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

Strain improvement plays a potential role in the

commercial development 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 xn, 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 15°C 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 precipitation (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 bridges (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

70°C. In addition, the enzyme from an alkalophilic Bacillus sp. B18 showed an exceptionally

high optimum temperature of 85°C.

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-mitroariflide 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 70°C, 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 60°C 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 specificity find application in molecular

biology for the structural analysis of complex glycopeptides, 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 proteolytic 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

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