “made the German housewives suspicious; the product had to be reformulated and
sold in larger packages”. The real breakthrough of enzymes occurred with the introduction of
microbial proteases into washing powders. The first commercial bacterial Bacillus protease
was marketed in 1956, under the trade name BIO-40 by the Swiss Company Gebrüder Schnyder.
In 1962, Novo Nordisk, in Denmark, was the first company to mass-produce an alkaline protease
suitable for wash conditions; they introduced Alcalase, produced by Bacillus licheniformis
and commercially named BIOTEX. This was followed by Maxatase, a detergent made by Gist-
Brocades [336]. The biggest market for detergents is in the laundry industry, amounting to a
worldwide production of 13 billion tons per year. The ideal detergent proteases have broad
substrate specificity to facilitate the removal of a large variety of stains (food, blood, grass, and
body secretions). Activity and stability at high pH and temperature, and compatibility with other
chelating and oxidizing agents added to the detergent are among the major prerequisites for the
use of proteases in detergents. The key parameter for the best performance of a protease in a
detergent is its pI (ionic strength). It is known that a protease is more suitable for this application
if its pI coincides with the pH of the detergent solution. Esperase and Savinase T (Novo
Industry) produced by alkalophilic Bacillus sp., are two commercial preparations with very high
isoelectric points (pI 11.0) hence, they can withstand higher pH ranges. Due to the present energy
crisis and the awareness for energy conservation, it is desirable to use proteases that are
active at lower temperatures. A combination of lipase, amylase, and cellulose is expected to
enhance the performance of protease in laundry detergents. All detergent proteases currently
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used in the market are serine proteases produced by Bacillus strains. Fungal alkaline proteases
are advantageous due to the ease of downstream processing to prepare a microbe-free enzyme.
An alkaline protease from Conidiobolus coronatus was found to be compatible with commercial
laundry detergents used in India, it retained 43 per cent of its activity at 50ºC for
50 min., in the presence of 25 mM Ca+2 and 1 M glycine [337].
Proteolytic enzymes have dominated the detergent market since ancient times. In fact,
approximately 89% share of detergent enzymes is captured by alkaline proteases, with Novo
Nordisk and Genencor International being the major suppliers [371]. Nonetheless, there is
always a need for newer enzymes with novel properties that can further widen the scope of
enzyme-based detergents. Keratinases have the ability to bind and hydrolyze solid substrates like
feather. This is an important property of detergent enzymes as they are required to act on protein
substrates attached to solid surfaces, making them attractive additives for hard-surface cleaners.
They could also help in the removal of keratinous soils that are often encountered in the laundry,
such as collars of shirts, on which most proteases fail to act. An extended application of
keratinases in detergents is their use as additives for cleaning up of drains clogged with
keratinous wastes [371]. Table 2.5: Protease in industry
Industry Enzyme Application
Leather Trypsin, Other protease Bating of leathers, Dehairing and dewooling of skins
Food processing Several proteases Modification of protein rich material i.e. soy protein or wheat gluten
Baking Neutral protease Dough conditioners
Dairy
Calf rennet and other trypsin, chymotrypsin, ficin, Fungal protease,
Chymosin
Coagulation of milk protein (cheese production), production of enzyme modified cheese; whey processing Replacement
of calf rennet Active component of calf rennet; also production by genetically engineered microbes developed
Detergent Alkaline protease Extensive use in laundry detergents for protein stain removal
Meat Papain Meat tenderization
Beverages Papain Removal of turbidity
Confectionery Thermolysin Reverse hydrolysis in aspartame synthesis Removal of dead tissues and dissolution of blood clots Treatment of certain
types of hemia
Pharmaceutical Trypsin, Chymopapain,
Carboxypeptidase, Trypsin
Conversion of hog insulin, Production of human insulin
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2.4.3.2 Leather Industry
Leather processing involves several steps such as soaking, dehairing, bating and tanning. The
major building blocks of skin and hair are proteinaceous. Leather industry contributes to one of
the major industrial pollution problems, and effluent disposal; the pollution causing chemicals,
sodium sulfide, salt, solvents, lime, etc. arise mainly from the pre-tanning processes of leather
processing [338]. In order to overcome the hazards caused by the tannery effluents, the use of
enzymes as a viable alternative to chemicals has successfully resorted to in improving
leather quality and in reducing environmental pollution [339, 340]. Proteases are used for
selective hydrolysis of noncollageneous constituents of the skin and for removal of nonfibrillar
proteins such as albumins and globulins, in the several pretanning operations. The purpose of
soaking is to swell the hide [341]. Traditionally, this step was performed with alkali. Currently,
microbial alkaline proteases are used to ensure faster absorption of water and to reduce the time
required for soaking by 10-20 hours [342, 343]. The use of non-ionic and, to some extent,
anionic surfactants to accelerate the process is compatible with the use of enzymes. The
conventional method of dehairing and dewooling consists of development of an extremely
alkaline condition followed by treatment with sulfide to solubilize the proteins of the hair root
(the severe alkaline condition was a health hazard for the workers). At present, alkaline
proteases with hydrated lime and sodium chloride are used for dehairing, resulting in a
significant reduction in the amount of wastewater generated. Earlier methods of bating were
based on the use of animal feces as the source of proteases; these methods were unpleasant, unhy-
gienic and unreliable, and were replaced by methods involving pancreatic trypsin
[339]. Currently, trypsin is used in combination with others Bacillus and Aspergillus proteases for
bating. The selection of the enzyme depends on its specificity for matrix proteins such as elastin
and keratin, and the amount of enzyme needed depends on the type of leather (soft or hard) to be
produced. Increased usage of enzymes for dehairing and bating not only prevents pollution
problems but is also effective in saving energy. Novo Nordisk manufactures three different
proteases, Aquaderm®, NUE®, and Pyrase®, for use in soaking, dehairing, and bating, re-
spectively (Novo Nordisk website).
Leather processing technology involves a series of operations, amongst which pre-tanning
contributes to the major amount of pollution (approximately 70%). Sodium sulfide, lime and
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solid wastes generated as a result of pre-tanning are mainly responsible for increased
biochemical oxygen demand (BOD), chemical oxygen demand (COD) and total dissolved solids
(TDS) [317]. Biocatalytic leather processing involves the use of a mixture of enzymes, among
which proteases, lipases and carbohydrases are well exploited for various pre-tanning stages
[317]. In addition, keratinolytic proteases lacking collagenolytic and having mild elastolytic
activities are increasingly being explored for the dehairing process. They would help in the
selective breakdown of keratin tissue in the follicle, thereby pulling out intact hairs without
affecting the tensile strength of leather [28]. A few reports that indicate that keratinases could be
useful depilating agents are available [372, 54]. In fact, a keratinase from B. subtilis S14 Macedo
et al. 2005 [28] was reported to completely eliminate the need for toxic sodium sulfide. Thus,
sulfide-based “hair-destroying dehairing” processes that pose an environmental threat by
increasing the BOD could be replaced by keratinase-based cleaner “hair-saving dehairing”
technology.
2.4.3.3 Food industry
The use of proteases in the food industry dates back to antiquity. They have been routinely used
for various purposes such as cheese making, baking, preparation of soy hydrolysates, and meat
tenderization.
2.4.3.3.1 Dairy industry
The major application of proteases in the dairy industry is in the manufacture of cheese. The
milk-coagulating enzymes fall into four main categories: (a) animal rennets, (b) microbial
milk coagulants, (c) vegetable rennet and (d) genetically engineered chymosin [14]. The
first commercial application of agricultural biotechnology approved by the FDA (Food and
Drug Administration) in 1990 was the development of fermentation-derived chymosin, an
enzyme used in cheese production to coagulate milk. Because natural chymosin must be
extracted from the lining of the stomachs of slaughtered 04-day-old calves, supplies were
limited. Advances in biotechnology have enabled scientists to create an unlimited, cheaper, and
more consistent supply of chymosin by using a genetically engineered microorganism to
produce the enzyme through fermentation. This technology is now used in over 90 per cent
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of all cheese manufacturing industries in U.S., creates chymosin that is 40 to 50 per cent
less expensive than the natural enzyme (Tomorrows Bounty webpage). The microbial
enzymes exhibited two major drawbacks, i. e., (a) the bitterness in cheese, after storage due to
the presence of high levels of nonspecific and heat-stable proteases; and (b) a low yield. The
exhaustive research on this matter has resulted in the production of enzymes that are
completely inactivated at normal pasteurization temperatures and contain very low levels of
nonspecific proteases.
2.4.3.3.1.1 Milk
Milk is a translucent white liquid produced by the mammary glands of mammals. Approximate
composition of milk can be given (Fig. 2.13):
• 87.3% water (range of 85.5% - 88.7%)
• 3.9 % milkfat (range of 2.4% - 5.5%)
• 8.8% solids-not-fat (range of 7.9 - 10.0%):
protein 3.25% (3/4 casein)
lactose 4.6%
minerals 0.65% - Ca, P, citrate, Mg, K, Na, Zn, Cl, Fe, Cu, sulfate, bicarbonate,
many others
acids 0.18% - citrate, formate, acetate, lactate, oxalate
enzymes - peroxidase, catalase, phosphatase, lipase
gases - oxygen, nitrogen
vitamins - A, C, D, thiamine, riboflavin, others
The nitrogen content of milk is distributed among caseins (76%), whey proteins (18%), and non-
protein nitrogen (NPN) (6%)
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Figure 2.13: Milk Composition as percent total volume
2.4.3.3.1.2 Casein Casein is the predominant phosphoprotein that accounts for nearly 80% of milk proteins. The
principal casein fractions are alpha (s1) and alpha (s2)-caseins, ß -casein, and kappa-casein. The
distinguishing property of all caseins is their low solubility at pH 4.6. The common
compositional factor is that caseins are conjugated proteins, most with phosphate group(s)
esterified to serine residues. These phosphate groups are important to the structure of the casein
micelle. Calcium binding by the individual caseins is proportional to the phosphate content.
Casein consists of a fairly high number of proline peptides, which do not interact. There are also
no disulfide bridges. As a result, it has relatively little tertiary structure. Because of this, it
cannot denature. It is relatively hydrophobic, making it poorly soluble in water. It is found in
milk as a suspension of particles called casein micelles which show some resemblance with
surfactant-type micelle in a sense that the hydrophilic parts reside at the surface. The caseins in
the micelles are held together by calcium ions and hydrophobic interactions.
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Figure 2.14: Casein micelle and casein submicelle
Figure 2.15 : κ- casein
2.4.3.3.1.2 Casein Types
Table 2.6: Four different types of bovine casein exist, each with several genetic variants.
Type Mol. wt. pI Phosphates/mole E1% g protein/L in skim milkα-s1 22,068-23,724 4.2-4.76 8-10 10.0-10.1 12-15 α-s2 25,230 10-13 3-4 β 23,944-24,092 4.6-5.1 4-5 4.5-4.7 9-11 κ 19,007-19,039 4.1-5.8 1 10.5 2-4
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2.4.3.3.1.2.1 α-s1 Casein α-s1 Casein is the most prevalent form of casein in bovine milk. It has been reported to exhibit
antioxidant and radical scavenging properties. It has also been reported to be involved in the
transport of and casein from the endoplasmic reticulum to the Golgi apparatus. It has two
hydrophobic regions, containing all the proline residues, separated by a polar region, which
contains all but one of eight phosphate groups. It can be precipitated at very low levels of
calcium.
Figure 2.16: α-s1 Casein
2.4.3.3.1.2.2 α-s2 Casein:
Proteolytic fragments of α-s2 Casein have been shown to exhibit antibacterial activity.
Specifically the 39 amino acid casocidin-1 peptide fragment has been shown to inhibit E.
coli and Staph. carnosis growth. It has concentrated negative charges near N-terminus and
positive charges near C-terminus. It can also be precipitated at very low levels of calcium.
70
Figure 2.17: α-s2 Casein
2.4.3.3.1.2.3 β-Casein:
β-Casein and its fragments have been implicated in a number of biological functions. The
casoparan peptide has been reported to activate macrophage phagocytosis and peroxide release.
Casohypotensin and casoparan may be involved in bradykinin regulation. Casohypotensin has
also been shown to be a strong inhibitor of endo-oligopeptidase A, a thiol-activated protease
capable of degrading bradykinin and neurotensin, and hydrolyzing enkephalin-containing
peptides to produce enkephalins. β-Caseins are also a source of casomorphin peptides which
exhibit opioid activity binding to opioid receptors. Casomorphins may be the hydrolysis product
of dipeptidyl peptidase IV. It has highly charged N-terminal region and a hydrophobic C-
terminal region. Very amphiphilic protein acts like a detergent molecule. Self association is
temperature dependant; will form a large polymer at 20° C but not at 4° C. Less sensitive to
calcium precipitation.
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Figure 2.18: β-Casein
2.4.3.3.1.2.4 κ-Casein
It is a mammalian milk protein involved in a number of important physiological processes. κ-
Casein's orientation on the surface of the casein micelle functions as an interface between the
hydrophobic interior caseins and the aqueous environment. During clotting of milk, hydrolysis
by chymosin or rennin releases the water soluble fragment, para-k-casein and the hydrophobic
caseinomacropeptide. Very resistant to calcium precipitation, stabilizing other caseins. Rennet
cleavage at the Phe105-Met106 bond eliminates the stabilizing ability, leaving a hydrophobic
portion, para-kappa-casein, and a hydrophilic portion called kappa-casein glycomacropeptide
(GMP), or more accurately, caseinomacropeptide (CMP).
Figure 2.19: κ-Casein
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2.4.3.3.1.3 Rennet Rennet is usually a natural complex of enzymes produced in any mammalian stomach to digest
the mother's milk, and is often used in the production of cheese. Rennet contains many enzymes,
including a proteolytic enzyme (protease) that coagulates the milk, causing it to separate into
solids (curds) and liquid (whey). The active enzyme in rennet is called chymosin or rennin but
there are also other important enzymes in it, e.g., pepsin or lipase. There are non-animal sources
for rennet that are suitable for vegetarian consumption.
Figure 2.20: preparation of Rennet
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2.4.3.3.1.4 Renin/Chymosin
Renin belongs to a family of enzymes referred to as aspartic proteases, which also includes the
enzymes pepsin, cathepsin, and chymosin. Renin is a mono specific enzyme that displays
remarkable specificity for its only known substrate, angiotensinogen.
Figure 2.21: 3D network κ- Casein
Chymosin causes cleavage of a specific linkage - the peptide bond between phenylalanine and
methionone in the κ- Casein. If this reaction applies to milk, the specific linkage between
the hydrophobic (para-casein) and hydrophilic (acidic glycopeptides) group of casein inside milk
would be broken, since they are joined by phenylalanine and methionine. The hydrophobic group
would unite together and would form a 3D network to trap the aqueous phase of the milk. The
resultant product is calcium phosphocaseinate. Due to this reaction, rennin is used to bring about
the extensive precipitation and curd formation in cheese making.
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Figure 2.22: specific linkage the peptide bond between phenylalanine and methionone in the κ- Casein
Chymosin (rennin) is essential for the manufacture of good quality cheeses. Found in the fourth
stomach of suckling calf's. Very expensive and “inhumane” to process now so it has been
engineered into bacteria that mass produces. It has a very specific activity- Hydrolyzes only one
bond in к-casein, one of the many proteins that make up the milk casein protein complex (к-, α-,
β-casein). This breaks up the casein complex (micelle) and it aggregates leading to a clot, the
first step in cheese production. Most other proteases can initiate a milk clot like chymosin but
they would continue the casein hydrolysis producing bitter peptides and eventually breaking the
clot.
Figure 2.23: Schematic representation of events in clotting of milk. The αs-, β-, and κ-caseins are shown by
striped, stippled and white balls, respectively.
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2.4.3.3.1.5 Milk Clotting
The milk-clotting process consists of 3 main phases:
1. Enzymatic degradation of κ-casein
2. Micellar flocculation
3. Gel formation
Each step follows a different kinetic pattern, the limiting step in milk-clotting being the
degradation rate of κ-casein. The kinetic pattern of the second step of the milk-clotting process is
influenced by the cooperative nature of micellar flocculation whereas the rheological properties
of the gel formed depend on the type of action of the proteases, the type of milk, and the patterns
of casein proteolysis The overall process is influenced by several different factors, such as pH or
temperature.
The conventional way of quantifying a given milk clotting enzyme employs milk as the substrate
and determines the time elapsed before the appearance of milk clots. However, milk clotting may
take place without the participation of enzymes because of variations in physicochemical factors,
such as low pH or high temperature.
As early as 1970 milk-clotting enzyme from Mucor pusillus was isolated by Kei Arima [272].
Milk coagulation is a basic step in cheese manufacturing. For a long time calf rennet, the
conventional milk-clotting enzyme obtained from the fourth stomach of suckling calves is the
most widely used coagulant in cheese making all over the world to manufacture most of the
cheese varieties. The worldwide reduced supply of calf rennet and the ever increase of cheese
production and consumption have stimulated the research for milk-clotting enzyme (MCE) from
alternative sources to be used as calf rennet substitutes. Various animals, plants and microbial
proteases have been suggested as milk coagulants. However, attention has been focused on the
production of milk-clotting enzymes (MCEs) from crude extract of spices since no work has
been done till date and they are part of food since historic times.
Work done on production of milk clotting enzymes from culture of Bacillus Subtilis natto [379]
and from some Pakistani Plants [157] has been taken into account and the same procedures for
different assays are followed.
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Protease, also known as proteolytic enzymes or proteinases, are enzymes that break down protein
into amino acids so the body can use them. Protease can also destroy viruses and bacteria,
because they are proteins. There are also commercial uses for protease. Rennet is a type of
protease originating from the fourth stomach of calves. Rennet is used in the process of making
cheese from milk.
2.4.3.3.2 Baking industry
The safety of the source organism is an important consideration in the safety assessment for
recombinant enzymes. Aspergillus oryzae, not considered to be pathogenic, is widely
distributed in nature and is commonly found in foods. Enzymes from Aspergillus oryzae are
extensively used in production of a variety of foods such as syrups, alcohol, fruit juices, brewing,
chocolate syrup, baking and meat tenderizing [368]. The enzyme is used in the baking industry as
a processing aid to strengthen gluten in dough systems. It causes a more elastic and stronger
gluten network similar to that obtained by traditional oxidizing agents such as potassium
bromate or ascorbic acid. The enzyme is active in the dough and the leavening of the unbaked
bread, but normally inactivated by high temperatures during the baking. The enzyme is used as a
processing aid only, and is not expected to be present in the final food. Any residue would be in
the form of inactivated enzyme, which would be metabolized like any other protein [369].
2.4.3.3.3 Soy sauce production
Soybeans serve as a rich source of food, due to their high content of good-quality protein.
Proteases have been used from ancient times to prepare soy sauce and other soy products. The
alkaline and neutral proteases of fungal origin play an important role in the processing of soy
sauce. Proteolytic modification of soy proteins helps to improve their functional properties.
Treatment of soy proteins with alcalase at pH 8 results in soluble hydrolysates with high
solubility, good protein yield, and low bitterness. The hydrolysate is used in protein-fortified soft
drinks and in the formulation of dietetic feeds.
2.4.3.3.4 Brewing industry
The brewing industry is a major user of proteases. In the production of brewing wort Bacillus
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subtilis protease are used to solublize protein from barley adjuncts, thereby releasing peptides and
amino acids which can fulfill the requirement of the nitrogen supply. The proteolytic enzymes are
used in chill proofing, a treatment designed to prevent the formation of precipitates during cold
storage. In beer, hazes are formed due to the presence of proteinanceous substances which also
precipitate the polyphenols and oligosaccharides. Hydrolysis of the protein components prevents
aggregation of the insoluble complex.
2.4.3.3.5 Meat tenderization
India is endowed with the largest buffalo population in the world. It accounts for 59.08 % of the
world buffalo population. About 10.66 million buffaloes are slaughtered annually producing
1.47 million MT buffalo meat. They are slaughtered mainly for meat. The byproducts from
slaughtered animals are also of good value. Buffalo tripe is one of the important edible offal and
weighs about 4.36 to 5.45 kg per animal. Commercial exploitation of tripe for development of
processed product manufacture is very limited because of its poor functional properties and
inherent toughness due to high collagen content. It is essential to develop technologies for
utilization of tripe into processed product manufacture by reducing its toughness. In order to
improve tenderness of meat, a number of methods have been tried. Tenderization may be
achieved by use of chemical or proteolytic enzymes. Proteolytic enzyme, papain is used to
tenderize tough meat cuts. Papain is very powerful in hydrolyzing fibrous protein and
connective tissue [370]. In general, uniform penetration of tenderizer enzyme has always
posed problem during tenderization treatment [371].
2.4.3.4 Synthesis of aspartame
The use of aspartame as a noncalorific artificial sweetener has been approved by the Food and
Drug Administration. Aspartame is a dipeptide composed of L-aspartic acid and the methyl
ester of L-phenylalanine. The L configuration of the two amino acids is responsible for the sweet
taste of aspartame. Maintenance of the stereospecificity is crucial, but it adds to the cost of
production by chemical methods. Enzymatic synthesis of aspartame is therefore, preferred.
Although proteases are generally regarded as hydrolytic enzymes, they catalyze the reverse
reaction under certain kinetically controlled conditions. An immobilized preparation of
78
thermolysin from Bacillus thermoprotyolyticus is used for the enzymatic synthesis of aspartame.
Toya Soda (Japan) and DSM (The Netherlands) are the major industrial producers of aspartame.
2.4.3.5 Pharmaceutical industry
The wide diversity and specificity of proteases are used to great advantage in developing
effective therapeutic agents. Oral administration of proteases from Aspergillus oryzae (Luizym
and Nortase) has been used as a digestive aid to correct certain lytic enzyme deficiency syndromes.
Clostridial collagenase or subtilisin is used in combination with broad-spectrum antibiotics in the
treatment of burns and wounds. An asparginase isolated from E. coli is used to eliminate
aspargine from the bloodstream in the various forms of lymphocytic leukemia. Alkaline protease
from Conidiobolus coronatus was found to be able to replace trypsin in animal cell cultures [372].
Curcain a plant protease was purified from the latex of Jatropha curcus was found to be active in
wound healing agent.
2.4.3.6 Therapeutic uses
The most obvious use of proteolytic enzymes is to assist digestion. Injection of some foreign
proteases into human reduces tissue inflammation and pain. Use of proteolytic enzymes helped
reduce the discomfort of breast engorgement in lactating women. Proteolytic enzymes were
reported in reducing pain, swelling and inflammation caused by sugary and injury.
2.4.3.7 Photography industry
The photography industry uses large quantity of silver in the light sensitive emulsion that it
produces. When such film is processed, to recover the expensive silver, the procedure involves
separating the silver containing gelatin from the film base. The aqueous solution that results
contain both gelatin and silver, but the presence of protein hinders the separation of silver.
Addition of proteolytic enzymes at a temperature of 500C and pH 8.0 rapidly degrades the
gelatin and allows the silver particles to separate out.
.
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2.4.3.8 Production of films, coatings and glues
Recently there has been an increased interest in the production of biodegradable films, coatings
and glues from keratinous waste products like hair, feathers, skin, fur, animal hooves, horns etc.
for compostable packaging, agricultural films or edible film applications. Keratin structure is
chemically modified and hydrolyzed to produce stable dispersions for such applications.
Alternatively controlled hydrolysis of keratin using keratinases could offer an environment-
friendly technology.
2.4.3.9 Animal feed
Feathers represent over 90% protein, the major component being β-keratin, a fibrous and
insoluble structural protein extensively cross-linked by disulfide, hydrogen and hydrophobic
bonds. Owing to their insoluble nature, feathers are resistant to degradation by common
microbial proteases, viz. trypsin, pepsin and papain. Thus, the several million tons of feathers
generated annually by the livestock industry leads to troublesome environmental pollution and
wastage of a protein-rich reserve [64, 330]. Until recent years, feathers were baked at high
temperature and pressure and used as animal feed supplement in the form of feather meal. The
hydrothermal treatment, in addition to being expensive, resulted in the destruction of certain
essential amino acids, viz. methionine, lysine and tryptophan, yielding a product with poor
digestibility and variable nutrient quality [360]. The drawbacks of the high-temperature
treatment impel the use of microbial keratinases that serve as attractive alternatives to hydrolyze
feather into nutritionally rich animal feed [64]. The application of keratinases/keratinolytic
microbes for improvement of feather as poultry feed has been extensively reviewed by Onifade
et al. 1998 [64]. The bulk of information on feather meal production using microorganisms is
provided by Shih and coworkers at North Carolina University. It is well documented that
supplementing feather meal/ raw feather with crude keratinase enzyme PWD1 modifies the
structure of keratin, leading to improved digestibility and bolstered growth of poultry [370].
Feather meal is relatively inexpensive and is shown to be superior to soybean meal in terms of
total cysteine, valine and threonine content, and the hydrolyzed meal can replace soybean meal at
7% dietary level. The crude enzyme can also serve as a nutraceutical product, leading to
significant improvement in broiler performance. In addition, nutritional enhancement can be
80
achieved by hydrolysis of feather meal/raw feather using keratinolytic microorganisms.
Fermentation significantly increases the levels of essential amino acids (methionine, lysine and
arginine), and the microbial biomass contributes as a rich source of protein. Feeding trials of the
feather lysate produced by B. licheniformis PWD1 revealed growth curves identical to that
observed with standard soybean meal [17]. In order to produce sufficient quantities of keratinase
PWD1 for application in feather meal production, scaling up of the enzyme has been
accomplished in a 150-l fermenter [70]. Furthermore, cloning, overexpression and bio-
immobilization of the enzyme have been successfully carried out to meet the demands of the
animal feed industries [324, 70]. The technology for production of keratinase PWD1 is licensed
to BRI and is being developed under the trade name Versazyme.
2.4.3.10 In fertilizer
The protein-rich concentrate feather meal generated for poultry feed can also be applied for
organic farming as a semislow- release nitrogen fertilizer [356]. Organic farming relies on the
use of nitrogen-rich organic amendments that serve the dual purpose of improving plant growth
and intensifying microbial activity in soil. Traditionally, guano has been widely used as a
fertilizer for organic farming. However, owing to high expenses, there is a need to search for
more suitable alternatives. Feather meal being nitrogen rich (15% N), inexpensive and readily
available source serves as a potential substitute to guano. It not only supplies nitrogen to plants
and promotes microbial activity, but also structures the soil and increases water retention
capacity. The microbially hydrolyzed feather meal can further edge over the steamed meal as
fertilizer due to its high nutritive value, easy production and economic feasibility.
2.4.3.11 Degradation of prion proteins
Prions are proteinaceous particles responsible for fatal neurodegenerative diseases called
transmissible spongiform encephalopathies (TSE) that include the dreaded mad cow disease,
scrapie, kuru and Creutzfeld–Jakob disease. Infectivity by prions is accompanied by the
conversion of harmless PrPc to infectious PrPsc, facilitated by PrPsc itself. These β-keratin-rich
PrPsc forms wad together into dementia- causing clumps. Shih and coworkers at BRI have
reported that the broadspectrum keratinase PWD1 (Versazyme) is capable of completely
81
degrading prions from brain tissue of bovine spongiform encephalopathy (BSE)- and scrapie-
infected animals in the presence of detergents and heat treatment. The enzymatic breakdown of
prions would most importantly help revive the use of animal meal as feed, which faced much
criticism by the European Union despite its high nutritive value due to risk of TSE. It would also
prove useful for decontaminating medical instruments, lab equipment and interchangeable items
like contact lenses and dentistry tools.
2.4.3.12 Miscellaneous
Besides their industrial and medicinal applications, proteolytic enzymes play an important role in
basic research. Proteases are used to clarify the structure function relationship, in selective
cleavage of proteins for sequence determination, or peptide mapping and synthesis. Protein
engineering and directed evolution strategies are exploited to create and screen for novel
protease variants that can be used in protein hydrolysis and synthesis applications
Other potential applications of keratinases include the anaerobic digestion of poultry waste to
generate natural gas for fuel, modification of fibers such as silk and wool, in medicine and
pharmaceuticals for elimination of acne or psoriasis, elimination of human callus for preparation
of vaccines for dermatophytosis and additives in skin-lightening agents as they stimulate keratin
degradation.
2.4.4 New Technology
A number of reports on the homology of proteases are available in literature. Studies of DNA
and protein sequence homology are important for a variety of purposes and have therefore
become routine in computational molecular biology. They serve as a prelude to phylogenetic
analysis of proteins and assist in predicting the secondary structure of DNA and proteins. The
nucleotide and amino acid sequences of a number of proteases have been determined, and their
comparison is useful for elucidating the structure-function relationship [105]. The homology
of proteases with respect to the nature of the catalytic site has been studied [114]. Accordingly,
the enzymes have been allocated to evolutionary families and clans. It has been suggested that
there may be as many as 60 evolutionary lines of peptidases with separate origins. Some of
these contain members with quite diverse peptidase activities, and yet there are some striking
82
examples of convergence [97].
Liggieri et al., [92] purified Asclepain c I from the latex of Asclepias curassavica which is 87%
homologous with Funastrain c II [89], 86% with Asclepain f [76] and 75% with cysteine
proteinase I purified from Carica candamarcensis [373]. Papain likes endopeptidases Morrenain
b I was purified from the latex of Morrenia brachystephana [85]. It shows 73% sequence
homology with plant protease araujiain h II [374], 64% with cathepsin K purified from Mus
musculus [375] and 60% with cathepsin L-like protease isolated from Leishmania major [361].
Endopeptidase named asclepain f, purified from the latex of Asclepias fruticosa [76] has 81%
sequence homology with plant protease of Morrenia odorata and 76.2 % with asclepain B isolated
from Asclepias syriaca [376].
Takagi et al, [302] found that the thermostable proteases of Bacillus stearothermophilus
and Bacillus thermoproteolyticus are 85% homologous and the thermo labile proteases of
Bacillus subtilis and Bacillus amyloliquefaciens are 82% homologous, whereas the
thermostable protease of Bacillus stearothermophilus is only 30% homologous to the
thermo labile protease of Bacillus subtilis. Koide et al., 1986 compared the amino acid
sequences of intracellular serine proteases from Bacillus subtilis with those of subtilisin
Carlsberg and subtilisin BPN’ and showed that they were 45% homologous [94].
The amino acid sequence of an extracellular alkaline protease, subtilisin J, is highly
homologous to that of subtilisin E and shows 69% identity to that of subtilisin Carlsberg,
89% identity to that of subtilisin BPN’, and 70% identity to that of subtilisin DY. The amino
acid sequence of subtilisin J is completely identical to that of the protease from Bacillus
amylosacchariticus except for two amino acid substitutions, Thr130 to Ser130 and Thr162 to
Ser162, in addition to one amino acid substitution in the signal peptide and two in the propeptide
region. The probable active-site residues of subtilisin J, i.e., Asp32, His64, and Ser221, are
identical to those of other subtilisin from Bacillus. Therefore, it was concluded
that the alkaline protease from Bacillus stearothermophilus is a subtilisin. Similarly, the various
Bacillus serine alkaline proteases, such as bacillopeptidase F, subtilisin, Epr, and ISP-1, show
considerable homology and conserved amino acids around the active site residues, i.e., Ser, Asp,
and His [197]. Alkaline proteases from various species of Aspergillus also show a high degree of
homology [241]. Alp from Aspergillus oryzae shows considerable homology (29 to 44%) to
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the members of the subtilisin family with conserved active-site residues. However, Alp
exhibits little homology to mammalian serine proteases such as trypsin and chymotrypsin
[311]. Studies of the homology of proteases have shown that the residues involved in the
substrate and metal ion binding, catalysis, disulfide bond formation and active-site formation are
conserved. Analysis of sequence homology is used in deciphering the structure-function
relationship of proteases.
2.5 Media Optimization When developing an industrial fermentation, designing fermentation medium is of critical
importance because medium composition can significantly affect product concentration, yield
and volumetric productivity. For commodity products, medium cost can substantially affect
overall process economics. Medium composition can also affect the ease and cost of downstream
product separation, for example in the separation of protein products from medium containing
protein.
There are many challenges associated with medium design. Designing the medium is a laborious,
expensive, open-ended, often time-consuming process involving many experiments. In industry,
it often needs to be conducted frequently because new mutants and strains are continuously being
introduced. Many constraints operate during the design process, and industrial scale must be kept
mind when designing the medium.
A medium design campaign can involve testing hundreds of different media. One of the more
difficult aspects of the medium design process is dealing with this flow data. In reality, often the
information generated from design experiments is difficult to assess because of its sheer volume.
Beyond about 20 experiments with five variables it very difficult for a researcher to maintain
medium component trends mentally, especially when more than one variable changes at a time.
Data capture and data mining techniques are crucial in this situation.
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Table 2.7: Summary of medium design strategies [349]
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2.5.1 Artificial neural network
An artificial neural network (ANN), usually called "neural network" (NN), is a mathematical
model or computational model that tries to simulate the structure and/or functional aspects of
biological neural networks. It consists of an interconnected group of artificial neurons and
processes information using a connectionist approach to computation. In most cases an ANN is
an adaptive system that changes its structure based on external or internal information that flows
through the network during the learning phase. Modern neural networks are non-linear statistical
data modeling tools. They are usually used to model complex relationships between inputs and
outputs or to find patterns in data.
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These networks are also similar to the biological neural networks in the sense that functions are
performed collectively and in parallel by the units, the term Artificial Neural Network (ANN)
tends to refer mostly to neural network models employed in statistics, cognitive psychology and
artificial intelligence. Neural network models designed with emulation of the central nervous
system (CNS) in mind are a subject of theoretical neuroscience (computational neuroscience).
2.5.2.1 Tools Used
The project requires the knowledge of the following tool which was used to carry out the
analysis.
2.5.2.1.1 MATLAB 7.0
MATLAB 7.0 is a numerical computing environment and fourth generation programming
language. Developed by The Math Works, MATLAB allows matrix manipulation, plotting of
functions and data, implementation of algorithms, creation of user interfaces, and interfacing
with programs in other languages. Although it is numeric only, an optional toolbox uses the
MuPAD symbolic engine, allowing access to computer algebra capabilities. An additional
package, Simulink, adds graphical multidomain simulation and Model-Based Design for
dynamic and embedded systems.
In this project we are using the neural network tool box of the software to model our networks
for the validation of literature work.
2.5.1.1 Back propagation Neural Network
2.5.1.2.1 Introduction
Backpropagation was created by generalizing the Widrow-Hoff learning rule to multiple-layer
networks and nonlinear differentiable transfer functions. Input vectors and the corresponding
target vectors are used to train a network until it can approximate a function, associate input
vectors with specific output vectors, or classify input vectors in an appropriate way as defined by
us.
Standard backpropagation is a gradient descent algorithm, as is the Widrow-Hoff learning rule,
in which the network weights are moved along the negative of the gradient of the performance
function. The term backpropagation refers to the manner in which the gradient is computed for
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nonlinear multilayer networks. Properly trained backpropagation networks tend to give
reasonable answers when presented with inputs that they have never seen. Typically, a new input
leads to an output similar to the correct output for input vectors used in training that are similar
to the new input being presented. This generalization property makes it possible to train a
network on a representative set of input/target pairs and get good results without training the
network on all possible input/output pairs.
Figure 2.24: working of BPNN
2.5.2.2.2 Algorithm
• The network learns a predefined set of input output example pairs by using a two phase
propagate adapt cycle.
• The networks begin with learning a predefined set of input-output example pairs by using
a two phase propagate-adapt cycle.
• After an input pattern has been applied as a stimulus to the first layer of networks units, it
is propagated through each upper layer until an output is generated.
• The output pattern is then compared to the desired output, and an error signal is computed
for each output unit.
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• A part of error signal was transmitted from output layer to each node in intermediate
layer that contributes directly to the output.
• The number of passes decides how many times our data is processed through the hidden
unit before giving the final predicted value and error from the desired output.
2.5.2.2.3 Procedure
1. The input and output feature was defined and the other parameters were set
2. The network are then initiliazed by the inputs,their maximum values, minimum values,
the wieghts were set.
3. The network was then simulated by providing the inputs and output layer.
4. The results of simulation are stored in simulation results network1_outputs and
network1_errors
5. The others were parameters were set. The epoch value was our variable which we have
recorded from 10 to 300.rest parameters remained same.
6. The network outputs obtained were then exported to ms.excel to calculate the mean
square error and correlation coefficient of a particular epoch value
7. The mean of RMSE and corr coeff were taken corresponding to epoch values 10 to 300.
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