PREFACE

Proteases or proteolytic enzymes are degradative enzymes which catalyze

the hydrolysis of proteins and specifically act on the internal peptide bonds of proteins and
peptides (Bayoudh et al. 2000). Proteases are of the most important industrial enzymes
accounting for nearly 60% of total worldwide sale(Ward OP et al. 1985; Kalisz HM, 1988).
Among the proteases alkaline proteases find wide industrial applications in the detergent, food,
pharmaceutical and leather industries as they have a high level of activity over a broad range of
pH. For this reason considerable attention has been paid to the isolation of alkaliphilic
microorganisms and study of their proteases.
Actinomycetes are biotechnologically important as they are prolific
producers of secondary metabolites like antibiotics. Recently they are being investigated for their
potential

to

produce

bioactive

compounds

have

anti

inflammatory,

hypertensive,

immunosuppressive and other bioactivities.

Actinomycetes are abundant in terrestrial soils and other natural habitats.
Actinomycetes producing alkaline proteases have been isolated from alkaline soils and extreme
environments such as soda lakes in Ethiopia ( Amare Gessesse et al. 2003).
In the present investigation an attempt has been made to isolate protease
producing actinomycetes from soil samples collected from milk processing units and other soils,
optimize the bioprocess variables for submerged fermentation and finally to characterize and
evaluate the potential of the isolated enzyme for industrial use.

INTRODUCTION
Proteases are hydrolytic enzymes found in every organism to undertake
important physiological functions. These include: Cell division, regulating protein turnover,
activation of zymogenic performs, blood clotting, lysis of blood clot, processing and transport of
secretory proteins across membrane, nutrition, regulation of gene expression and in pathogenic
factors. Proteases differ in their specific activity, substrate specificity, pH and temperature
optima and stability, active site and catalytic mechanisms. All these features contributed in
diversifying their classification and practical applications in industries involving protein
hydrolysis.
Proteases are classified based on chemical nature of the active site, the
reaction they catalyze, their structure and composition (Rao et al. 1998). The major classes are
again classified into subclasses based on pH, catalytic site on polypeptide, occurrence and so on.

Based on the catalytic site on the substrate, proteases are mainly classified
into endoproteases and exoproteases. Endoproteases preferably act at the inner region of the
polypeptide chain. By contrast, exoproteases preferentially act at the end of the polypeptide.
Exoproteases are further classified into aminopeptidases (those proteases which act at the free Nterminus of the polypeptide substrate) and the carboxypeptidases are those proteases which act at
the free C-terminal of the polypeptide chain.

OBJECTIVE OF THE PRESENT INVESTIGATION

To isolate Protease producing actinomycetes from different terrestrial and
water samples, strain improvement and optimization of medium constituents and process
variables for maximizing protease production.
The objectives of the present study include:
1. Isolation of actinomycetes from different terrestrial and natural substrates.
2. Primary screening of the isolates to evaluate their protease producing capability.
3. Secondary screening of the isolates showing promising activity for selection of the
production media and determining the concentration of the constituents.
(a). Taxonomical studies for determination of morphological and biochemical properties,
cell wall composition and 16S rRNA.
4. Strain improvement studies using physical and chemical mutagens and selection of the
highest protease producing mutants.
5. Utilization of statistical methods for optimization of medium constituents and process
variables in submerged fermentation.
6. Purification and Characterization of the enzyme and determination of its structure.

ENZYME TECHNOLOGY
With the development of the science of biochemistry, has come, a fuller
understanding of the wide range of enzymes present in living cells and their mode of action.
Although enzymes are formed only in living cells, many can be isolated without loss of catalytic
function in vitro. This unique ability of enzymes to perform their specific chemical
transformations in isolation has led to an ever-increasing use of enzymes in industrial processes,
collectively termed enzyme technology.
Microbial enzymes and coenzymes are widely used in several industries,
notably in detergent, food processing, brewing and pharmaceuticals. They are also used for
diagnostic, scientific and analytical purposes. Since ancient times they have been used in the
preparation of fermented foods, especially in oriental countries (Reed, 1975). At present the
economically most important enzymes are proteases, glucoamylases, glucose isomerase, and
pectinases. Over 300 tons of each enzyme is being produced annually. Some of the microbial
enzymes used industrially are shown in Table1.1 (Kumar, 1991). It may be noted that most of
these are hydrolases.
Most industrially important enzymes are extra cellular i.e secreted by the
cells into the ambient medium, from where they have to be recovered by removal and separation
from the cellular and other solid material.
Determination of enzyme activity
The enzyme activity is determined by the concentrations of enzyme, substrate,
cofactors , allosteric effectors, the concentration and type of inhibitors, ionic strength, pH, temperature
and initial reaction time etc.

Table 1.1 Microbial Sources of some representative enzymes used industrially

Enzyme

Source

Amylase

Aspergillus oryzae, B. licheniformis, B.cereus. |B. megaterium, B.polymyxa

Cellulase

Aspergilus niger, trichoderma reeesi

Dextranase

Penicillium sp., Trichoderma sp.

Glucoamylase

A.niger, Rhizopus sp.

Glucose

Bacillus coagulans, Actinoplanes sp., Arthrobacter sp, Streptomyces sp,

Isomerase

Some other Bacillus sp.

Invertase

Saccharomyces Cerecisiae

Lactase

Kluyveromyces fragilis, K.lactis, a. Niger

Lipase

Rhizopus sp, Candida lipolytica, Geotrichum candidum.

Pectinase

Aspergillus sp.

Protease

Aspergillus ap., Bacillus sp., Streptomyces griseus

Rennet

Mucor pusillus, Endothia Parasitica.

Many assay procedures for measurement of enzyme activity are

available. The rate of substrate conversion serves as a measure of the activity. The knowledge of
enzyme activity is necessary: to follow the production and isolation of enzymes, to understand
and determine the properties of commercial preparations and to ascertain the correct amount of
enzyme to be added to a particular commercial process.
The first step in deciding on a suitable assay is to choose the appropriate
substrate. Some of the substrates that have been used for the assay of hydrolases are as follows
(Collier, 1970).
Amylases and amyloglucosidases: Raw or soluble and modified starch of known dextrose
equivalent.
Cellulases: Cellulose power, cellular phosphate, filter paper and ground bran.
Pectinases: Pectic acid, pectin, pectinic acid and freeze-dried fruit purees.
Proteases: Casein, egg albumin, gelatin, hemoglobin, milk powder and raw meat.
Once the substrate is selected, the assay is carried out under
predetermined temperature, pH and incubation period. At the end of incubation period, the
reaction is readily stopped by the use of pH change or heat or by adding sufficient enzyme
inhibitor. The extent of reaction is then determined by a suitable chemical or physical method.
Chemical methods
In assay of proteases (where casein or azocasein or any other suitable
substrate is used), the addition of trichloro acetic acid (TCA) causes the precipitation of large
peptide molecules, which can be removed by filtration or centrifugation. Then the supernatant
can be read at 280 nm.

 The unit of enzyme activity is defined in different ways by different authors. For example
One unit of enzyme activity represents the amount of enzyme required to liberate one g of
tyrosine per min per ml under standard assay conditions, where enzyme activity is calculated
by measuring g of tyrosine released, by comparing with the standard (Tsuchida et al. 1986;
Nehra et al. 1998).
An increase of 0.1 absorbance unit against blank without enzyme under standard assay
conditions is considered as one unit of proteolytic activity. (Romero et al. 2001).
One unit of enzyme activity corresponds to the increase in optical density by 0.1 against a
blank, where enzyme is first treated with trichloro acetic acid and then substrate is added under
the standard assay conditions (Chrzanowdka, 1993).
Sometimes, it is desirable to prepare a control blank, where enzyme
solution is first mixed with TCA and casein is added next. This is done to correct any color
formation due to undigested proteins or incomplete termination of the reaction by TCA.
Physical Methods
Most proteases cause milk to clot, the clotting time being inversely
proportional to the enzyme activity. The substrate is usually prepared from dried milk, which is
easier to standardize than fresh milk. Enzyme is added to the solution of fat free milk, which is
already at the correct temperature, pH and calcium concentration.
The time taken to form visible curd flake is noted (It should be between
1 to 5 minutes). The initial production of these flakes is accurately seen if the reaction solution is
well mixed and if only a thin film of this continually blowing liquid is observed.

Sources of Enzymes
Enzymes can be obtained from plant, animal and microbial sources.
Plant source: -amylase, papain, bromelain, urease, ficin, polyphenol oxidase (tyrosinase),
lipoxygenase etc.
Animal Source: Pepsin, lipase, lysozyme, rennin, trypsin, phosphor-mannase, chymotrypsin etc.
Microbial Source: -amylase, pencillin acylase, protease, invertase, lactase, dextranase,
pectinase, pullulanase etc.
In general, the enzymes from plant and animals are considered to be more
important than those from microbial sources, but for both technical and economical reasons,
microbial enzymes are considered to be more important. Therefore increasing efforts are being
persuaded to produce enzymes by microbial fermentation.
Advantages of microbial enzymes
Animal sources for enzymes are very limited. Microorganisms are attractive because of their
biochemical diversity.
They have short generation time and require smaller area, 20kg of rennin is produced in 12 hrs
by B. subtilis with liter fermentor where as one calf stomach gives 10 kg after several months.
Feasibility of bulk production and ease of extraction.
Use of inexpensive media.
Ease of developing simple screening procedures.
Strain development by genetic engineering to produce abnormal amounts.
Synthesis of foreign enzymes by genetically engineered microorganisms.
Absence of seasonal variations.

Until 1985, about 2500 enzymes were known, out of which only 250
enzymes find commercial applications and another 200 were available for use in genetic
engineering. These include restriction endonucleases, ligases and editing enzymes (editases).
Only a handful enzymes have attained the status of being industrially
significant by virtue of their role in well established and well defined commercial applications
(e.g bacterial -amylase, amyloglucosidase, alkaline protease, urease, papain, pencillin acylase,
glucose isomerase etc.). While some other enzymes are awaiting the status of significant
enzymes (e.g. lipase fungal -amylase, acid protease etc.).
With the advent of biotechnological methods in the manipulation of
proteins, the classical biocatalysts, enzymes have metamorphosed into an important tool, finding
wide range of industrial applications. The advantage of adopting enzymes as industrial reagents
is because of their efficiency, precision, specificity, convenience and economics. They are
replacing chemical catalysts in many reactions where value added products are produced.
The prospects for enzymes application have improved due to developments in the
following areas:
High yields can be obtained by genetic manipulation. Hansenula polymorpha, yeast has
been genetically modified, so that 35% of its total protein consists of the enzyme alcohol
oxidase.
Optimization of fermentation conditions via induction of enzymes production, use of low
cost nutrients and introduction of fed batch fermentation.
Release of enzymes from cells by means of new cell breaking methods.
Modern purification methods such as affinity chromatography, ion-exange chromatography
and precipitation.

 Development of processes for the immobilization of enzymes and for their re-cycling. The
proportion of enzyme cost in some processes becomes only a few percent.
Continuous enzyme production in special reactors, which minimizes the cost for a new
system in continuous operations.
The following enzymes are currently produced commercially: amylase, protease, penicillin
acylase, isomerases, catalases etc.
Analytical enzymes used for analytical purpose: glucose oxidase, cholesterol oxidase etc.
Enzymes used in medicine: proteases, streptokinase, asparaginase etc.

10

Table 1.2.The current applications of enzymes and their sources.

Enzyme

Source

Region of application

Dextranase

Penicillium sp. Trichoderma sp.

Dental hygiene (cosmetic/healthcare)

Papain

Papaya Latex

Meat tenderization (food industry)

Latex of ficus

Latex of Ficus carica

Dissolves scrap film to recover the

Proteases

silver.
Trypsin

Mucolytic action, wound cleaning

Beef pancreas

(therapeutics)
Chymotrypsin

Beef pancreas

Along with trypsin treatment

Pepsin

Beef stomach

Digestive agent (therapeutics)

Renin

Beef stomach, Bacterial- B.subtilis, Curdling

of

milk

for

cheese

Fungal- A . Oryzae

manufacture (food & food processing)

Pectinases

Aspergillus niger, A. wentii

Fruit juices, (food & drink industry)

Lipases

Rhizopus sp., Candida lipolytica.

Fat synthesis (food & drink industry).

Penicillin acylase

E.coli. Penicillium sp.

In the preparation of semisynthetic

penicillins
Confectionary (food & drink industry)

Invertase

Saccharomyces cerevisiae

Cellulase

A.niger, Trichoderma reesei.

Glucose Oxidase

A.niger., P.amagasakiense

Cellulase production (food industry).

Blood

Glucose

estimation

(diagnostics), antioxidant (food &

11

drink industry)
Amylases

Maltose production, baking (food

B.amyloliquifaciens

processing).
Paper making, alcohol production
B.subtilis, B. polymyxa
(chemical industry)
Degumming of silk (textile industry)
A.oryzae

Glucose

Bacillus

sp.,

isomerase

Actinoplanes sp.,

B.

coagulans, Fructose production (food & drink

industry)

12

PRODUCTION OF ENZYMES
There are three basic techniques by which enzymes can be produced:
1. Semi-solid culture
2. Submerged culture
3. Multi- stage continuous submerged culture.
Submerged batch culture is more important of these two, since most
commercially import enzymes are growth associated .Multistage culture is only applicable to
those cases where product formation is non-growth- associated.
The following are the factors of importance in enzyme production:

Microbial strain and its metabolic behaviour.

Growth rate.

Medium components.

Culture conditions: temperature, pH aeration and addition of surfactants.

Regulatory mechanisms: Induction, feedback repression and catabolic repression.

13

REFERENCES:
Amare Gessesse, Hatti-Kaul, R., Berhanu A. Gashe and Mattiasson, B. (2003). Novel Alkaline
protease fom alkaliphilic bacteria grown on chicken feather. Enz. Microb. Technol.32:519-527.
Bayoudh, a., Gharsallah, N., Chamkha, M.Dhouib, A.; Ammar, S. and Nasri, M. (2000):
Purification and characterization of an alkaline protease from Pseudomonas aeruginosa MNI. J.
of Industrial Microbiology & Biotechnology. 24: 291-295.
Chrzanowdka, J., Kolaezkowak, M. And Polanowski, A.( 1993). Production of exocellular
proteolytic enzymes by various species of Penicillium Enzyme Microb.Technol. 15:140-143.
Collier, b.(1970), Thiol-dependent dissociation of a fraction of toxin into enzymically active and
inactive fragments.Process Biochem.5:39-40.
Kalisz, HM.(1983). Microbial Proeinasses. Adv Biochem. Eng. Biotechnol. 36:1-65.
Kumar, H.D. (1991). A text Book of Biotechnology Affilaited East-West. Press(P)Ltd, ed ,New
Delhi.
Nehra, K.S., Santhosh, D., Kamala, C. and Randhir, S.(1998). Production of alkaline protease by
Aspergillus sp. under submerged and solid substrate fermentation .Indian J. Microbio. 38:153.
Rao, M.B., Tanksale, A.M., Ghatge, M.S and Deshpande, V.V , (1998). Molecular and
Biotechnological aspects of microbial proteases. Microbiol, Mol.Bol. Rev.62:597-635.
Reed, G.(1975). Enzymes in Food Processing., (2nd edition), Academic Press, Orlando.
Romero, F.J., Garcia, L.A., Salas, J. A., Diaz, M. And Quiors, L. M.(2001). Production,
purification and partial characterization of two extracellular proteases from Serratia
marcescens grown in whey .Process Biochem.36:507.

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Tsuchida, O., Yamagota, Y., Ishizukz, J., Arai, J., Yamada, J., Takeuchi, M.and Ichishima,
E.(1986). An alkaline proteinase of an alkalophilicBacillus sp. Curr. Microbiol. 14:7.
Ward, O. P. (1985) Proteolytic Enzymes., In: Blanch HW, Drew S, Wang DI,
Comprehensive Biotechnology, Oxford, UK: Pergamon Press. 3: 789-818.

15

eds.