16

PROTEASES
Among the large number of microbial enzymes, proteases occupy a
pivotal position owing to their wide applications. The current estimated value of the
worldwide sales of microbial enzymes is$ I billion and proteases alone account for about
60% of the total sales and they were the first enzymes to be produced in bulk (Meenu et al.
2000; Neurath, 1989; Manject Kaur et al. 1998).Milk clotting enzymes have been used to
transform milk into products such as cheese since about 5000 BC. Pancreatic proteases were
used for dehairing of hides and as pre-soak detergents since about 1910.Now; pancreatic
proteases are largely replaced by microbial proteases.
Alkaline proteases are a physiologically and commercially important
group of enzymes which are primarily used as detergent additives. They play a specific
catalytic role in the hydrolysis of proteins. In 1994, the total market for industrial enzymes
account for approximately $400 million, of which enzymes worth $112 million were used for
detergent purposes (Hodgson, 1994).In Japan, 1994, alkaline proteases sales were estimated
at15,000million yen (equivalent to $116million )(Horikoshi,1996).This enzyme accounts for
40%of the total worldwide enzyme sales. It is expected that there will be an upward trend in
the use of alkaline proteases in the future.
Proteases are broadly classified into two groups peptidases and
proteinases. Peptidases hydrolyze peptide bonds from either N or C terminal end of the
protein chain, or in other words, hydrolyze bonds of amino acids, which are outside.
Peptidases were formerly called as exopeptidases. The proteinases hydrolyze peptide bonds
within the protein chain or in other chain or in other words, hydrolyze amino acids in the
middle of the chain. They were formerly referred to as endopeptidases.


17
A more rational system of proteases classification is based on a
comparison of active sites, mechanism of action and 3-D structure (Rawlings and Barret,
1993).
Proteases can also be classified on the basis of
a) pH
b) Substrate specificity
c) Similarity in action to well characterized enzymes like trypsin, chymotrypsin and
elastase
d) Active site amino acid residue and catalytic mechanism.
More conventionally, proteases are classified into 4 important groups
like serine, cysteine, aspartic and metallo proteases.
Serine proteases
Serine proteases are the most widely distributed group of proteolytic
enzymes of both microbial and animal origin (salvesen and Nagase, 1983). The enzymes
have a reactive serine residue in the active site and are generally inhibited by diisopropyl
fluorophosphate (DFP) and phenyl methyl sulphonyl fluoride (PMSF). Most of the proteases
are also inhibited by some thiol reagents, such as P- chloromercuric benzoate(pCMB).These
are generally active at neutral and alkaline pH, with an optimum pH, between 7-11. They
have broad substrate specificities, including considerable esterolytic activity towards many
ester substrates, and are generally of low molecular weight (18.5-35 kDa).




18
Cysteine proteases
Cysteine proteases are sensitive to sulphydryl reagents, such as pCMB,
Tosyllysine Chloromethyl Ketone (TLCK), iodoacetic acid, iodoacetamide, heavy metals,
and are activated by reducing agents such as potassium cyanide or cysteine,dithiotheitol, and
ethylene diamine traacetic acid (EDTA). The occurrence of cysteine proteases has been
reported in only a few fungi (Kalisz, 1988).Intracellular enzymes with properties similar to
cysteine proteinase have been reported in Trichosporn species, Oidiodendron kalrai and
Nannizzia fulva. Extracellular cysteine proteases have been observed in Microsporium
species, Aspergillus oryzazae, and Sporotrichum pulverulentum .
Aspartic proteases
Aspartic proteases are characterized by maximum activity at low pH
(3-4) and insensitivity to inhibitors of the other three groups of enzymes.They are widely
distributed in fungi, but are rarely found in bacteria or protozoa. Most aspartic proteases
have molecular weights in the range 30-45 kDa and their isoelectric points are usually in the
pH range of 3.4-4.6.
Metalloproteases
All these enzymes have pH optima between pH 5-9 and are sensitive to
metal chelating reagents, such as EDTA, but are unaffected by serine protease inhibitors or
sulphydryl agents (Salvesen and Nagase, 1983). Many of the EDTA-inhibited enzymes can
be reactivated by ions such as zinc, calcium, and cobalt. These are widespread, but only a
few have been reported in fungi. Most of the bacterial and fungal metalloproteases are zinc-
containing enzymes, with one atom of zinc per molecule of enzyme. The zinc atom is
essential for enzyme activity. Calcium is required to stabilize the protein structure.


19
Based on their optimal pH proteases are also classified as:
1) Acid proteases
Acid proteases are proteases which are active in the pH ranges of 2-6 (Rao et al.
1998) and are mainly of fungal in origin (Aguilar et al. 2008). Common examples in
this subclass include aspartic proteases of the pepsin family. Some of the
metalloprotease and cystein proteases are also categorized in as acidic proteases.
2) Neutral proteases
Neutral proteases are proteases which are active at neutral, weakly alkaline or weakly
acidic pH .Majority of the cystein proteases, metalloproteases, and some of the serine
proteases are classified under neutral proteases. They are mainly of plant in origin,
except few fungal and bacterial neutral proteases (Aguilar et al. 2008).
3) Alkaline proteases
Alkaline proteases are optimally active in the alkaline range (pH 8-13), though they
maintain some activity in the neutral pH range as well (Horikoshi, 1996). They are
obtained mainly from neutralophilic and alkaliphilic microorganisms such as Bacillus
and Streptomyces species. In most cases the active site consists of a serine residue,
though some alkaline proteases may have other amino acid residue in their active site
(Rao et al. 1998).






20
ALKALOPHILIC MICROORGANISMS
All microorganisms follow a normal distribution pattern based on the
pH dependence for their maximum growth, and the majority of these microorganisms are
known to proliferate well at near neutral pH values. As the pH moves away from this neutral
range the number of microorganisms decreases. However, some neutrophilic organisms are
capable of growth even at extreme pH conditions. This is primarily due to the special
physiological and metabolic systems, enabling their survival and multiplication under such
adverse conditions (Krulwich et al. 1990; Krulwith and Guffanti, 1989). Such
microorganisms may also be referred to as pH dependent extremophiles.
Alkalophilic microorganisms constitute a diverse of group that thrives
in highly alkaline environments. They have been further categorized into two broad groups,
namely alkalophiles and alkalotolerants. The term alkalophiles is used for those organisms
that were capable of growth above pH 10, with an optimal growth around pH 9, and are
unable to grow at pH 7 or less (Krulwich, 1986). On the other hand, alkalotolerant organisms
are capable of growing at pH values 10, but have an optimal growth rate nearer to neutrality
(Hodgson, 1994). The extreme alkalophiles have been further subdivided into two groups,
namely facultative and obligate alklophiles. Facultative alkalophiles have optimal growth at
pH 10 or above but can grow well at neutrality, while obligate alkalophiles fail to grow at
neutrality .
Isolation and Screening
Vonder (1993) has reported the isolation of obligate alkalophilic
organisms from human and animal feces in 1993. He briefly described these organisms and


21
proposed the name Bacillus alcalophilus for his strains and also stated that he had been able
to prove that life exists that not only tolerates, but also depends on, a highly alkaline pH.
Today, many of these alkalophilic Bacillus strains and other alkalophiles are of considerable
industrial importance, particularly for use of their proteases in laundry detergents (Aunstrup
et al. 1972). Normal garden soil was reported to be a preferred source for isolation,
presumably because of the various biological activities that generate transient alkaline
conditions in such environment (Grant et al. 1990). These organisms were also isolated from
nonalkaline habitats, such as neutral and acidic soils, and thus appear to be fairly widespread.
One of the most important and noteworthy features of many
alkalophiles is their ability to modulate their environment. They can convert neutral medium
or high alkaline medium to optimize external pH for growth (Krulwich and Guffanti, 1983).
In natural environments, sodium carbonate is generally the major
source of alkalinity. Its addition to the isolation media enhances the growth of alkalophilic
microorganisms (Grant et al. 1979). The addition of sodium carbonate to the medium for the
isolation of alkalophilic. Actinomycetes results in brown color and cracking of the medium
(Kitada et al.1987). At temperatures >70 C, agar based media usually lose their gel strength,
making them useless for isolation of thermophiles . As a result, the need for gelling agents
with good thermal stability led to the discovery of agents, such as Gelrite TM (Deming and
Baross,1986) and an optimized concentration (3 &w/v) of bacteriological grade agar.





22
Isolation media
The primary stage in the development of an industrial fermentation
process is to isolate strain (s) capable of producing the target product in commercial yields.
This approach results in intensive screening programs to test a large number of strains to
identify high producers having novel properties. In the course of designing a medium for
screening proteases, it is essential that the medium should contain likely inducers of the
product and be devoid of constituents that may repress enzyme syntheses. Normally,
alkalophilic organisms are isolated by surface plating on a highly alkaline medium and
subsequent screening for the desired characteristics. The organisms are further grown on
specific media for estimating proteolytic activities using appropriate substrates such as
skimmed milk or casein. The isolates, exhibiting desired level of activity are chosen and
maintained on slants for further use. The most commonly used general medium for the
isolation of alkalophiles has been described by Horikoshi (1971). Several types of defined
media have also been used for their isolation, which include nutrient agar (Jashi and Ball,
1993),glucose-yeast extract-asparagine agar (GYA) (Sen and Satyanarayana, 1993),MYGP
agar (Srinivasa et al. 1983),peptone- yeast extract-glucose (PPYG)media (Gee et al. 1980),
wheat meal agar (Fujiwara and Yamamoto, 1987).the medium composition was varied by
several workers to isolate microorganisms of choice, such as those with high proteolytic
activity or those that were thermostable. For any type of medium, a high pH value is essential
to isolate the obligate alkalophiles (Grant and Tindall, 1980).
Extra cellular alkaline protease producing Streptomyces species is an
isolated from soil which was characterized and tentatively identified as Sterptomyces


23
aurantiogriseus EGS-5 was cultivated in production medium investigated by Rao and
Narasu (2007).
Alkalophilic microorganisms which have been screened for use in
various industrial applications, predominantly, members of the genus Bacillus and other
species were found to be prolific source of alkaline proteases. The different alkaline protease-
producing Bacillus species and strains are summarized in Table 2.1 (Steele et al. 1992).
Several fungi have also been reported to produce extracellular
alkaline proteases (Matsubara and Feder, 1971). The different alkaline proteases producing
fungal species are summarized in Table 2.2 similarly, some yeasts were also reported to
produce alkaline protease, which include Candida lipolytica (Tobe et al. 1976); Yarrowia
lipolytica (Ogrydziak, 1993), and Aureobasidium Pullulans (Donaghy and McKay, 1993).
Halophiles that were described to produce alkaline proteases included
Holobacterium sp. Alkaline proteases are also produced by some rare actinomycetes. Some
of the commercially exploited microorganisms for alkaline proteases are shown in Table 2.3.










24

Table 2.1 Some alkaline protease producing Bacillus species
Bacillus sp.and their strains References
B.firmus
B.alcalophilus
B.alcalophilus subsp.halodurans KP1239
B.amyloliquefaciens
B.licheniformis
B.proreolyticus
Bacillus alcalophilus ATCC 21522
(Bacillus sp.No. 221)
B.subtilis
B.thuringiensis
Bacillus sp.Ya-B
Bacillus sp.B21-2
Bacillus sp. Y
Bacillus sp. KSM-K16
Moon and parulekar, 1991;
Sharma et al. 1994
Takii et al. 1990
Malathi and Chakoshi,1991
Horikoshi,1987
Boyer and Byng,1996
Horiloshi,1996

Chu et al. 1992
Hotha and Banik,1997
Tsai et al. 1983
Fujiwara and Yamamoto, 1987
Shimogaki et al. 1991
Kobayashi et al. 1996














25
Table 2.2 Alkaline protease producing fungal species
Fungal species References
A.flavus
A.fumigatus
A. melleus
A.sulphureus
A.niger
A.oryzae
Cephalosporium sp. KSM 388
Chrysosporium Keratinophilum
Entomophthora coronata
Fusarium graminearum
Penicillium griseofulvum
Fusarium sp.
P.lilacinus

Chakraborty and Srinivasan, 1993
Monod et al. 1991; Larcher et al. 1996
Luisetti et al. 1991
Danno, 1970
Barthomeuf et al. 1992
Nakadai et al. 1973
Tsuchiya et al. 1987
Dozie et al. 1994
Jonsson, 1968
Phadatare et al. 1993
Dixit and Verma, 1993
Kitano et al. 1992
Den Belder et al. 1994



















26
Table 2.3 Commercial producers of alkaline proteases

Organism

Trade name

Manufacturer

Bacillius licheniformis

Protein engineered variant
Of Savinase
Protein engineered variant
Of alklophilic Bacillus sp.

Alkalophilic Bacillus sp.

Alkalophilic Bacillus sp.

Alkalophilic Bacilus sp.

Alkalophilic Bacillus sp.

Aspergillus sp.


Alcalase

Durazym

Maxapem


Savinase, Esperase

Maxacal, Maxatase

Opticlean, ptimase

Proleather

Protease P

Novo Nordisk, Denmark

Novo Nordisk, Denmark

Solvay Enzymes GmbH, Germany


Novo Nordisk,Denmark

Gist-Brocades, The Netherlands

Solvsy Enzymes GmbH,,Germany

Amano Pharmaceuticals Ltd. Japan

Amano pharmaceuticals Ltd. Japan




















27
APPLICATIONS OF ALKALINE PROTEASES

Alkaline proteases are robust enzymes with considerable industrial
potential in detergents, leather processing, silver recovery, and medical purposes, food
processing, feeds, and chemical industries, as well as waste treatment. The different areas of
applications currently using alkaline proteases are:
Detergent Industry
The detergent industry has now emerged as the single major consumer
of several hydrolytic enzymes acting in the alkaline pH range. Detergents containing
different enzymes: proteases, amylases and lipases are available in the international markets
under several brand names. The use of different enzymes as detergent additives arises from
the fact that proteases can hydrolyze proteinaceous stains and amylases are effective against
starch and other carbohydrate stains while lipases are effective against oily or fat at alkaline
pH and it should also be compatible with detergents. (Aunstrup and Andersen, 1974)
The interest in using alkaline enzymes in automatic dishwashing
detergents has also increased recently (Charyan, 1986; Glover, 1985). The enzyme detergent
preparations presently marked for cleaning of membrane systems are Alkazym (Novodan
A/S, Copenhagen, Denmark), Terg-A-Zyme (Alconox, Inc, New York, USA) and Ultrasil 53
(Hankel kGaA, Dusseldorf, Germany). In addition, contact lens cleaning solution containing
alkaline protease derived from a marine shipworm bacterium was used for the cleaning of
contact lens at low temperatures.In India, one such enzyme based optical cleaner (available
in the form of tablets containing Subtilopeptidase is presently marketed by M/S Bausch and
Lomb (India) Ltd.



28
Leather Industry
Another industrial process, which has received attention, is the
enzyme-assisted dehairing of animal hides and skin in the leather industry. Traditionally, this
process is carried out by treating animal hides with a saturated solution of lime and sodium
sulphide, besides being expensive and particularly unpleasant to carry out, a strongly
polluting effluent is produced. The alternative to this process is enzyme-assisted dehairing.
Enzyme- assisted dehairing is preferentially possible if proteolytic enzymes can be found that
are stable and active under the alkaline conditions (pH 12) of tanning.
Early attempts using a wide variety of enzyme were largely
unsuccessful, but proteases from certain bacteria which are alkalophilic in nature have been
shown to be effective in assisting the hair removal process (Taylor et al. 1987).several
alkaline proteases from alkalophilic actinomycetes have also been investigated for this
purpose. Some as hair, feather, wool, etc. at alkaline pH and may have commercial
applications (Horikoshi and Akiba, 1982).
Silver recovery
Alkaline proteases find potential application in the bioprocessing of
used X-ray films for silver in its gelatin layers. The conventional practice of silver recovery
by burning film causes a major environment pollution problem. Thus, the enzymatic
hydrolysis of the gelatin layers on the X-ray film enables not only silver, but also the
polyester film base, to be recycled.
Medicinal uses
Collagenases with alkaline protease activity are increasingly used for
therapeutic application in the preparation of slow- release dosage forms. A new semi-alkaline


29
protease with high collagenolytic activity was produced by Aspergillus niger LCF9.the
enzyme hydrolyzed various collagen types without amino acid release and liberated low
molecular weight peptides of potential therapeutic use (Barthomeuf et al. 1992).
Food Industry
Alkaline proteases can hydrolyse proteins from plants fish or animals
to produce hydrolysates of well- defined profile. The commercial alkaline protease, Alcalase
has a broad specificity with some preference for terminal hydrophobic amino acids. Using
this enzyme, a less bitter hydrolysate (Adler Nissen, 1986) and a debittered enzymatic whey
protein hydrolysate (Nakamura et al. 1993) were produced.
Very recently, another alkaline protease from B.amyloliquefaciens
resulted in the production of a methionine-rich protein hydrolysate from chickpea protein
(George et al. 1997).The protein hydrolysates commonly generated from casein, whey
protein and soya protein find major application in hypoallergenic infant food formulations
( American Academy of pediatrics Committee on Nutrition, 1989). They can also be used for
the fortification of fruit juices or soft drinks and in manufacturing of protein rich therapeutic
diets (Adamson and Reynolds, 1996; Parrado et al. 1991).
In addition, protein hydrolysates having angiotensin-1 converting
enzyme inhibitory activity were produced from sardine muscle by treatment with a
B.lichemformis alkaline protease. These protein hydrolysates could be used effectively as a
physiologically functional food that plays an important role in blood pressure regulation
(Matsui et al. 1993)
Further, proteases play a prominent role in meat tenderization;
especially of beef. An alkaline elastase (Takagi et al. 1992) and alkaline protease (Wilson et


30
al. 1992) have proved to be successful and promising meat tenderizing enzymes, as they
possess the ability to hydrolyze connective tissue proteins as well as muscle fiber proteins. A
method has been developed in which the enzyme is introduced directly in the circulatory
system of the animal, shortly before slaughter (Bernholdi, 1975) or after stunning the animal
to cause brain death (Warren, 1992).
A potential method used a specific combination of neutral and
alkaline proteases for hydrolyzing raw meat. The resulting meat hydrolysate exhibited
excellent organoleptic properties and can be used as a meat flavoured additive to soup
concentrates. Hydrolysis of over 20% did not show any bitterness when such combinations of
enzymes were used. The reason for this may be that the preferential specificity was favorable
when metalloproteinase and serine proteinase were used simultaneously (Pedersen et al.
1994).
Waste treatment
Alkaline proteases provide important application for the management
of wastes from various food processing industries and household activities. These proteases
can solubilize wastes through a multistep process to recover liquid concentrates or dry solids
of nutritional value for fish or livestock (Shoemaker, 1986;Shih and Lee,1993).
Dalev (1994) reported an enzymatic process using a B.subtilis alkaline
protease in the processing of waste feathers from poultry slaughter houses. The end product
was a heavy, grayish powder with a very high protein content, which could be used as a feed
additive.
Similarly, many such other keratinolytic alkaline proteases were used
in food technology (Dhar and Sreenivasulu,1984; Chandrasekharan and Dhar,1986; Bockle


31
and Miller,1997) for the production of amino acids or peptides(Kida et al.1995),for
degrading waste keratinous material in household refuse (Mukhopadhay and Chandra,1992),
and as a depilatory agent to remove hair in bath tub drains, which caused bad odors in houses
and in public places(Takami et al.1992).
Chemical Industry
It is now firmly established that enzymes in organic solvents can
expand the application of biocatalysts in synthetic chemistry. However, a major drawback of
this approach is the strongly reduced activity of enzymes under anhydrous conditions. Thus,
it is of practical importance to discover ways to activate enzymes in organic solvents. Some
studies have demonstrated the possibility of using alkaline proteases to catalyze peptide
synthesis in organic solvents (Chen et al. 1991; Nagashima et al.1992; Gololobov et al.
1994). In addition, many efforts to synthesize peptides enzymatically have employed
proteases immobilized on insoluble supports (Wilson et al. 1992).
A sucrose-polyester synthesis was done in anhydrous pyridine using
Proleather, a commercial alkaline protease preparation from Bacillus sp. (Patil et al.1991).
The Proleather also catalyzes the transesterification of D-glucose with various acyl donors in
pyridine (Watanabe et al. 1995).
Further, the enzyme Alcalase acted as catalyst for resolution of N-
protected amino acid esters (Chen et al. 1991) and alkaline proteases from Conidiobolus
coronatus was found to replace subtilisin Carlsberg in resolving the racemic mixtures of DL-
phenylalanine and DL-phenylglycine (Sutar et al;1992).




32
PRODUCTION OF ALKALINE PROTEASES
In industrial strain development, strain potential is certainly the most
important factor, but not the only one to consider. The best potential of a strain is realized
only under the best regulated process regimen. In the absence of the latter, it is possible to
get the best strain, but end up with mediocre fermentation performance. Thus production of a
metabolite in excess of normal is also determined by the nutritional and environmental
conditions during the growth.
Media development
The appropriate selection of medium components based on both
aspects of regulatory effects and economy is the goal in designing the chemical composition
of the fermentation media, where the nutritional requirement for growth and production must
be met. Fast formation and high concentration of the desired product are the criteria for the
qualitative and quantitative supplement of nutrients and other ingredients.
Further a continuing study of fermentation conditions should be done
as an important part of a strain development program as new mutant strains will be obtained
that may perform better, under conditions other than those originally developed from the
parent culture. Thus in any enzyme fermentation, the principle aim would be to minimize the
cost of manufacture by optimizing both the fermentation and recovery processes using high
producer.
This it is important to recognize that the development of strain for
fermentation process requires a triangular interaction among culture improvement,
development of media and optimization of process conditions. Any improvement made in
one of these areas will suddenly lead to numerous opportunities in the other two areas .This


33
triangular interaction on is s an endless cycle. The reward of running this cycle is increased
productivities, decreased costs and a more readily available supply of health and life-saving
pharmaceuticals.
Most alkalophilic microorganisms produce alkaline proteases, though
interest is limited only to those that yield substantial amounts. It is essential that these
organisms be provided with optimal growth conditions to increase enzyme production. The
Culture conditions that promote protease production were found to be significantly different
from the culture conditions promoting cell growth (Moon and Parulekar, 1991). In the
industrial production of alkaline proteases, technical media were usually employed that
contained very high concentrations (100-150 g dry wt of complex carbohydrates, pro-
teins, and other media components). With a view to improve an economically feasible
technology, research efforts are mainly focused on: (1) Improvement in the yields of alkaline
proteases and (ii) Optimization of the fermentation medium and production conditions.
Improvement of Yield
St rai n i mprovement pl ays a pot enti al rol e i n t he
commerci al devel opment of microbial fermentation processes. As a rule the wild strains
usually produce limited quantities of the desired enzyme to be useful for commercial
application (Glazer and Nikaido, 1995). However,
in
most cases, by adopting simple selection
methods, such as spreading of the culture on specific media, it is possible to pick colonies
that show substantial increase in yield (Aunstrup, 1974). Conventional physical and chemical
mutagens are used for screening of high yielding strains (Sidney and Nathan, 1975).
Strain improvement to overproduce a given product relies heavily on
random mutagenesis and the subsequent selection of, or screening for overproducing


34
mutants. The development of mutants by actinomycetes has long been recognized. These
were considered as special type of variants. The formation of new strains through the
mutation of a culture, however, is more fundamental and hereditary. White strains were
obtained from blue-pigmented forms; strains free from aerial mycelium, from those
producing such mycelium; red strains from orange-yellow forms. These mutations were
accompanied by changes in morphological, cultural and physiological characters which
differentiated the new strains from the parent cultures. The difference thus obtained may be
so distinct as to give the new strain a characteristic of a species. In recent years, extensive
use has been made of the mutagenic effects of irradiation and of certain chemical agents.
These have found extensive application in obtaining special strains of organisms.
Jensen reported that, under the influence of ultraviolet
rays, strains of Nocardia, isolated from Australian soils, gave rise to new forms, some of
these resembled typical species of Streptomyces and others were closely related to the
mycobacteria.A strain of Streptomyces griseus kept for a long time (more than 30 years) in

the culture collection and which was inactive antibiotically was induced to form a
mutant that produced streptomycin .The exposure of spores of Streptomycin sp. to
ultraviolet and gamma rays results in hereditary changes affecting colony morphology and
pigmentation. These changes are largely associated with instabilities that result in further
variation during colony growth and spore formation, These instabilities persist indefinitely,
giving rise to new variants having their own patterns of instability .These changes differ from
gene mutations in that they can be induced with much greater frequency UV-sensitive
mutants were isolated from Streptomyces coelicolor and S. clavuligerus .Which showed a
hyper mutable phenotype.


35
Optimization Of Fermentation Medium
Nutritional and environmental conditions optimization by the classical
method of changing one independent variable (nutrient, antifoam, pH, temperature, etc.)
while fixing all the others at a certain level can be extremely time consuming and expensive
for a large number of variables. To make a full factorial search, which would examine each
possible combination of independent variable at appropriate levels, could require a large
number of experiments x
n
, where x is the number of levels and n is the number of variables.
Other alternative strategies of conventional medium optimization must, therefore, be
considered which allow more than one variable to be changed at a time. These methods have
been discussed by several investigators (Greasham and Inamine, 1986; Hicks, 1993; Bull et
al. 1990 ; Veronique et al. 1983; Nelson, 1982; Hendrix, 1980; Stowe and Mayer 1966 ).
When more than five independent variables are to be investigated, the
Plackett and Burman (1946) design may be used to find out the most important variables in a
system, which are then optimized in further studies. Das and Giri (1996) studied the effects
and interactions of the factors in factorial experiments using response surface design. Dunn et
al. (1994) used modeling expressed in sets of mathematical equations.
Alkaline proteases are mostly produced by submerged
fermentation. In addition, solid state fermentation processes have also been exploited to a
lesser extent for production of these enzymes (George et al.1995;Chakraborty and
Srinivasan, 1993) .Efforts have been directed mainly towards:(i)Evaluation of the effects of
various carbon and nitrogenous nutrients as cost effective substrates on the yield of enzymes,
(ii) Requirement of divalent metal ions in the fermentation medium; and (iii) Optimization of
environmental and fermentation parameters such as pH, temperature, aeration, and agitation.


36
In addition, no defined medium has been established for the best
production of alkaline proteases from different microbial sources. Each organism or strain
has its own special conditions for maximum enzyme production.
Carbon source
Studies have indicated a reduction in protease production due to catabolite
repression by glucose (Kole et al. 1988; Frankena et al. 1986; Frankena et al. 1985; Hanlon et
al. 1982). On the other hand, (Zamost et al. 1990) have correlated the low yields of protease
production with the lowering of pH brought about by the rapid growth of the organism. In
commercial practice, high carbohydrate concentrations repressed enzyme production.
Therefore, carbohydrate was added, either continuously or in small amounts through out
the fermentation to supplement the exhausted component and keep the volume minimum
and thereby reduce the power requirements (Aunstrup, 1980).
Increased yields of alkaline proteases were reported by several
workers who used different sugars such as lactose (Malachi and Chakraborty, 1991), maltose
(Tsuchiya et al. 1991), sucrose (Phadatare et al. 1993), and fructose (Seri and Satyanarayana,
1993). However, a repression in enzyme synthesis was observed with these ingredients at high
concentrations. Whey, a waste byproduct of the dairy industry containing mainly lactose and
salts, has been demonstrated as a potential substrate for alkaline protease production (Donaghy
and McKay, 1993). Various organix acids, such as acetic acid ( lKeda et al. 1974), methyl
acetate ( Kitada and Horikoshi, 1976), and citric acid or sodium citrate (Kumar et al. 1997;
Takii et al. 1990) have been demonstrated to increase the production of proteases at alkaline
pH. The use of these organic acids was interesting in view of their economy as well as their
ability to control pH variations.


37
Nitrogen source
In most microorganisms, both inorganic and organic forms of
nitrogen are metabolized to produce amino acids, proteins, and cell wall components. The
alkaline protease comprises 15.6% sources in the medium (Kole et al. 1988). Althogh
complex nitrogen sources are usually used for alkaline protease production, the requirement
for a specific nitrogen supplement differs from organism to organism. Low levels of alkaline
protease production were reported with the use of inorganic nitrogen sources in the
production medium (Sen and Satyanarayana, 1993). Enzyme synthesis was found to be
repressed by rapidly metabolizble nitrogen sources, such as amino acids or ammonium ions
in the medium (Frankena et al. 1986), indicated repression in the protease activity with the
use of ammonium salts (Nehete et al. 1986). Sinha and Satyanarayana (1991) have observed
an increase in protease production by the addition of ammonium sulphate and potassium
nitrate. Similarly, sodium nitrate (0.25%) was found to be stimulatory for alkaline protease
production (Banerjee and Bhattacharyya, 1992b). On the contrary, several reports have
demonstrated the use of organic nitrogen sources leading to higher enzyme production than
the inorganic nitrogen sources. Fujiwara and Yamamoto (1987) have recorded maximum
enzyme yields using a combination of 3 % soyabean meal and 1.5 %bonito extract. Soyabean
meal was also reported to be a suitable nitrogen source for protease production (Cheng et al.
1995; Sen and Satyanarayana, 1993; Tsai et al. 1988; Chandrasekharan and Dhar, 1983).
Corn steep liquor (CSL) was found to be a cheap and suitable source
of nitrogen by some workers (Sen and Satyanarayana, 1993; Fujiwara and Yamamoto, 1987).
Tryptone (2 %) and casein (1-2 %) also serve as excellent nitrogen sources (Phadatare et al.
1993; Ong and Gaucher, 1976). Addition of certain amino compounds was shown to be


38
effective in the production of extracellular enzymes by alkalophilic Bacillus sp. However,
glycine appeared to have inhibitory effects on both amylase and protease production.
Casamino acids were also found to inhibit protease production (Ong and Gaucher, 1976). Oil
cakes (as nitrogen source) were found to stimulate the production of enzymes. In some
studies, use of oil cakes did not favor enzyme production (Sen and Satyanarayana, 1993;
Sinha and Satyanarayana, 1991).
Metal ion requirement
Divalent metal ions, such as calcium, cobalt, copper, boron, iron,
magnesium, manganese, and molybdenum are required in the fermentation medium for
optimum production of alkaline proteases. However the requirement for specific metal ions
depends on the source of enzyme. The use of AgNO3

at a concentration of 0.05 mg/100ml or
ZnSO4 at a concentration of 0.1.25 mg/100 ml resulted in an increase in protease activity by
RhiZopus oryzae (Banerjee and Bhattacharyya, 1992b). Potassium phosphate has been used
as a source of phosphate in most studies (Mao et al. 1992; Moon and Parulekar, 1991). This
was shown to be responsible for buffering the medium. Phosphate at a concentration of 2 g/1
was found to be optimal for protease production. However, amounts in excess of this
concentration showed an inhibition in cell growth and repression in protease production
(Moon and Parulekar, 1991). When the phosphate concentration was 4 g/1, precipitation of
the medium on autoclaving was observed (Moon
-
and Parulekar, 1993). This problem,
however, could be overcome by the supplementation of the disodium salt of EDTA in the
medium (Chaloupka, 1985). In at least one case the salts did not have any effect on the
protease yields (Phadatare, 1993).



39
pH and temperature
The important characteristic of most alkalophlic microorganisms is
their strong dependence on the extracellular pH for cell growth and enzyme production. For
increased protease yields from these alkalophiles, the pH of the medium must be maintained
above 7.5 throughout the fermentation process (Aunstrup, 1980). The culture pH also
strongly affects many enzymatic processes and transport of various components across the
cell membrane (Moon and Parulekar, 1991). When ammonium ions were used the
medium turned acidic, while it turned alkaline when organic nitrogen, such as aminoacids or
peptides were consumed (Moon and Parulekar, 1993). The decline
.
in the pH may also be due
to the production of acidic products (Moon and Parulekar, 1991). In view of a close
relationship between protease synthesis and the utilization of nitrogenous compounds, pH
variations during fermentation may indicate kinetic information about the protease
production, such as the start and end of the protease production period.
Temperature is yet another critical parameter that has to be controlled
and varied from organism to organism. The mechanism of temperature control of
enzyme production is not well understood (Chaloupka, 1985). However, studies by
Frankena et al. (1986) have shown that a link existed between enzyme synthesis and
energy metabolism in Bacilli, which was controlled by temperature and oxygen uptake.
Aeration and agitation
During fermentation the aeration rate indirectly indicates the dissolved
oxygen level in the fermentation broth. Different dissolved oxygen profiles can be obtained
by: (i) Variations in the aeration rate, (ii) Variations in the agitation speed of the bioreactor;
or (iii) Use of oxygen rich or oxygen deficient gas phase (appropriate air oxygen or air-
nitrogen mixtures) as the oxygen source (Moon and Parulekar, 1991; Michalik et al.


40
1995). The variation in the agitation speed influences the extent of mixing in the shake flasks
or the bioreactor and also affects the nutrient availability.
Optimum yields of alkaline protease are produced at 200 rpm for
B. subtilis ATCC 14416 (Chu et al. 1992) and B. licheniformis (Sen and Satyanarayana,
1993). In one study, Bacillus sp.B21-2 produced increased enzyme titres when agitated at
600 rpm and aerated at 0.5 vvm (Fujiwara and Yamamoto, 1987). Similarly, Bacillus firmus
exhibited maximum enzyme yields at an aeration rate of 7.0 l/min (Mao et al. 1992) and
an agitation rate of 360 rpm. However, lowering the aeration rate to 0.1 1/min caused a
drastic reduction in the protease yields (Moon and Parulekar, 1991). This indicates that a
reduction in oxygen supply is an important limiting factor for growth as well as protease
synthesis.





















41
ISOLATION AND PURIFICATION OF ALKALINE PROTEASES
When isolating enzymes on industrial scale for commercial
purposes the prime consideration is the cost of production in relation to the value of
the end product. Crude preparations of alkaline' proteases are generally employed for
commercial use. Nevertheless the purification of alkaline proteases is important from the
perspective of developing a better understanding of the functioning of the enzyme .
Recovery
After successful fermentation, when the fermented medium leaves the
controlled environment of the fermenter, it is exposed to a drastic change in
environmental conditions. The removal of the cells, solids, and colloids from the
fermentation broth is the primary step in enzyme downstream processing, for which
vacuum rotary drum filters and continuous disc centrifuges are commonly used. To prevent
the losses in enzyme activity caused by imperfect clarification or to prevent the clogging of
filters, it is necessary to perform some chemical pretreatment of the fermentation broth
before commencing separation ( Mukhopadhyay et al. 1990; Aunstrup, 1980). Changes in pH
may also be suitable for better separation of solids (Tsai et al. 1983). Furthermore the
fermentation broth solids are often colloidal in nature and are difficult to remove
directly. In this case, addition of coagulating or flocculating agents becomes vital
(Boyer and Byng, 1996). Flocculating agents are generally employed to effect the
formation of larger flocs or agglomerates, which, in turn, accelerate the solid-liquid
separation. Cell flocculation can be improved by neutralization of the charges on the
microbial cell surfaces, which includes changes in pH and the addition of a range of
compounds that alter the ionic environment.


42
The flocculating agents, commonly used are inorganic salts,
mineral hydrocolloids, and organic polyelectrolytes. For example the use of a
polyelectrolyte Sedipur TF 5 proved to be an effective flocculating agent at 150 ppm and pH
7.0-9.0, and gave 74 % yield of alkaline protease activity (Sitkey et al. 1992). In some cases,
it becomes necessary to add a bioprocessing filter aid, such as diatomaceous earth, before
filtration (Boyer and Byng, 1996).
Concentration
Because the amount of enzyme present in the cell free filtrate is
usually low, the removal of water is a primary objective. Recently, membrane separation
processes have been widely used for downstream processing (Strathmann, 1990).
Ultrafiltration (UF) is one such membrane process that has been largely used for the recovery
of enzymes (Bohdziewicz, 1994; Bohdziewicz, 1996) and formed a preferred alternative
to evaporation. This pressure driven separation process is expensive, results in tittle loss
of enzyme activity, and offers purification and concentration (Sullivan et al. 1984), as well
as diafiltration, for salt removal or for changing the salt composition (Boyer and Byng,
1996). However, a disadvantage underlying this process is the fouling or membrane
clogging due to the precipitates formed by the final product. This clogging can usually be
alleviated or overcome by treatment with detergents, proteases, or acids and alkalies. Han et
al (1995) used a temperature-sensitive hydrogel ultrafiltration for concentrating an alkaline
protease. This hydrogel comprised poly (N- isopropylacrylamide), which changed its volume
reversibly by the changes in temperature. The separation efficiency of the enzyme was
dependent on the temperature and was 84 % at temperatures of 15C and 20T. However, at
temperatures above 25T, a decrease in the separation efficiency was observed.



43
Precipitation
Precipitation is the most commonly used method for the isolation and
recovery of proteins from crude biological mixtures (Bell et al. 1983). It also performs both
purification and concentration steps. It is generally affected by the addition of reagents such
as salt or an organic solvent, which lowers the solubility of the desired proteins in an aqueous
solution. Although precipitation by ammonium sulphate has been used for many years, it is
not the precipitating agent of choice for detergent enzymes. Ammonium sulphate was found
wide utility only in acidic and neutral pH values and it developed ammonia under alkaline
conditions (Aunstrup, 1980). Hence, the use of sodium sulphate or an organic solvent
was the preferred choice. Despite better precipitating qualities of sodium sulphate over
ammonium sulphate, the poor solubility of the salt at low temperatures restricted its use for
this purpose (Shih et al. 1992).
Many reports revealed the use of acetone at different volume
concentrations: 5 volumes (Horikoshi, 1971), 3 volumes (Kim et al. 1996; Tsujibo et al.
1990), and 2.5 volumes (Kumar et al. 1997), as a primary precipitation agent for the recovery
of alkaline proteases. Precipitation was also reported by various workers with acetone
at different concentrations: 80 % (v/v) (Kwon et al. 1994), 66 % (v/v) (Yamagata et al. 1995)
or 44, 66, and 83 % (v/v) (El-Shanshoury et al. 1995), followed by centrifugation and/or
drying. Precipitation of enzymes can also be achieved by the use of water soluble, neutral
polymers such as polyethylene glycol (Larcher et al. 1996).
Ion-exchange chromatography (IEC)
Alkaline proteases are generally positively charged and are not bound
to anion exchangers (Tsai et al. 1983; Kumar et al. 1997; Fujiwara et al. 1993).


44
However, cation exchangers can be a rational choice and the bound molecules are eluted
from the column
,
by an increasing salt or pH gradient.
Affinity chromatography
Reports on the purification of alkaline proteases by different
affinity chromatographic methods showed that an affinity adsorbent hydroxyapatite was used
to separate the neutral protease (Keay and Wildi, 1970) as well as to purify the alkaline
protease from a Bacillus sp. (Kobayashi et al. 1996). Other affinity matrices used were
Sephadex-4-phenylbutylamine (Ong and Gaucher, 1976), casein agarose (Bockle et al. 1995;
Manachini et al. 1998), or N-benzoyloxycarbonyl phenylalanine immobilized on agarose
adsorbents (Larcher et al. 1996). However, the major limitations of affinity chromatography
are the high cost of enzyme supports and the labile nature of some affinity ligands, which
make them unrecommendable for use as a process scale.
Aqueous two-phase systems
This technique has been applied for purification of alkaline proteases
using mixtures of polyethylene glycol (PEG) and dextran or PEG and salts such as H3PO4,
MgSO4 (Lee and Chang, 1990; Sharma et al. 1994; Sinha et al. 1996, Hotha and Banik,
1997). In addition, other methods, such as the use of reversed micelles for liquid-liquid
extraction (Rahman et al. 1988), affinity precip
i
tat
i
on (Pecs et al. 1991), and foam
fractionation (Banerjee et al. 1993) have also been employed for the recovery of alkaline
proteases.
Stabilization
The enzyme preparations used commercially are impure and are
standardized to specified levels of activity by the addition of diluents and carriers. Further,
the conditions for maximum stability of crude preparations may be quite different than for


45
purified enzymes. Because loss of activity is encountered during storage m the factory,
shipment to chent(s) and /or storage in client's facilities, storage stability is of prime concern
to enzyme manufacturers. Protease solutions are subjected to proteolytic and autolytic
degradation that results in rapid inactivation of enzymatic activity. To maintain the enzyme
activity and provide stability, addition of stabilizers like calcium salts, sodium formate,
borate, propylene glycol, glycerine or betaine polyhydric alcohols, protein
preparations, and related compounds has proved successful;; (Weijers and Van't, 1992;
Eilertson et al. 1985; Schmid, 1979). Also, to prevent contamination of the final
commercial crude preparation during storage, addition of sodium chloride at 18-20 %
concentration has been advised (Shetty et al.1993; Aunstrup, 1980). The handling of dry
enzymes possesses potential health hazards and therefore, it is customary to maintain the
enzyme preparations in stabilized liquid form.
The stabilization of alkaline proteases and/or subtilisins has also been
made possible through use of protein engineering and numerous examples have been
illustrated in literature. The alkaline and thermal stabilities of subtilisin BPN9 were improved
by random mutagenesis followed by application of proper screening assays (Cunningham
and Wells, 1987; Bryan et al. 1986). Site-directed mutagenesis is often based on specific
protein design strategies, including change of electrostatic potential (Erwin et al.
1990;Pantaliano et al. 1987), introduction of disulfide brid
g
es (Mitchinson and Wells, 1989;
Takagi et al. 1990), replacement of oxidation labile residues (Estell et al.1985), modification
of side chain interactions (Braxton and Wells, 1991), improvement of internal packaging
(Imanaka et al. 1986), strengthening of metal ion binding (Pantaliano et al. 1988), reduction
in unfolding entropy (Pantaliano et al. 1989; Mattews et al. 1987), residue substitution or


46
deletion based on homology (Yonder et al. 1993 ;Takagi et al. 1992) and modification of
substrate specificity (Takagi et al. 1.997; Takagi et al. 1996).
Properties of Alkaline Proteases
The enzymatic and physicochemical properties of alkaline proteases
from several microorganisms have been studied extensively.
Optimum pH and temperature
The optimum pH range of alkaline proteases is generally between
pH 9 and 11, with a few exceptions of higher pH optima of 11.5 (Yum et al. 1994; Tobe et
al. 1975, Takami et al. 1990), pH 11-12 (Horikoshi, 1996; Kumar, 1997), and pH 12-13
(Fujiwara et al. 1993). They also have high isoelectric points and are generally stable
between pH 6 and 12. The optimum temperatures of alkaline proteases range from 50 to
70C. In addition, the enzyme from an alkalophilic Bacillus sp. B18 showed an exceptionally
high optimum temperature of 85C.
Molecular masses
The molecular masse of alkaline proteases range from 15 to 30 kDa
(Fogarty et al. 1974) with few reports of higher molecular masses of 31.6 kDa (Freeman
et al. 1993), 33 kDa (Larcher et al. 1996), 36 kDa (Tsujibo et al. 1990) and 45 kDa (Kwon et
al. 1994). However, an enzyme from Kurthia spiroforme had an extremely low
molecular weight of .8 kDa. (Steele et al. 1992). In some Bacillus sp. multiple
electrophoretic forms of alkaline proteases were observed (Kumar, 1997; Kobayashi et al.
1996; Zuidweg et al. 1972). The multiple forms of these enzymes were the result of
nonenzymatic, irreversible deamination of glutamine or asparagine residues in the protein
molecules, or of autoproteolysis (Kobayashi et al. 1996).



47
Metal ion requirement and inhibitors
Alkaline proteases require a divalent cation like Ca
+2
Mg
+2
, and Mn
+2
or a combination of these cations, for maximum activity. These cations were also found to
enhance the thermal stability of a Bacillus alkaline protease (Palowal et al. 1994). It is
believed that these cations protect the enzyme against thermal denaturation and play a vital
role in maintaining the active conformation of the enzyme at high temperatures .In
addition, specific ca 2+ binding sites that influence the protein activity and stability apart
from the catalytic site were described for protease K (Bajorath et al. 1988).
Inhibition studies give insight into the nature of the enzyme, its
cofactor requirements, and the nature of the active site (Sigma and Moser, 1975). In some of
the studies, catalytic activity was inhibited by Hg
+2
ions (Shimogaki et al. 1991). In this
regard, the poisoning of enzymes by heavy metal ions has been well documented in the
literature (Vallee and Ulmer, 1972).
Alkaline proteases are completely inhibited by phenylmethylsulfonyl
fluoride (PMSF) and diisopropyl fluorophosphate (DFP). In this regard, PMSF sulfonates the
essential serine residue in the active site and results in the complete loss of activity (Gold and
Fahmey, 1964). This inhibition profile classifies these proteases as serine hydrolases
(Morihara, 1974). In addition, some of the alkaline proteases were found to be metal ion
dependent in view of their sensitivity to metal chelating agents, such as EDTA (Steele et al.
1992; Dhandapani and Vijayaragavan, 1994; Shevchenko et al. 1995). Thiol inhibitors have
little effect on alkaline proteases of Bacillus sp. although they do affect the alkaline enzymes
produced by Streptomyces sp. (Yum et al. 1994; El-Shanshoury et al. 1995).



48
Substrate specificity
Although alkaline proteases are active against many synthetic
substrates and native proteins, reaction rates vary widely. The alkaline proteases and
subtilisins are found to be more active against casein than against haemoglobin or
bovine serum albumin. Alkaline proteases are specific against aromatic or hydrophobic
amino acid residues such as tyrosine, phenylalanine, or leucine at the carboxyl side of the
splitting point, having a specificity similar to, but less stringent than a chymotrypsin
(Morihara, 1974). With the B-chain of insulin as substrate, the bonds most frequently cleaved
by a number of alkaline proteases were Glu 4 - His 5, Ser 9 -- His 10, Leu. 15 -Tyr 16, Tyr
16 -- number of alkaline proteases were Glu 4 - His 5, Ser 9 -- His 10, Leu. 15 -Tyr 16,
Tyr 16 --Leu 17, Phe 25 -Tyr 26, Tyr 26 -Thr 27 and Lys 29 -Ala 30(Yamagata et al. 1995;
Larcher et al. 1996; Peek et al. 1992; Matsuzawa et al. 1988;Tsai et al. 1988; Tsuchiya et
al. 1993). In addition to elucidated that an alkaline elastase from Bacillus sp. Ya-B
cleaved both the oxidized insulin A- and B-chains in a block cutting manner.
Tsai et al (1984) observed that the alkaline elastase from Bacillus sp.
Ya-B also hydrolysed elastin and elastase specific substrates like succinyl-Ala3-p-
nitroanilide and succinyl-Ala-Pro- Ala-p-m
i
troariflide at a faster rate. This enzyme showed a
preference for aliphatic amino acid residues, such as alam*ne,, that are present in elastin. It is
considered that the elastolysis was initiated by the formation of an enzyme substrate complex
through electrostatic interaction between positively charged residues of the elastase and
negatively charged residues of the elastin in a pH range below 10.6 (Tsai et al. 1984). In
keratin, the disulfide bonds form an important structural feature and prevent the
proteolytic degradation of the most compact areas of the keratinous substrates. A


49
thermostable alkaline protease from an alkalophific Bacillus sp. no. AH101 exhibiting
keratinolytic activity showed degradation of human hair keratin with 1 % thioglycolic acid at
pH 12 and 70C, and the hair was solubilized within 1 hr (Takami et al. 1992). Similarly,
enhanced keratin degradation after addition of DTT has also been presented for alkaline
proteases of Streptomyces sp. (Bockle et al. 1995).














50
ACTINOMYCETES
Actinomycetes are widely distributed in nature. Soils and
composts are particularly favourable for their development, where they are found in great
abundance, both in numbers and in kinds. Globig,(1988) was among the first to draw
attention to the occurrence of actinomycetes in the soil. Beijerinck (1900)
established that actinomycetes occur in great abundance in the soil. They found that the
season of year and soil treatment had a great influence upon the numbers of these organisms.
This was followed by the works of numerous other investigators, notably that of Waksman
(1920). The role of actinomycetes in the breakdown of organic residues in the soil, methods
for determining their presence and abundance in the soil, and the recognition of the presence
of numerous types of actinomycetes received considerable attention in these works. A large
number of studies reported the abundance of Streptomyces and Micromonospora, the two
actinomycete genera
in
soil. They have proved to be prolific sources of antibiotics,
enzymes and enzyme inhibitors. They are relatively easy to isolate and can be included with
little difficulty in high throughput screening programs which evolved accordingly
(Cross, 1982).
Some general properties of actinomycetes ascribing their fungal as
well as bacterial properties were reviewed earlier (Becker et al .1965). Like fungi,
actinomycetes form hyphae with true branching. True bacteria have no vegetative thallus and
actinomycetes are morphologically similar to filamentous fungi, which have a vegetative
thallus during at least part of their life cycle. Bacterial endospores are not known to be
formed by actinomycetes but the formation of endospore-like structures may occur in
mycobacteria. The actinomycetes have many bacterial properties as well. The diameter of


51
their hyphae falls within the bacterial order of magnitude of 1um Cytological similarities
between true bacteria and actinomycetes include the types of flagella formed.
Eucaryotic organisms have flagella formed of 11 fibrils, each of which has about the
diameter of a bacterial or actinomycete flagella. Other bacterial properties shared by
actinomycetes include lack of sterols, sensitivity to antibacterial antibiotics and
phages, lysine synthesis through the diaminopimelic acid pathway and cell walls
containing mucopeptides (Becker et al .1965).
Proteolytic actinomycetes
Waksman first established that various actmomycetes, mostly
members of the genus Streptomyces possess strong, proteolytic activities. Some cultures are
able to decompose very efficiently proteins in gelatin, egg white and blood serum. This is
equally applicable for both saprophytic and pathogenic types. They vary greatly, in this
respect, both qualitatively and quantitatively, as tested by the process of gelatin liquefaction
or casein decomposition in ordinary plates. The degree and rapidity of proteolysis varied
with individual species. Stapp found that out of 477 freshly isolated cultures of
streptomycetes, only one failed to liquefy gelatin.The liquefying actions of the others were
characterized by varying degrees of rapidity. The quantitative ability to secrete
proteolytic enzymes could be measured by the degrees of gelatin of gelatin liquefaction
and of casein hydrolysis.
The proteolytic activities of the various species of actinomycetes are
so marked that waksman.A (1920) suggested the use of this

property for diagnostic purposes.
However, Lieske stated that proteolysis is not a constant property and cannot be used for
characterization of the organisms. Various forms of gelatin were used for the test. The


52
results were always identical. A strain that dissolved gelatin rapidly when first isolated
continued to do so after 1, 2, 3, 4 and 5 years of cultivation. The proteolysis enzymes of
actinomycetes are more resistant to the effect of higher temperatures than are
corresponding animal enzymes. Sterile culture filtrates of certain species of actinomycetes
were found to exert a marked effect not only upon animal proteins but also upon proteins
derived ftom, soybeans, peanut meal, and corn meal. According to Simon (1955),
Streptomyces griseus produced a protease in

a medium containing 2 percent soybean
meal. An active enzyme preparation with potency equal to that of pancreatin was obtained in
the culture. Casein, soybean, fibrin and peptone could be used as substrates for the
enzymes. The optimum pH reaction for the enzyme activity was found to be pH 8.2. An
aqueous solution of the enzyme was inactivated at 60C in 30 min.
Enzymes produced by actinomycetes seem to be very promising as
immobilized preparations for use in routine clinical diagnostic tests. Using
enzymes from actinomycetes a number of methods have been developed for rapid
enzymic determinations of clinically important compounds in biological fluids, such as the
determination of
,
the total level of cholesterol using cholesterol oxidase and cholesterol
esterase, and uric-acid and L-glutainate by application of urate oxidase and L-glutamate
respectively and phospholipids by the concerted action of phospholipase D and choline
oxidase. There are also possibilities for the use of such enzymes as L-asparginase, pronase
and urate oxidase as therapeutic agents There is an ever increasing interest in the use of
actinomycete enzymes in bio-organic chemistry. For example, synthesis of biologically
active oligopeptides have been performed by means of some proteases, while resolution
of racemic mixtures have been achieved by acylases and chiral components obtained


53
by stereospecific reduction of appropriate substrates performed by oxidoreductases.
Actmomycete enzymes with high substrate specificit
y
find application in molecular
biology for the structural anal ysis of complex gl ycopept ides, polysaccharides and
proteins.
Actinomycetes produce a large number of proteolytic enzymes.
Proteolytic enzymes are produced by different species of Streptomycetes. Among the
Streptomyces strains studied which showed varying degree of proteolytic activities were S.
griseus (5strains), S. griseoflavus (2strains), S.cellulosae (5strains), S. fulvissimus (3strains),
S.olivaceous (5strains), S.violaceous niber (3strains), S.diastatochromogenus (3strains),
S.bobiliae (2strains), S.aureus (2strains), S.pheochromogenus and S. erythrochromogenus.
Bechtereva et al (1958) studied the course of accumulation of active
proteolytic enzymes by Streptomyces violaceus and S. lavendulae. The period of
intensive accumulation of proteolyt
i
c enzymes in a simple synthetic medium and in a corn-
extract medium, was found to be related to the decomposition of the cells .
Actinomycete proteases are applied in laboratory practice in

the
structural determination of protein-composed macromolecules in removing proteinaceous
material during purification of certain biopreparations. For commercial purposes they
are routinely obtained as by products formed during biosynthesis of antibiotics mostly from
the fermentation broths of Streptomyces fradiae,Streptomyces griseus and Streptomyces
rimosus(Morihara et al, 1967) .Of all the actinomycete protease complexes available, the
enzyme pronase has gained considerable interest in

the recent past. Pronase is a mixture of
several peptidases, ten of which have been purified to homogeneity and characterized. Most
of the components of Pronase are serffie- and metalloproteases. The molecular weights of


54
pronase enzymes were estimated to vary from 15,000 to 30,000 daltons and they were found
to display proteolytic activity under alkaline conditions. It may also serve as a highly
specific reagent for the preparation of optically active amino acids . In the pharmaceutical
industry, immobilized pronase is used to remove impurities ftom, preparations of 6-amino
peniciflanoic acid . Highly purified preparations of Pronase, lipase and phospholipase
injected into the lens capsule liquefies hardened material present in age-related cataracts prior
to their surgical removal .Some mesophilic and thermophilic actinomycete proteases are
proved to be homologous with well-known microbial and mammalian endopeptidases.
These proteases classified according to their substrate specificity exhibit a collagenase-like,
elastase-like, fibrinolytic, keratiase-like, rennin-like or trypsin-like activity. A trypsinlike
activity was found not only in the enzymatic complex produced by Streptomyces erythraeus
and also by Streptomyces fradiae (Morihara, 1974). These enzymes were most active at
about pH 8 and their molecular weights were estimated to be about 20,000 daltons. Enzymes
having collagenase-like activity have been isolated from culture filtrates of pathogenic
actinomycetes such as Actinonmadura .
The production of collagenase was induced by insoluble collagen
and its macromolecular fragments, as well as by gelatin and peptone. A soil streptomycete
was reported to degrade collagen isolated from bovine achiles tendon, calf skin, human
placenta, carp swim bladder and rat-tail tendon and release appreciable quantities of
hydroxyproline (Mukhopadhyay and Chandra, 1996). A keratinase-like activity was
detected in the culture filtrates of Streptomyces fradiae. The enzyme was strongly
alkalophilic having an optimum pH of 13.0 .



55
Actinomycete proteases are similar in action to mammalian proteases
and find major application in the food industry for protein liquefation, milk clotting or as
meat tenderizers. Attempts have also been made to introduce actinomycete proteases as
fibrinolytic and thrombolytic agents in medial treatment. Since actinomycete proteases are
easy to obtain in a highly purified form, these can be used in model reaction studies and for
enzymatic synthesis of biologically active peptides. Protease isolated from Streptomyces
cellulosae) have been used to obtain biologically active peptides. Some proteases of
actinomycete origin are highly resistant to heat and denaturing agents. The high
thermostability of proteases isolated &om thermophilic actinomycetes is well known.
Protease isolated from Streptomyces rectos var. proteolyticus, Thermoactinomycesalbus,
(Mordarski et al, 1976), Thermomonospora vulgris and Thermomonospora fusca .
Several other actinomycetes are used on an industrial scale. Recently, a newly isolated
Streptomyces diastaticus strain SS I was reported to produce a dier-mostable alkaline
metalloprotease .
Proteolytic enzymes find widespread application in many industries.
A recent application of proteolytic enzymes in the leather industry is their use as dehairing or
depilation agents. Dehairing or depilation of hides and skids is an important and
unavoidable step for the manufacture of leather in the tannery. '171-te conventional chemical
method of dehairing hides and skins is the lime-sulphide process, which is environmentally
objectionable. The treatment is liable to damage the hair or wool, which is valuable
by-products of the leather In addition; dissolved sulphide and pulped hair contribute
to high C.O.D and B.O.D. of the effluent. Hence, there is a need for an alternative method
of dehairing. Enzymatic dehairing has been widely accepted as a suitable alternative.


56
Proteolytic enzymes cause depilation of skins and hides by degrading the
component globular and non-fibrous proteins of the basement membrane at the epidermal
junction.
The first successful enzymic unhairing process, teimed as Arazym
process, was achieved by Rohm in Germany . Among the mold proteases, protease from
Aspergillus flavus, A.oryzae, A.parasiticus, Afiiniigatus, A.effusus, A.ochraceous,
Awentil, Penicillium griseofulvum and rhizopus oryzae exhibited marked depilatory
activities on hides and skins.Proteolytic enzymes derived from a large number of
Bacillus species were reported to be used in dehairing and bating of hides and skins in earlier
times.
Among the actinomycetes, proteolytic enzymes from Streptomyces
sp. were reported to effectively dehair hides and skins. Recently, keratinolytic
activity of Streptomyces sp. SKI-02, was reported (Leuchtenberger et al.1983).
Enzymes from Streptomyces sp. Such as S. moderatus NRRL 3150, S.
hygroscoplcus. S. froadiae and S. griseus have potential application in dehairing of hides
and skins.








57
REFERENCES
Adamson, N. J. and E. C. Reynolds.(1996).Characterization of casein phosphopeptides
prepared using alcalase: Determination of enzyme specificity. Enzyme Microb. Tech. 19:202.
Adler-Nissen, J.(986). Enzymic hydrolysis of food proteins. Elservier Applied Science
Publishers, New York, USA.
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