16

INTRODUCTION AND REVIEW OF LITERATURE

Biotechnology is an applied science, aimed at harnessing the

natural biological capabilities of microbial, plant and animal cells for the

benefit of mankind. Biotechnology couples scientific and engineering

principles with commercial considerations to develop and improve

products and processes made from living systems.

In his book entitled „Mega trends‟ internationally known futurist

John Naisbitt observed that record history has taken industrial

civilization through a series of technology based eras from the chemical

age (plastics) to atomatic age (energy) and a microelectronics age

(computers) and now we are at the age of biotechnology.

Biotechnology is a fast growing applied science. It has been defined

by the European Federation of Biotechnology as “an important

application of knowledge and techniques and capabilities of

microorganisms, animal and plant cell cultures and offers the

possibilities of producing substances and compounds essential to life and

to the greater well being of man”.

The FDA defines biotechnology as a technique that uses living

organisms, or a part of living organism to produce or modify a product, to

improve a plant or animal or to develop a microorganism to be used for a

specific purpose.

Biotechnology is the controlled used of biological agents such as

microorganism or cellular components for beneficial use. Biotechnology is

the integrated use of knowledge of biochemistry, microbiology and

17

engineering sciences in order to achieve technological application of

capabilities of microorganisms and cultured tissue cells.

The domain of biotechnology integrates most modern and highly

specific technologies on one hand and traditional fermentation process

on the other.

Unlike a single scientific discipline, biotechnology can draw upon a

wide array of relevant fields such as microbiology, biochemistry,

molecular biology, cell biology, immunology, protein engineering,

enzymology, classified breeding techniques, and the full range of

bioprocess technologies Fig. 1.1. Biotechnology is not itself a product or

range of products, rather it should be regarded as a range of enabling

technologies involving the practical application of organisms or their

cellular components to manufacturing and service industries and

environmental management.

One major impact of the new technology has been the ability to

convert the cells into factories to synthesizing compounds that were

previously available only in limited quantities. Examples of such

compounds are peptide hormones, antiviral and antitumor proteins and

growth factors. This technology also provides new routes to the

achievement of traditional goals, for example in the production of

antigens and vaccines.

The unprecedented development and growth of biotechnology has

been an outcome of discrete milestone discoveries and events in basic

biological research.

18

The advances in the field of biotechnology have altogether

influenced many fields of applied sciences. This has led to the

introduction of many branches of biotechnology as agricultural

biotechnology, pharmaceutical biotechnology, textile biotechnology, paper

biotechnology etc.

The modern biotechnology has been fully sparkled by the advent of

recombinant DNA technology.

Pharmaceutical Biotechnology:

It is a major branch of biotechnology undergoing fast development.

The concepts based on biotechnology, in the production of therapeutic

proteins and hormones, fermentation products like the antibiotics,

specially designed vaccines or drug design using the receptor hypothesis,

gene correction, drug delivery to specific tissues (targeted delivery),

production control using the biosensors, standardization of

chemotherapeutic agents and diagnostic aids using the gene cloning

technology, recombinant DNA technology, enzyme immobilization,

monoclonal antibodies and mutagenesis have been exploited and

attempted for possible use.

Obviously, the products which occur naturally are of

microbiological or biological origin having applicable potential in

pharmaceutical industry in human therapeutics, in disease diagnosis as

well as clinical monitoring of patient may lastly be covered under the

discipline of pharmaceutical biotechnology. The major areas which could

be considered include antibiotics as microbial secondary metabolites,

monoclonal antibodies, genetic engineering and related products, enzyme

19

products, microbial steroid conversion, recombinant vaccines, single cell

protein, animal and plant cell cultures for production of pharmaceuticals,

immunomodulators, blood products, tissue banks, protein hydrolysates

and glandular products. Furthermore, other products which too belong to

biologicals may include sera, diagnostic agents, organic acids, vitamins,

nucleotides, oligonucleotides, plasma expanders, alkaloids, and other

microbiological products used in diagnostic and biological assays.

Some of the applications of biotechnology include:

Bioprocess technology: Historically, the most important areas in

biotechnology are brewing, antibiotics, mammalian cell culture, including

processing of new products like polysaccharides, medicinally important

drugs, solvents, protein-enhanced foods and designing of novel fermentor

to optimize productivity.

Enzyme technology: Used for the catalysis of extremely specific

chemical reactions; immobilization of enzymes; to create specific

molecular converters (bioreactors). Products formed include L-amino

acids, high fructose syrup, semi-synthetic penicillins, starch and

cellulose hydrolysis and their hydrolyzed products , enzyme probes for

bioassays etc.

Waste technology: Long historical importance but more emphasis now

is being made to couple these processes with the conversion and

recycling of resources, foods, fertilizers and biological fuels.

Environmental technology: Great scope exists for the application of

biotechnological concepts for solving many environmental problems:-

20

pollution control, removing toxic wastes, recovery of metals from mining

wastes and low-grade ores.

Renewable resources technology: The use of renewable energy sources,

in particular, lignocellulose to generate new sources of chemical raw

material and energy, ethanol, methane and hydrogen. Total utilization of

plant and animal material.

Plant and animal agriculture: Genetically engineered plants to

improve nutrition, disease resistance, keeping quality, improved yields

and stress tolerance will become increasingly commercially available.

Improved productivity, etc., for animal farming, improved food quality,

flavour, taste and microbial safety.

Healthcare: New drugs and better treatment for delivering medicines to

diseased parts, improved disease diagnosis and understanding of the

human genome.

Scope and importance of Biotechnology in India:

Biotechnology has its newest roots in the science of molecular

biology and microbiology. Advances in these two areas have been

exploited in a variety of way both for production of industrially important

biochemicals (including enzymes) and for basic studies in molecular

biology.

Biotechnology is the emerging era for India. By now, everybody

recognized that sustainable development is possible only through

biotechnology. The main application areas of biotechnology, particularly

in India can be classified into three categories.

21

1. Agriculture

2. Medicine

3. Industrial products

Indian biotechnology industry came a long way and holds

promising future in all these three segments. Achievement of

biotechnology of agriculture is laudable. Confederation of Indian

Industries (CII) has projected the growth of biotechnology industry to

the turn of US $ 4.5 billion by the year 2010. During the last 5 years,

genetic engineering, immunological techniques, cell culture methods

and hybridoma technology are increasingly used. Manufacture of new

products and local research in these areas has intensified. Health care

products will dominate the science and may account for about 40% of

the market by 2010.

Biotechnology in India:

In 1982, Government of India, has set upon official agency, the

National Biotechnology Board (NBTB) which functions under

Department of Science and Technology (DST). In 1986, NBTB was

replaced by a full-fledged department, the department of

biotechnology (DBT). International center for genetic engineering and

biotechnology (IGEB) has established its center in New Delhi and is

started its full-fledged activity from 1988.

In addition, the other centers for biotechnology in India are:

1) Indian Agricultural Research Institute (IARI), Delhi

2) Central Food Technology and Research Institute (CFTRI), Mysore

3) Regional Research Laboratory (RRL), Jammu

22

4) Central Drug Research Institute (CDRI), Lucknow.

5) Jawaharlal Nehru University (JNU), New Delhi

6) Anna University, Chennai

7) Central Institute of Medicinal and Aromatic Plants (CIMAP),

Lucknow

8) Andhra University, Visakhapatnam.

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 co-enzymes 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 economically the most important enzymes are

proteases, glucoamylases, glucose isomerase, and pectinases. Some of

the microbial enzymes used industrially are shown in Table 1.1 (Kumar,

1991). It may be noted that most of these are hydrolases.

23

Most industrially important enzymes are extracellular 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

materials.

Determination of enzyme activity

The enzyme activity is determined by the concentrations of enzyme,

substrates, co-factors, allosteric effectors, the concentration and type of

inhibitors, ionic strength, pH, temperature and initial reaction time etc.

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 starch and modified

starch of known dextrose equivalent.

Cellulases: Cellulose powder, cellular phosphate, filter paper and ground

bran.

Pectinases: Pectic acid, pectin, pectinic acid and freeze-dried fruit purée.

24

Proteases: Casein, egg albumin, gelatin, haemoglobin, 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.

Lowry Method

This is the most common method for protein analysis. Here, the

Biuret reaction is quickly followed by reaction with Folin & Ciocalteu‟s

phenol reagent and comparing the color obtained with the color values

derived from a standard curve of a standard protein (usually BSA). The

extinction is read at 700 nm. This sensitive method detects both peptide

bonds and aromatic amino acids.

Bradford Method

This method depends on quantitating the binding of a dye,

Coomassie blue (brilliant) to an unknown protein and comparing this

binding to that of different amounts of a standard protein, usually BSA.

Enzymes and industrial applications

Food production

Enzymes are used in wide range of applications which include

proteases in meat tenderization and hydrolysis of whey proteins,

rennet in cheese production, pectinases in wine and fruit juice

25

clarification and production and amylases in starch processing and

production of fruit juices especially in production of apple juice.

Enzymes in detergents

Enzymes such as proteases, lipases, catalases and amylases are

some of the active ingredients with great potential in washing

products or detergent preparations. Recent developments in this

sector reveal that enzymes are also used in personal care products.

Enzymes in poultry industry

The advantages for the use of enzymes in poultry industry include

lower costs of commercial enzyme preparations, improved enzymes for

animal feeds and a better understanding of the composition of the

anti-nutritive compounds.

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, phospho-

mannase, chymotrypsin etc.

Microbial source: -amylase, penicillin 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

26

technical and economical reasons, microbial enzymes are considered to

be more important. Therefore increasing efforts are being pursued 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; 20 kg of

rennin is produced in 12 hr. by B. subtilis with 100 L fermentor

whereas 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 abnormally

huge 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.

27

Only a handful of 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, penicillin 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

fermentations.

Release of enzymes from cells by means of new cell breaking methods.

28

Modern purification methods such as affinity chromatography, ion-

exchange 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:

Enzymes used in industry: Amylase, protease, penicillin acylase,

isomerases, catalases etc.

Enzymes used for analytical purpose: Glucose oxidase, cholesterol

oxidase etc.

Medicinal enzymes: Asparaginase, proteases, streptokinase,

urokinase, etc.

The current applications of some enzymes are presented in the Table 1.2.

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 three, since

most commercially important enzymes are growth associated. Multistage

29

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

catabolite repression.

INTRODUCTION TO THERAPEUTIC ENZYMES

Therapeutic enzymes have a broad variety of specific uses: as

oncolytics, as anticoagulants or thrombolytics, and as replacements for

metabolic deficiencies. Typical examples of oncolytic enzymes are L-

asparaginase, L-glutaminase, etc.

The oncolytic enzymes fall into two major classes:

1. Those that degrade small molecules for which neoplastic tissues

have a requirement

2. Those that degrade macromolecules such as membrane

polysaccharides, structural and functional proteins or nucleic

acids.

Use of enzymes as therapeutic agents entails their

administration to tumor bearing subjects along with a pro-drug

30

conjugated to a functional group that is susceptible to attack by an

enzyme. To achieve the requisite selectivity, the following two features

are of considerable importance.

1. The acidic intracellular environment of many neoplasms as

compared to normal tissues

2. An enzyme with an acidic pH-activity optimum

Therapeutic enzymes are widely distributed in plant and animal tissues

and microorganisms including bacteria, yeast and fungi. Although

microorganisms are potential sources of therapeutic enzymes, utilization

of such enzymes for therapeutic purposes is limited because of their

incompatibility with the human body, Table 1.3. But there is an

increased focus on utilization of microbial enzymes because of economic

feasibility (A Sabu, 2003).

A major potential application of therapeutic enzymes is in the

treatment of cancer. Asparaginase has proved to be promising for the

treatment of acute lymphocytic leukaemia. Its action depends upon the

fact that tumor cells are deficient in aspartate-ammonia ligase activity,

which restricts their ability to synthesize the normally non-essential

amino acid L-asparagine. Because of which they are forced to depend on

body fluids. The action of the asparaginase does not affect the

functioning of normal cells which are able to synthesize enough for their

own requirements, but reduces the free exogenous concentration and so

it induces a state of fatal starvation in the susceptible tumour cells. Since

their biological action hinges on catalysis, a property that enhances

31

potency therapeutic enzymes cover a wide range of diseases and

conditions.

Therapeutically useful enzymes are required in relatively tiny

amounts but at a very high degree of purity and specificity. Factors

which reduce the potential utility of enzymes as therapeutic agents in

disease treatment are:

1. They are too large to be distributed intracellularly within the body

cells

2. Being foreign proteins to the body, they are antigenic and elicit an

immune response which causes severe and life-threatening allergic

reactions on prolonged use

3. Their effective life time within the circulation is very limited.

Microbial therapeutic enzymes play a major role in the biochemical

investigation, diagnosis, curing and monitoring of many dreaded

diseases as describe in the Fig. 1.2:

Current Options in Biotechnology

Enzymes as drugs have two important features that distinguish them

from the other types of drugs:

1. Enzymes often bind and act on their targets with great affinity and

specificity.

2. Enzymes are catalytic and convert multiple target molecules to the

desired products.

Therapeutic proteins are divided into various categories:

1. Hormones

2. Lymphokines

32

3. Blood proteins

4. Vaccines

INTRODUCTION TO L-ASPARAGINASE

L-asparaginase (L-asparagine amido hydrolase, E.C. 3.5.1.1)

belongs to an amidase group that catalyses the conversion of L-

asparagine to L-aspartic acid and ammonium.

HOOCCHNH2CH2CONH2+H2O → HOOCCHNH2CH2COOH+NH3

Asparagine is an amino acid required by cells for the

production of protein. Asparagine is not an essential amino acid in

normal cells and they synthesize this amino acid by the catalytic activity

of asparagines synthetase from aspartic acid and glutamine.

However, neoplastic cells cannot produce L-asparagine due to

the absence of L-asparagine synthetase (Keating et al., 1993) and they

depend on cellular pools of L-asparagine for their growth. Tumor cells,

more specifically, lymphatic tumor cells require huge amounts of

asparagines for their rapid and malignant growth. L-asparaginase

exploits the unusually high requirement tumor cells have for the amino

acid asparagine.

This enzyme has been isolated, purified and experimentally

studied in detail as an antileukaemia agent in human patients (Clavell et

al., 1986; Story et al., 1993) and observed its high potential against

childhood acute lymphoblastic leukaemia during the induction of

remission or the intensification phases of treatment (Hill et al., 1967;

Oettgen et al., 1967).

33

Acute lymphoblastic leukaemia (ALL) is a malignant

transformation of a clone of cells from the bone marrow where early

lymphoid precursors proliferate and replace the normal cells of the bone

marrow. It can be distinguished from other malignancies of lymphoid

tissue by the immuno-phenotype of the cells.

The chemical name for L-asparaginase enzyme is mono

methoxy polyethylene glycol succinimidyl L-asparaginase. L-asparaginase

is modified by covalently conjugating unit of mono methoxy polyethylene

glycol (PEG), forming the active ingredient PEG-L-asparaginase (derived

from Escherichia coli). Asparaginase catalyzes the hydrolysis of

asparagine to aspartic acid and ammonia. Pegasparginase a pegylated

form of the enzyme L-asparaginase derived from E.coli is an oncolytic

agent used in combination with chemotherapy for the treatment of

patients with acute lymphoblastic leukemia who are hypersensitive to

native forms of L-asparaginase.

The importance of microorganisms as L-asparaginase sources

has been focused since the time it was first discovered from Escherichia

coli and its antineoplastic activity demonstrated in guinea pig serum

(Broome, 1961; Mashburn and Wriston 1964, Roberts et al., 1966; Boyse

et al., 1967). Since then several research groups have extensively

involved in isolation of microbial strains such as pseudomonas

fluorescens (Degroot and Lichtenstein, 1960), Serratia marcescens

(Rowley and Wriston, 1967), Escherichia coli ( Mashburn and Wriston,

1964; Kozak and Jurga, 2002), Erwinia carotovora (Wade et al., 1968),

Proteus vulgaris (Tosa et al., 1972), Saccharomyces cerevisiae,

34

Streptomyces karnatakensis, Streptomyces venezuelae and several fungal

genera like Aspergillus, Penicillium and Fusarium (Curran et al., 1985;

Gulati et al., 1997; Boss, 1997; Gallagher et al., 1999; Sarquis et al.,

2004) from various xenobiotic sources producing L-asparaginase enzyme.

Comparative evaluation of L-asparaginase for its potential

activity from different microbial sources revealed that biochemical and

therapeutic properties differ with source of strain in addition to enzyme

production properties. Erwinia asparaginase is considered to be

comparably less toxic and is frequently employed in the event of allergic

reactions to Escherichia coli asparaginase although Erwinia asparaginase

has a shorter half life than E.coli asparaginase (Konecna et al., 2004). In

addition, long term administration of enzyme protein produces the

corresponding antibody in the living bodies and the antibody causes an

anaphylactic shock or neutralization of the drug effect (Khan and Hill,

1969). Therefore, a search for new L-asparaginase immunologically

different from that existing has been greatly desired.

L-asparaginase production is highly influenced by carbon

and nitrogen sources in Staphylococci and repressed by L-asparagine and

L-aspartic acid (Mikuchi et al., 1997) while the enzyme production was

inhibited by the presence of glutamine and urea in Aspergillus tamari and

Aspergillus terreus (Sarquis et al., 2004). A typical L-asparaginase

production pattern was noticed by Escherichia coli, where conventional

aerobic environment yielded in large quantities of cells with minimum

enzyme while anaerobic fermentation reversed cell and enzyme

production yields (Boeck et al., 1970).

35

Clinical Pharmacology o f L-asparaginase

Leukemic cells are unable to synthesize asparagine due to a

lack of asparagine synthetase and are dependent on an exogenous source

of asparagine for survival. Rapid depletion of asparagine which results

from treatment with the enzyme L-asparagine, kills the leukemic cells.

Normal cells are less affected by the rapid depletion due to their ability to

synthesize asparagines.

The following reactions have been observed in patients with

acute lymphoblastic leukemia (approximately 75%):

1. Hypersensitivity reactions

2. Pancreatic function

3. Liver function

4. Hematologic

5. Metabolic

6. Neurologic

SYMPTOMS:

The most common symptoms seen with ALL reflect the

abnormal blood cell production in the bone marrow.

Excessive fatigue and weakness caused by lack of healthy red cells

Pain in bones and joints of the arms and legs caused by the bone

marrow filling with leukemia cells

Excessive bruising, bleeding and petechiae caused by lack of

platelets

Persistent infections and fever caused by lack of healthy white cells

36

SIDE EFFECTS:

Mild headache

Loss of appetite

Nausea or vomiting

Mild stomach cramps

Weight loss

TOXIC EFFECTS INCLUDE:

Cramping abdominal pain

Increase in blood sugar

Many of the side effects of asparaginase are due to the fact that is a

protein. Its most important side effect is the possible occurrence of a

severe and occasionally fatal allergic reaction.

L-asparaginase is an enzyme commercially produced by bacteria. It

is inherently a foreign protein and can produce an anaphylactic reaction.

L-asparaginase may interfere with blood clotting, may raise blood sugar

levels, may raise liver enzyme blood tests, and may cause liver disease in

some patients.

Some Asparaginase Interations With Other Drugs

Methotrexate is another common anti-tumor drug. L-

asparaginase and methotrexate work against each other & treatment is

associated with acute side effects that include unpredictable toxicities

such as allergy (20%), thromboembolic events (2 to 11%) and severe

pancreatitis (4 to 7%). The interactions of L-asparaginase with other

drugs are shown in Table1.5.

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Table 1.1: Microbial sources of some important enzymes used

industrially

Enzyme Source

Amylase Aspergillus oryzae, B. licheniformis, B. cereus, B. megaterium,

B. polymyxa.

Cellulase Aspergillus niger, Trichoderma reesei

Dextranase Penicillium sp., Trichoderma sp.

Glucoamylase A. niger, Rhizopus sp.

Glucose isomerase

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

Invertase Saccharomyces cerevisiae

Lactase Kluyveromyces fragilis, K. lactis, A. niger

Lipase Rhizopus sp., Candida lipolytica, Geotrichum candidum.

Pectinase Aspergillus sp.

Protease Aspergillus sp., Bacillus sp., Streptomyces griseus

Rennet Mucor pusillus, Endothia parasitica.

38

Table 1.2: Current applications of enzymes and their sources

Enzyme Source Region of application

Dextranase Penicillium sp., Trichoderma

sp.

Dental hygiene (cosmetic /

health care)

Proteases: -

Papain

Papaya latex

Meat tenderization (food

industry)

Latex of ficus Latex of Ficus carica Dissolves scrap film to

recover the silver.

Trypsin Beef pancreas Mucolytic action, wound

cleaning (therapeutics)

Chymotrypsin Beef pancreas Along with trypsin

treatment

Pepsin Beef stomach Digestive agent

(therapeutics)

Renin Beef stomach, Bacterial -B. subtilis,

Fungal - A. oryzae

Curdling of milk for cheese

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 semi synthetic penicillins

Invertase Saccharomyces cerevisiae Confectionary (food & drink

industry)

Cellulase A. niger, Trichoderma reesei. Cellulase production (food

industry)

Glucose Oxidase A. niger, P. amagasakiense Blood glucose estimation

(diagnostics), antioxidant

(food & drink industry)

Amylases B. amyloliquifaciens

B. subtilis, B. polymyxa

Maltose production, baking

(food processing)

Paper making, alcohol

production (chemical

industry)

A. oryzae Degumming of silk (textile

industry)

Glucose isomerase Bacillus sp., B. coagulans,

Actinoplanes sp. Fructose production (food & drink industry)

39

Table 1.3: Microbial sources of some therapeutic enzymes

Enzyme Source

L-glutaminase Beauveria bassiana, Vibrio

costicola, Zygosaccharomyces rouxii

L-asparaginase Pseudomonas acidovorans,

Acinetobacter sp.

Β-Lactamase Citrobacter freundii, Serratia

marcescens, Klebsiella pneumoniae

Serratia peptidase Serratia marcescens

Alginate lyase Pseudomonas aeruginosa

L-arabinofuranosidase Aspergillus niger

Penicillin acylase Penicillium sp.

Laccase Trametes versicolor

40

Table 1.4: Some important therapeutic enzymes and their uses

Enzyme EC

Number

Reaction Use

Asparaginase 3.5.1.1 L-asparagine+H2O→aspartate+NH3 Leukaemia

Collagenase 3.4.24.3 Collagen hydrolysis Skin ulcers

Glutaminase 3.5.1.2 L-Glutamine + H2O→L-

glutamate+NH3

Leukemia

Hyaluronidase 3.2.1.35 Hyaluronate hydrolysis Heart attack

Lysozyme 3.2.1.17 Bacterial cell wall hydrolysis Antibiotic

Rhodanase 2.8.1.1 S2O3 Cyanide

poisoning

Ribonuclease 3.1.26.4 RNA hydrolysis Antiviral

β-Lactamase 3.5.2.6 Penicillin→penicilloate Penicillin

allergy

Streptokinase 3.4.22.10 Plasminogen →plasmin Blood clots

41

Table 1.5: Interactions of l-asparaginase with other drugs

Agent Effect Mechanism

Methotrexate Decreased effect of methotrexate when

asparaginase is given immediately prior to

or with methotrexate; enhanced effect of

methotrexate when asparaginase is given

after methotrexae

Suppression of

asparagine

concentrations

Methotrexate Increased hepatotoxicity Additive

Prednisone Increased hyperglycemia Additive

Serum

thyroxine

binding

globulin

Decreased total serum thyroxine

concentration

Decreased synthesis

of thyroxine-binding

globulin in liver

Vincristine Increased vincristine neurotoxicity Unknown

42

Genetics

Microbiology

Biochemistry/

Chemistry

Electronics Food

Science

Biotechnology

Biochemical Food

technology

Engineering

Engineering

Chemical Mechanical

Engineering Engineering

Fig 1.1. The interdisciplinary nature of biotechnology (Higgings et al., 1985)

43

Fig 1.2: Therapeutic enzymes used in Biotechnology

44

REVIEW OF LITERATURE

Distribution of L-asparaginase among microorganisms

L-asparaginases are distributed throughout the animal, plant and

microbial kingdoms. Considerable research has been under taken for the

production of L-asparaginase (both extracellular and intracellular) by

variety of microorganisms. Among all these system, L-asparaginase

derived from bacterial and fungal sources have dominant application in

pharmaceutical sector (Yasser et al., 2002). Atteompts have been made to

specify the cultural conditions and selection of superior strains of the

bacteria for large scale production. The advantages of using

microorganisms for the production of L-asparaginase include:

1. Bulk production capacity

2. Economical

3. Microbes are easy to manipulate to obtain enzymes with desired

characteristics.

The major bacterial species that produce this enzyme include

Escherichia coli (Khushoo et al., 2004; Derst et al., 1994, Erwinia

cartovora (Aghaiypour et al., 2001; Borisova et al., 2003), Serratia

marcescens, Pseudomonas acidovoras, Pseudomonas aerginosa (El-

Bessoumy et al., 2003), Erwinia chrysanthemi (Kotzia and Labrou, 2007),

Enterobacter aerogenes (Mukherjee et al., 2000), Candida Utilis (Kil et al.,

1995), Thermus thermophilus (Prista and Kyridio, 2001) and

Staphylococcus aureus ( Muley et al., 1998). Certain L-asparaginase

producing fungal species also isolated and studed include Aspergillus

tamari, A. terrus, A. pencillium, Hypomyces solani, Nectria haematococca

45

(Maria Inez de Moura Sarquis et al., 2004; K. Nakahama et al., 1973),

and Fusarium species like Fusarium roseum, F. saloni. L-asparaginase

production is also reported by a yeast species Saccharomyces cerevisae

(Maria Inez de Moura Sarquis et al., 2004) and an algal species named

Chlamydomonas microalgae (John H. Paul, 1982).

Biochemical aspects of L-asparaginase

Biochemical aspects play a vital role in enzyme production studies.

Erwinia asparaginase is considered to be less toxic compared to E. coli

asparaginase and hence is employed in the events of allergic reactions

inspite of having a shorter half life than E. coli asparaginase (Koninca et

al., 2004). This enzyme was characterized by X-ray scattering (saxs)

pattern of homo tetrameric asparaginase-II from E.coli was measured in

solution in conditions resembling those in which its crystal form was

obtained and compared. The resultant crystallographic model proved that

the overall quaternary structure in crystal and in solution were similar

but homo tetramer is less compact in solution than in the crystal form

(Maciej kozak et al., 2002).

Transformation in Bacillus subtilis 168 with two differently encoded

regulated genes has proven to produce better enzyme activity (Susan et

al., 2002). L-asparaginase enzyme can be efficiently produced by E.coli

through recombinant techniques (Mukherjee, 2004; Valeria

Gabrielasavoius, 2000).

An organism identified as Enterobacter cloacae produced L-

asparaginase (intracellularly which was resistant to a temperature range

46

of 39-42ºC and a good yield was obtained utilizing L-Fructose, D-

Galactose, Saccharose or Maltose (Nawaz et al., 1998).

Importance of L-asparaginase

The important application of the L-asparaginase enzyme is in the

treatment of acute lymphoblastic leukemia (mainly in children), Hodgkin

disease, acute myelocytic leukemia, acute myelomonocytic leukemia,

chronic lymphocytic leukemia, lymphosarcoma treatment,

reticulosarbom and melanosarcoma (Stecher et al.,; Verma et al., 2007).

The role of L-asparaginase in lymphocytic leukemia cells treatment is

based on the fact that these cells are not capable of synthesis L-

asparagine and are rely on the exogenous sources to get hold of L-

asparagine (Lee et al., 1989). On the contrary, normal cells are protected

from L-asparagin starvation due to their ability to generate this essential

amino acid (Duval et al., 2002). The neoplastic activity attributed to the

depletion of L-asparagine by the action of L-asparaginase (Lee et al.,

1989). Through many species producing L-asparaginase as mentioned

above only E.coli and Erwinia cartovora asparaginases are currently in

medical use as efficient as drugs in the lymphocytic leukemia, because of

high substrate affinity (Verma et al., 2007; Schqartz et al., 1966) and

factors affecting the clearance of the enzyme from the media of the

reaction (Stecher et al., 1999; Broome, 1965).

Determination of L-asparaginase activity

Enzymatic assay of L-asparaginase is done by Nesslerization

method. The reaction is monitored by measuring the amount of ammonia

released during reaction. The ammonia released is complexed with

47

Nessler‟s reagent and the resultant reddish brown solution‟s optical

absorbance is measured at 436 nm using UV-Visible spectrophotometer

(F.S.Liu et al., 1972). Using ammonium standard curve, the enzyme

activity will be calculated.

Another method for the determination of asparaginase enzyme

activity is Indophenol method (Tetsuya Tosa et al., 1971), Boering

mannheinkit (Maria Inez de Moura Sarquis et al., 2004) and previously

(Paul & Cooksey 1979) which is done using 5Mm L-asparatic acid

(sodium salt pH 7.0) or in the presence of 1, 10, 25 mM NH4Cl and the

activity was determined using the radiochemical assay described by

(Prusine milner, 1970) Cambell et al method12.

Recently Gulati et al., 1997 developed an assay method for L-

asparaginase which is also based on the production of ammonia during

hydrolysis of L-asparagine degraded by glutamate de-hydroginase

consequently with the oxidation of β-NADH. Depletion of β-NADH is then

monitored spectrometerically at 340 nm (Victor M. Balcal et al., 2001).

Among all the methods studied, modified Nesslerization method was

chosen to be the best method for the assay of L-asparaginase activity.

48

Production of L-asparaginase

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 programme, 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.

49

Thus 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 (Holt and

Saunders, 1986). This triangular interaction is 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.

Several microbial strains were isolated and characterized for the

enzyme L-asparaginase production yields. Production levels of L-

asparaginase varied with the organism to organism. In general, the

production yields are not observed to be more than 10 IU except a few.

50

The following table depicts literature report on this enzyme production by

various microbial strains.

S.No. Microbe type Microbial strain Ref. No.

1 Bacteria Pseudomonas aeuriginosa

Yasser et al., 2002

2 Bacteria Proteus vulgaris Tetsuya Tosa et al., 1971

3 Bacteria E.coli Calina Petruta

Cornea, 2000

4 Bacteria Erwinia aroideae

5 Bacteria Serratia macerans Bernard Heinemann,1969

6 Fungi Aspergillus pencillium

Maria Inez de Moura Sarquis, 2004

7 Fungi Fusarium roseum K. Nakahama et al, 1973

8 Fungi Fusarium saloni K. Nakahama et al, 1973

9 Fungi Hypomyces solani K. Nakahama et al, 1973

10 Fungi Nectria haematococca

K. Nakahama et al, 1973

11 Yeast Saccharomyces cerevisiae

Patricia C Dunlop, 2004

Physiology of L-asparaginase production:

The production of L-asparaginase by submerged fermentation (SMF)

and solid-state fermentation (SSF) were studied. Certain physiological

factors which had an effect on the production include:

1. The composition of the growth medium

2. pH of the medium

3. Phosphate concentration

4. Inoculum age

5. Temperature

6. Aeration

51

7. Agitation

8. Carbon source

9. Nitrogen source

10. Mineral sources

Improvement of Yield

Strain improvement plays a key 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 a

substantial increase in yield (Aunstrup, 1974). Conventional physical and

chemical mutagens are used for screening of high yielding strains (Sidney

and Nathan, 1975).

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 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

52

allow more than one variable to be changed at a time. Several

investigators have discussed these methods (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.

L-asparaginase is generally produced by submerged fermentation.

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. In addition, no defined medium has been established for the

best production of L-asparaginase from different microbial sources. Each

organism or strain has its own special conditions for maximum enzyme

production.

Different methods have been adopted for the improvement of L-

asparaginase production. The statistical methods which were based on

an experimental design were applied to optimize the solid state

fermentation in Pseudomonas aeriginosa 50071 using Placket-Burman

53

factorial design followed by Box-Behnken design which was used to

improve the production of L-asparaginase enzyme (Abdel Fattah et al.,

2002).

Better mutant strain of Bacillus subtilis 168 encoded with regulated

genes improved the production level (Susan H Fisher, 2002).

Development and characterization of some L-asparaginase producting

recombinant E.coli strains increased the enzyme by 2-3 folds compared to

the parenteral strain (Valeria Gabrieal savoi et al., 2000).

Effect of carbon sources on L-asparaginase production

L-asparaginase is an inducible enzyme and is generally induced in

the presence of the glucose. 0.2% glucose is used as carbon source in

(Gulati et al 1997) which proved to be a better isolation method for the

microbal cultures. 0.1% Maltose, Saccharose, Fructose and Galactose

showed better enzyme production (Nawaz et al., 1998). 20.3% sorbital

showed 10 fold increase of enzyme activity. However, reports are also

available that supplementation of glucose resulted in depressed

production of L-asparaginase (Tetsuya tosa et al.,1971). In another

investigation, 1% lactose is observed to be enhancing the production of L-

asparaginase (Liu & Zajic., 1972). Glucose influence on enzyme

production is found to be indirect and not related with growth of the

organisms. This is evidenced based on observation that when cells were

grown in presence of glucose under aerobic environment only cell growth

without enzyme production. Further continuation of the experiment after

54

late exponential phase resulted in L-asparaginase production (L.D. Boeck

et al., 1970)

Effect of nitrogen source on L-asparaginase production

Nitrogen sources have been preferred for enhancing the production of

L-asparaginase. 2% praline showed better production 58.00 IU/lit (Maria

Inez de Moura Sarquis., 2004) when compared with different nitrogen

sources like urea, glutamine etc. combined use of 1% sodium fumerate

and 5% corn steep liquor enhanced enzyme production levels (Tetsuya

Tosa et al., 1971). Use of 0.5% tryptone, 0.5% yeast extract produced 4.0

IU/ml of the enzyme (F.S.Liu and J.E. Zajic., 1972), Tripticase soya broth

was sued for better enzyme production (L.D. Boeck, et al., 1970). 5% corn

syrup (50%DW), 0.1g (NH4)2SO4, 0.15 % glutamic acid were also used for

the production (Calina Petruta., 2000), Casein hydrolysate (3.11%) and

corn steep liquor (3.68%) showed the best activity in Pseudomonas

aeruginosa in solid state fermentation. L-asparaginase as a sole nitrogen

source in Enterobacter cloacae showed better enzyme production.

L-asparaginase production was investigated in the filamentous fungi

Aspergillus tamari and Aspergillus terreus. The fungi were cultivated in

medium containing different nitrogen sources. A. terrus showed the

highest L-asparaginase activity production level (58 U/L) when cultivated

in a 2% praline medium. Both fungi presented the lowest level of L-

asparaginase production in the presence of glutamine and urea as

nitrogen sources. These results suggest that L-asparaginase production

by of filamentous fungi is under nitrogen regulation.

55

Role of Phosphate and other ions on L-asparaginase production

The role of the phosphate in production and regulation L-asparaginase

was well documented in the literature (Gulati et al., 1997) and the reports

indicate that the phosphates are the major contents of the medium i.e.,

M9 medium and it has better buffering capacity for the production of L-

asparaginase enzyme and so phosphate is considered as one of the major

contents of the medium.

All trace elements play a vital role in the production of L-asparaginase

enzyme. Gulati et al., 1997 explained tha K+, Na+, Ca+2, Mg+2, SO4-2 and

Cl- ions have an effect on L-asparaginase production in Erwinia

carotovora and bacterial species.

Role of temperature and pH

Most of L-asparaginase production studies have been done using

different bacterial & fungal strains. In these microbial strains the

optimum asparaginase production was noticed in the temperaturerange

of 28-370C (L.D. Boeck, et al., 1970; H. Geekil S., 2004; K. Nakahama et

al., 1973). however, production of L-asparaginase at elevated

temperature is also reported in thermophilic bacteria at 42oC and other

species with better production at elevated temperatures include

Enterobacter cloacae and Fusarium species (K. Nakahama et al., 1973).

The pH of the medium plays a vital role in the production of L-

asparaginase. The best enzyme production in so far reported strains was

at neutral pH. However slight variations of this also noticed for better

enzyme production at pH 6.2 (Maria Inez de Moura Sarquis., 2004; K.

Nakahama et al., 1973). The pH range of 7.4-7.5 showed better enzyme

56

activity for the production of L-asparaginase (.F.S.Liu and J.E. Zajic.,

1972; N.K. Maladkar., 1989)

Role of aeration and agitation

Several investigations demonstrated the impact of agitation intensity

on mixing, oxygen transfer and production formation in many bacterial

fermentation studies (L.D. Boeck, et al., 1970; M.S. Nawaz et al., 1998;

H. Geekil S., 2004; M.H.Bilimoria., 1969). It has been reported that a

higher agitation speed is sometimes detrimental to bacterial growth and

this may decrease the enzyme production.

Immobilization studies of L-asparaginase

Co-immobilization of L-asparaginase on to highly activated supports

also enhanced or improved the production levels. It is worthwhile to

report that an organism indentified asEnterobacter cloacae produced L-

asparaginase (intracellularly which was resistant to a temperature range

of 39-42oC and a good yield was obtained utilizing L-fructose, D-

galactose, Saccharose or Maltose (Nawaz et al., 1998).