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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
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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.
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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
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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:-
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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.
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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
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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.
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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.
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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
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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
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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.
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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.
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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
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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
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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
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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
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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).
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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,
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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).
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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
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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.
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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)
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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
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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
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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
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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)
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Fig 1.2: Therapeutic enzymes used in Biotechnology
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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).