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Review on latest overview of proteases

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INTERNATIONAL JOURNAL OF CURRENT LIFE SCIENCS

ISSN: 2249- 1465

REVIEW ARTICLE
International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12– 18, January, 2012

REVIEW ON LATEST OVERVIEW OF PROTEASES

*Kirti Rani, Rachita Rana and Sanchi Datt
Amity Institute of Biotechnology, Amity University, Noida, 201303 (U.P.), India

ART ICLE IN FO AB STR A CT

Article History:
Received, 10th ,Novemer,2011
Received in revised form,
10th,December,2011
Accepted, 30th ,Dcember,2011
Published online, 30th ,January ,2012
Proteases are enzymes with highly specialized proteolytic functions. They
are ubiquitous in occurrence, being found in all living organisms, and are
essential for cell growth and differentiation. They not only have several
physiological functions and roles in the living beings but are also of great
importance in various industries as well thus providing a lot of economic
benefits.

Key words: The aim of this review is to study and analyze the updated information on
various biological aspects of proteases highlighting their sources, types,
Chymotrypsin, Papain, Rennet, mode of action. Details on microbial production of the enzyme as well as
Subtilisin, Serine protease industrial applications are also included.

© Copy Right, IJCLS, 2011, Bret Research Journals. All rights reserved.

INTRODUCTION add considerable interest to an already important group of

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.
Proteases form a large group of enzymes belonging to the
class of hydrolases. They are ubiquitous in nature and
perform 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.
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, and processing and transport of
secretory proteins across the membranes. These properties
enzymes [Raghunath T. et al, 2010]. 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. Furthermore, a
study of proteolytic enzymes is valued because of their use
in various forms of medical therapies and for their
importance as reagents in laboratory, clinical, and
industrial processes. Proteases represent one of the three
largest groups of industrial enzymes and account for about
60% of the total worldwide sale of enzyme [Mala B. et al,
1998].
The vast diversity of proteases, in contrast to the
specificity of their action, has attracted worldwide
attention in an attempt to exploit their physiological and
biotechnological applications.
SOURCES
Proteases are widely distributed in each part of biological
source. Being necessary for living organisms to carry out
various physiological functions, they are ubiquitous, being
found in a wide diversity of sources such as plants,
animals and microorganisms.

*Corresponding author: +9-9990329492

Email: kirtisharma2k@rediffmail.com, krsharma@amity.edu
 International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12 – 18, January, 2012
Proteases from Plants:
The use of plants as a source of proteases is governed by
several factors such as the availability of land for
cultivation and the suitability of climatic conditions for
growth. Moreover, production of proteases from plants is a
time-consuming process. Papain, bromelain, keratinases,
and ficin represent some of the well-known proteases of
plant origin [Mala B. et al, 1998].
Papain is a traditional plant protease and has a long history
of use. It is extracted from the latex of Carica papaya
fruits, which are grown in subtropical areas of west and
central Africa and India. The crude preparation of the
enzyme has a broader specificity due to the presence of
several proteinase and peptidase isozymes. The enzyme is
active between pH 5 and 9 and is stable up to 80 or 90°C
in the presence of substrates. Bromelain is prepared from
the stem and juice of pineapples. The enzyme is
characterized as a cysteine protease and is active from pH
5 to 9. Its inactivation temperature is 70°C, which is lower
than that of papain. Some of the botanical groups of plants
produce proteases like keratinases which degrade hair.
A new thermostable serine proteases named ‘wrightin’
from the latex of the plant Wrightia tinctoria, ‘Carnein’
from the latex of the weed Ipomoeacarnea spp. fistulosa
(Morning glory) and ‘Milin’ from the latex of Euphorbia
milii, was purified and have potential applications in food
and other biotechnology industries. A neutral protease,
from Raphanus sativus leaves has also been purified
[Sanna T. et al, 2001]. An aspartic protease from potato
leaves was purified has different physiological roles
[Guevara M. G. et al, 2001] a Thiol Protease was purified
from Pineapple Crown Leaf [Singh L. et al, 2004]. A 70-
kDa serine protease was identified from artificially
senescing parsley leaves, this protease activity is low in
young leaves, was found to increase considerably in
parallel to the advance of senescence and the reduction in
the protein content of the leaves . Endoproteases were also
isolated from alfalfa, oat and barley senesced leaves which
are involved in the process of protein degradation during
foliar senescence [Nieri B. et al, 1998], [Miller B.L. et al,
1981], [Drivdahl R.H. et al, 1977].
Proteases from Animals
The most familiar proteases of animal origin are pancreatic
trypsin, chymotrypsin, pepsin, and rennins. These are
prepared in pure form in bulk quantities. However, their
production depends on the availability of livestock for
slaughter, which in turn is governed by political and
agricultural policies [Mala B. et al, 1998]. Trypsin (Mr
23,300) is the main intestinal digestive enzyme responsible
for the hydrolysis of food proteins. It is a serine protease
and hydrolyzes peptide bonds in which the carboxyl
groups are contributed by the lysine and arginine residues.
Based on the ability of protease inhibitors to inhibit the
enzyme from the insect gut, this enzyme has received
attention as a target for bio - control of insect pests.
Chymotrypsin (Mr 23,800) is found in animal pancreatic
extract. Pure chymotrypsin is an expensive enzyme and is
used only for diagnostic and analytical applications. It is
specific for 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. It
is used extensively in the de-allergenizing of milk protein
hydrolysates. It is stored in the pancreas in the form of a
precursor, chymotrypsinogen, and is activated by trypsin
in a multistep process.
Pepsin (Mr 34,500) is an acidic protease that is found in
the stomachs of almost all vertebrates. The active enzyme
is released from its zymogen, i.e., pepsinogen, by
autocatalysis in presence of hydrochloric acid. Pepsin is an
aspartyl protease and resembles human immunodeficiency
virus type 1 (HIV-1) protease, responsible for the
maturation of HIV-1. It exhibits optimal activity between
pH 1 and 2, while the optimal pH of the stomach is 2 to 4.
Pepsin is inactivated above pH 6.0. The enzyme catalyzes
the hydrolysis of peptide bonds between two hydrophobic
amino acids.
Rennet is a pepsin-like protease (rennin, chymosin) that is
produced as an inactive precursor, pro-rennin, in the
stomachs of all nursing mammals. It is converted to active
rennin (Mr 30,700) by the action of pepsin or by its
autocatalysis. It is used extensively in the dairy industry to
produce a stable curd with good flavour.
Proteases from Microbes
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.
Bacteria - Most commercial proteases, mainly neutral and
alkaline, are produced by organisms belonging to the
genus Bacillus. Bacterial neutral proteases are active in a
narrow pH range (pH 5 to 8) and have relatively low
thermo - tolerance. Due to their intermediate rate of
reaction, neutral proteases generate less bitterness in
hydrolyzed food proteins than do the animal proteinases
and hence are valuable for use in the food industry.
Neutrase, a neutral protease, is insensitive to the natural
plant proteinase inhibitors and is therefore useful in the
brewing industry. The bacterial neutral proteases are
characterized by their high affinity for hydrophobic amino
acid pairs. Their low thermotolerance is advantageous for
controlling their reactivity during the production of food
hydrolysates with a low degree of hydrolysis. Some of the
neutral proteases belong to the metalloprotease type and
require divalent metal ions for their activity, while others
are serine proteinases, which are not affected by chelating
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 International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12 – 18, January, 2012
agents. Bacterial alkaline proteases are characterized by
their high activity at alkaline pH, e.g., pH 10, and their
broad substrate specificity. Their optimal temperature is
around 60°C.
Pseudomonas is a gram-negative bacterium that
predominantly produces several proteolytic enzymes. The
predominant proteases secreted by this bacterium are
alkaline proteases. Pseudomonas aeruginosa has a number
of diverse proteases that have been purified and
characterized from multiple strains.
Fungi - Fungi elaborate a wider variety of enzymes than
do bacteria. For example, Aspergillus oryzae produces
acid, neutral, and alkaline proteases. The fungal proteases
are active over a wide pH range (pH 4 to 11) and exhibit
broad substrate specificity. However, they have a lower
reaction rate and worse heat tolerance than do the bacterial
enzymes. Fungal enzymes can be conveniently produced
in a solid-state fermentation process. Fungal acid proteases
have an optimal pH between 4 and 4.5 and are stable
between pH 2.5 and 6.0. They are particularly useful in the
cheese - making industry due to their narrow pH and
temperature specificities. Fungal neutral proteases are
metalloproteases that are active at pH 7.0 and are inhibited
by chelating agents.
Viruses - 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. Retroviral aspartyl proteases that
are required for viral assembly and replication are
homodimers and are expressed as a part of the polyprotein
precursor. The mature protease is released by autolysis of
the precursor.
TYPES
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. Based on the functional group
present at the active site, proteases are further classified
into four prominent groups, i.e., serine proteases, aspartic
proteases, cysteine proteases and metalloproteases. There
are a few miscellaneous proteases which do not precisely
fit into the standard classification, e.g., ATP-dependent
proteases which require ATP for activity [Rhagunath T. et
al, 2010].
Exopeptidases
The exopeptidases act only near the ends of polypeptide
chains. Based on their site of action at the N or C terminus,
they are classified as Aminopeptidase and
Carboxypeptidase, respectively.
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).
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. In general,
aminopeptidases are intracellular enzymes, but there has
been a single report on an extracellular aminopeptidase
produced by Aspergillus oryzae. Aminopeptidase I from
Escherichia coli is a large protease (400 kDa). It has a
broad pH optimum of 7.5 to 10.5 and requiresMg+2 or
Mn+2 for optimal activity. A leucine aminopeptidase was
purified about 670 fold from germinated grains of barley
(Hordeum vulgare). 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 and is activated by Zn+2, Mn+2, or Co+2 ions.
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. and Pseudomonas sp. 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.
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 [Mala B. et al,
1998]. The endopeptidases are divided into four subgroups
based on their catalytic mechanism as follows.
Serine Proteases - Serine proteases are characterized by
the presence of a serine group in their 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. They are numerous and
widespread among viruses, bacteria, and eukaryotes,
suggesting that they are vital to the organisms. Serine
proteases are found in the exopeptidase, endopeptidase,
oligopeptidase, and omega peptidase groups. 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.
Serine alkaline proteases are produced by several bacteria
(like Arthrobacter, Streptomyces, Flavobacterium), molds,
yeasts, and fungi. They hydrolyze a peptide bond which
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 International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12 – 18, January, 2012
has tyrosine, phenylalanine, or leucine at the carboxyl side
of the splitting bond.
Subtilisins - Subtilisins of Bacillus origin represent the
second largest family of serine proteases. Two different
types of alkaline proteases, subtilisin Carlsberg (produced
by Bacillus licheniformis) and subtilisin Novo or bacterial
protease Nagase, BPN9 (produced by Bacillus
amyloliquefaciens), have been identified.
Aspartic proteases - Aspartic acid proteases, commonly
known as acidic proteases, are the endopeptidases that
depend on two highly conserved aspartic acid residues for
their catalytic activity. 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. 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 - Pepsin-like enzymes
produced by Aspergillus, Penicillium, Rhizopus, and
Neurospora and Rennin-like enzymes produced by
Endothia and Mucor spp.
Cysteine/ Thiol Proteases - 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. Cysteine proteases
have neutral pH optima, although a few of them, e.g.,
lysosomal proteases, are maximally active at acidic pH.
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. Based on their side chain
specificity, they are broadly divided into four groups -
Papain-like, Trypsin - like with preference for cleavage at
the arginine residue, Specific to glutamic acid and others.
Papain is the best-known cysteine protease.
Metalloproteases - These proteases are the most diverse
of the catalytic types of proteases. They are characterized
by the requirement for a divalent metal ion for their
activity. They include enzymes from a variety of origins
such as collagenases from higher organisms, hemorrhagic
toxins from snake venoms, and thermolysin from bacteria.
Based on the specificity of their action, metalloproteases
can be divided into four groups - neutral, alkaline,
Myxobacter I, and Myxobacter II. The neutral proteases
show specificity for hydrophobic amino acids, while the
alkaline proteases possess a very broad specificity [Mala
B. et al, 1998].
Microorganisms elaborate a large array of proteases,
which are intracellular and/or extracellular. Intracellular
proteases are important for various cellular and metabolic
processes, such as sporulation and differentiation, protein
turnover, maturation of enzymes and hormones and
maintenance of the cellular protein pool. Extracellular
proteases are important for the hydrolysis of proteins in
cell-free environments and enable the cell to absorb and
utilize hydrolytic products. At the same time, these
extracellular proteases have also been commercially
exploited to assist protein degradation in various industrial
processes [Gupta R. et al, 2002].
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, tumour
growth and metastasis, activation of zymogens, release of
hormones and pharmacologically active peptides from
precursor proteins, and transport of secretory proteins
across membranes.
In general, extracellular proteases catalyze the hydrolysis
of large proteins to smaller molecules for subsequent
absorption by the cell, where as intracellular proteases play
a critical role in the regulation of metabolism. Some of the
major activities in which the proteases participate are
nutrition, germination, regulation of gene expression,
sporulation and conidial discharge, protein turnover.
PRODUCTION
Generally, proteases produced from microorganisms are
constitutive or partially inducible in nature and, under
most culture conditions, Bacillus species produce
extracellular proteases during post-exponential and
stationary phases. Extracellular protease production in
microorganisms is also strongly influenced by media
components, e.g. variation in C/N ratio, presence of some
easily metabolizable sugars, such as glucose, and metal
ions. Protease synthesis is also affected by rapidly
metabolizable nitrogen sources, such as amino acids in the
medium. Besides these, several other physical factors,
such as aeration, inoculum density, pH, temperature and
incubation, also affect the amount of protease produced. In
order to scale up protease production from microorganisms
at the industrial level, biochemical and process engineers
use several strategies to obtain high yields of protease in a
fermentor. Controlled batch and fed-batch fermentations
using simultaneous control of glucose, ammonium ion
concentration, oxygen tension, pH and salt availability and
chemostat cultures have been successfully used for
improving protease production for long-term incubations,
using a number of microorganisms. In a recent study by
our group, the overall alkaline protease yield from B.
Mojavensis was improved up to 4-fold under semi-batch
and fed-batch operations by separating biomass and
protease production phases, using intermittent de-
repression and induction during the growth of the
organism. In recent years, there has been a great amount of
research and development effort focusing on the use of
statistical approach methods, using different statistical
software packages during process optimization studies,
with the aim of obtaining high yields of alkaline protease
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 International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12 – 18, January, 2012
in the fermentation medium. The application of properly
designed approaches with multi-factor models allows
process and biochemical engineers to design scale-up
strategies for increasing enzyme production. Some other
methods have been used for improving protease
production from different microorganisms, such as cell
immobilization of B. firmus 44, aqueous two-phase
(ATPase) systems composed of polyethylene glycol
(PEG)-(4000, 6000, 9000) and potassium phosphate using
B. thuringiensis and the ATPase system composed of
PEG-6000 and dextran T500 using B. licheniformis solid
state fermentation methods and biphasic growth systems.
Also, it has been observed that rice bran and combinations
of rice and wheat brans can be used, for producing
proteases of Aspergillus oryzae by solid-state
fermentation.
INDUSTRIAL APPLICATIONS
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 prove to be of great importance in a wide variety
of industries. Some of their applications [Rhagunath T. et
al, 2010] are discussed in detail below.
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 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. Most of the detergent proteases
currently used in the market are serine proteases produced
by Bacillus strains. But fungal alkaline proteases are
advantageous due to the ease of downstream processing to
prepare a microbe-free enzyme. A combination of lipase,
amylase, and cellulose is expected to enhance the
performance of protease in laundry detergents.
Leather Industry
Leather processing involves several steps such as soaking,
dehairing, bating and tanning. The major building blocks
of skin and hair are proteinaceous. 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. Proteases are used for
selective hydrolysis of non-collageneous constituents of
the skin and for removal of non-fibrillar proteins such as
albumins and globulins, in the several pre-tanning
operations. The purpose of soaking is to swell the hide.
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. The use of non-ionic and, to
some extent, anionic surfactants to accelerate the process
is compatible with the use of enzymes.
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. Currently, trypsin is used in combination with
others Bacillus and Aspergillus proteases for bating. The
selection of the enzyme depends on its specificity for
matrix proteins such as elastin and keratin, and the amount
of enzyme needed depends on the type of leather (soft or
hard) to be produced. Increased usage of enzymes for
dehairing and bating not only prevents pollution problems
but is also effective in saving energy. Novo Nordisk
manufactures three different proteases, Aquaderm, NUE,
and Pyrase, for use in soaking, dehairing, and bating,
respectively.
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.
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 - animal rennets,
microbial milk coagulants, vegetable rennet and
genetically engineered chymosin. Both animal and
microbial milk coagulating proteases belong to a class of
acid aspartate proteases. The microbial enzymes exhibited
two major drawbacks - the bitterness in cheese, after
storage due to the presence of high levels of nonspecific
and heat-stable proteases and 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. In cheese making, the primary
function of proteases is to hydrolyze the specific peptide
bond (the Phe105-Met106 bond) to generate para - casein
and macropeptides. Chymosin is preferred due to its high
specificity for casein, which is responsible for its excellent
performance in cheese making.
Baking industry
Wheat flour is a major component of baking processes. It
contains an insoluble protein called gluten, which
determines the properties of the bakery dough. Endo- and
exoproteinases from Aspergillus oryzae have been used to
modify wheat gluten by limited proteolysis. Enzymatic
treatment of the dough facilitates its handling and
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 International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12 – 18, January, 2012
machining and permits the production of a wider range of
products. The addition of proteases reduces the mixing
time and results in increased loaf volumes. Bacterial
proteases are used to improve the extensibility and
strength of the dough.
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.
Brewing industry
The brewing industry is a major user of proteases. In the
production of brewing wort Bacillus 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 prevent aggregation of the insoluble
complex.
Meat tenderization:
Buffaloes are slaughtered mainly for meat. The by-
products from slaughtered animals are also of good value.
Buffalo tripe is one of the important edible offal.
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. 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. In general, uniform
penetration of tenderizer enzyme has always posed
problem during tenderization treatment.
Synthesis of aspartame
The use of aspartame as a non-calorific 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 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.
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. Curcain a plant protease
was purified from the latex of Jatropha curcus was found
to be active in wound healing agent.
Therapeutics
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.
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
contains both gelatin and silver, but the presence of protein
hinders the separation of silver. Addition of proteolytic
enzymes at a temperature of 500°C and pH 8.0 rapidly
degrades the gelatin and allows the silver particles to
separate out. The alkaline proteases of B. subtilis. 18′ and
B. coagulans PB-77 were also efficient in decomposing
the gelatinous coating on used X-ray films from which the
silver could be recovered.
Management of Industrial Waste:
Recently, the use of alkaline protease in the management
of wastes from various food processing industries opened
up a new era in the use of proteases in waste management.
Proteases solubilize proteinaceous waste and thus help
lower the biological oxygen demand of aquatic systems.
Waste feathers make up approximately 5% of the body
weight of poultry and are considered to be a high protein
source for food and feed, provided their rigid keratin
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 International Journal of Current Life Sciences - Vol.2, Issue, 1, pp. 12 – 18, January, 2012
structure is completely destroyed. The use of keratinolytic
protease for food and feed industry waste, for degrading
waste keratinous material from poultry refuse and as
depilatory agent to remove hair from the drains [Takami
H. et al, 1992] has been reported. A formulation
containing proteolytic enzymes from B. subtilis, B.
amyloliquefaciens and Streptomyces sp. and a disulfide
reducing agent (thioglycolate), that enhances hair
degradation and helps in clearing pipes clogged with hair
containing deposits, is currently available in market.
Degumming of Silk
Sericin, which is about 25% of the total weight of raw silk,
covers the periphery of the raw silk fibers, thus providing
the rough texture of the silk fibers. This sericin is
conventionally removed from the inner core of fibroin by
conducting shrink-proofing and twist-setting for the silk
yarns, using starch [Kanehisa K. et al, 2000]. One of the
least explored areas for the use of proteases is the silk
industry. The process of degumming is generally
expensive and therefore an alternative method suggested is
the use of enzyme preparations, such as protease, for
degumming the silk prior to dyeing. The silk-degumming
efficiency of an alkaline protease from Bacillus sp. RGR-
14 has been studied.
CONCLUSION
Proteases are a unique class of enzymes, since they are of
immense physiological as well as commercial importance.
They possess both degradative and synthetic properties.
Since proteases are physiologically necessary, they occur
ubiquitously in animals, plants, and microbes. However,
microbes are a goldmine of proteases and represent the
preferred source of enzymes in view of their rapid growth,
limited space required for cultivation, and ready
accessibility to genetic manipulation. Microbial proteases
have been extensively used in the food, dairy and
detergent industries since ancient times. There is a
renewed interest in proteases as targets for developing
therapeutic agents against relentlessly spreading fatal
diseases such as cancer, malaria, and AIDS. Advances in
genetic manipulation of microorganisms by cloning the
protease producing gene opens new possibilities for the
introduction of predesigned changes, resulting in the
production of tailor-made proteases with novel and
desirable properties. Attempts are also being made to
isolate proteases from extremophilic microorganisms.
*******
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Exploitation of biodiversity to provide microorganisms
that produce proteases well suited for their diverse
applications is considered to be one of the most promising
future alternatives.
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