Indian Journal of Biotechnology

Vol 2, July 2003, pp 334-341

Sources, Properties and Applications of Microbial Therapeutic Enzymes

A Sabu*
Biotechnology Division, Regional Research Laboratory, Thiruvananthapuram 695019, India

Received 22 January 2003; accepted 20 February 2003

Enzymes or biocatalysts are produced in the human body from amino acids that the body obtains by digesting
food proteins. Enzymes accelerate and control all biochemical processes in the body and in a single second several
millions of enzyme mediated chemical reactions occur in a human body. Each enzyme is programmed to carry out
one special task. The immense number of enzymes acts as a perfectly matched orchestra to ensure that enormously
complex life mechanisms and processes occur in a right direction. Sufficient amount and optimal function of enzymes
present in the human body is essential Cor life and health. Microbial enzymes play a major role in the diagnosis,
curing, biochemical investigation and monitoring of many dreaded diseases of the century. Information on this topic
is very meagre and thus the present review is an attempt to compile information on the sources, properties and
applications of important therapeutic enzymes.
Keywords: therapeutic enzymes, glutaminase, asparaginase, enzyme therapy, tumour, biodrug

Introduction use pancreatic enzymes to treat cancer in 1902

Enzymes are proteinaceous in character. Each
enzyme is programmed to carry out one special task.
Like a key in a lock each enzyme fits together with
one specific substrate modifying it in one proper way.
The manufacture or processing of enzymes for use as
drugs is an important facet of today's pharmaceutical
industry (Cassileth, 1998). Attempts to capitalize on
the advantages of enzymes as drugs are now being
made at virtually every pharmaceutical research
centre in the world. Since the later years of the 19th
century, crude proteolytic enzymes have been used
for gastrointestinal disorders, e.g., pepsin for
dyspepsia. In fact, other than as digestion aids,
enzymes were largely ignored as drugs until a group
of researchers observed that an extra cellular secretion
of Bacillus pyocyaneus was capable of killing anthrax
bacilli, and of protecting mice from otherwise lethal
inocula of the bacterium. They deduced that the
secretion in question was a nuclease, i.e. it was acting
by enzymatically degrading nucleic acids. This
milestone study gradually opened up the way for the
use of parenteral enzymes first in the treatment of
infections, then of cancer, and finally of a diverse
spectrum of diseases. Enzyme supplements are
available in pills, capsules and powders. Supplements
often consist of combinations of several different
enzymes. John Beard, an English scientist, was first to
*Tel: 91-471-2515339; Fax: 91-471-2491712
E-mail: sabuahameed@yahoo.com
(Gonzalez & Isaacs, 1999). He proposed in 1906 that
pancreatic proteolytic enzymes, in addition to their
well- known digestive function, represent the body's
main defence against cancer. He further proposed that
pancreatic enzymes would most likely be useful as
anticancer agents. During the first two decades of this
century, a number of physicians, both in Europe and
the USA, used injectable pancreatic enzymes to treat
advanced human cancer, often with great success.
There are several studies from the 1960s showing, in
an animal model, that orally ingested pancreatic
enzymes have an anticancer effect, and might work
through immune modulation. German researchers
later used enzyme therapy to treat patients with
multiple sclerosis, cancer, and viral infections. Dr
Edward Howell introduced the term enzyme therapy
to the United States in the 1920s. He believed that by
eating raw meat, people created an enzyme surplus,
which resulted in better health and increased
resistance to diseases (Cassileth, 1998).
Therapeutic enzymes have a broad variety of
specific uses: as oncolytics, thrombolytics or
anticoagulants, and as replacements for metabolic
deficiencies. Additionally, there is a growing group of
miscellaneous enzymes of diverse function.
Proteolytic enzymes have been widely used as anti-
inflammatory agents. Reduction of inflammation and
edema is ascribed to the dissolution of soft fibrin and
to the clearance of proteinaceous debris found in
inflammatory exudates.
 SABU: MICROBIAL THERAPEUTIC ENZYMES 335

Information on the utilization of microbial enzymes Table l--Some important therapeutic enzymes and their
for therapeutic purposes is scarce and the available applications
reports are largely on some anticancer enzymes and
Enzyme Application
others, which are active against cystic fibrosis.
Development of medical applications for enzymes has L-Asparaginase Antitumour
been at least as extensive as those for industrial L-Glutarninase Antitumour
applications, reflecting the magnitude of potential Superoxide dismutase Anti-oxidant, anti-inflammatory
rewards. The variety of enzymes and their potential Serratio peptidase Anti-inflammatory
therapeutic applications are considerable. A selection Penicillin acylase Synthetic antibiotic production
of those enzymes, which have realised this potential Collagenase To treat skin ulcers
to become important therapeutic agents, is given in Lipase Digests lipids
Table 1. At present, the most successful applications Streptokinase Anticoagulant
are extra cellular, purely topical uses, the removal of Urokinase Anticoagulant
cytotoxic substances and the treatment of life- Laccase Detoxifier
threatening disorders within the blood circulation L-arginase Antitumour
(Sabu, 2003). L- Tyrosinase Antitumour
Since the enzymes are specific biological catalysts, Glucosidase Antitumour
they should make the most desirable therapeutic Galactosidase Antitumour
agents for the treatment of metabolic diseases. 13-lactamase Penicillin allergy
Unfortunately, a number of factors severely reduce Ribonuclease Antiviral
this potential utility; enzymes are too large to be
distributed simply within the body's cells. This is the in aspartate-ammonia ligase activity, which restricts
major reason why enzymes have not yet been their ability to synthesise the normally non-essential
successfully applied to the large number of human amino acid, L-asparagine. Therefore, they are forced
genetic diseases. A number of methods are being to extract it from body fluids. The action of
developed in order to overcome this by targeting asparaginase does not affect the functioning of normal
enzymes; for example, enzymes with covalently cells, which are able to synthesise enough for their
attached external ~-galactose residues are targeted at own requirements, but reduce the free exogenous
hepatocytes and enzymes covalently coupled to concentration, and so induce a state of fatal starvation
target-specific monoclonal antibodies are being used in the susceptible tumour cells. A 60% incidence of
to avoid non-specific side-reactions. complete remission has been reported in a study of
In contrast· to the industrial use of enzymes, almost 6,000 cases of acute lymphocytic leukaemia.
therapeutically useful enzymes are required with very This enzyme is administered intravenously.
high degree of purity. The favoured kinetic properties
of these enzymes are low Km and high V max in order to Microbial Therapeutic Enzymes
be maximally efficient even at very low enzyme and Microbial enzymes are preferred over plant or
substrate concentrations. Thus, the sources of such animal sources due to their economic production,
enzymes are chosen with care to avoid any possibility consistency, ease of process modification and
of unwanted contamination by incompatible material optimization. They are relatively more stable than
and to enable ready purification. Therapeutic enzyme corresponding enzymes derived from plants or
preparations are generally offered for sale as animals. Further, they provide a greater diversity of
lyophilised pure preparations with only biocompatible catalytic activities. The majority of enzymes currently
buffering salts and mannitol diluents added. The costs used in industry are of microbial origin. But once we
of such enzymes may be quite high but still enter into the therapeutic applications of microbial
comparable to those of competing therapeutic agents enzymes, a number of factors severely reduce their
or treatments. potential utility. One of the major problems is the
A major potential therapeutic application of large molecular size of biological catalysts, which
enzymes is in the treatment of cancer. Asparaginase prevents their distribution within the somatic cells.
has proved to be particularly promising for the Investigations are on to overcome these problems by
treatment of acute lymphocytic leukaemia. Its action the technique of drug targeting. Another important
depends upon the fact that tumour cells are deficient problem related to enzyme therapy is the elicitation of
336 INDIAN J BIOTECHNOL, JULY 2003

immune response in the host cells after injecting the utilization of microbial enzymes because of economic
foreign enzyme protein. Modern medical science feasibility. Microbial sources of some therapeutic
could overcome this problem also by disguising the enzymes are given in Table 2.
enzyme as an apparently non-proteinaceous molecule
by covalent modification. L-glutarninase modified by Production
covalent attachment of polyethylene glycol, has been There are different methods of fermentation by
shown to retain its antitumour effect whilst possessing which we can produce these important enzymes. On
no immunogenicity. Other methods like entrapment of commercial scale, liquid cultures in huge bioreactors
the enzyme within artificial liposomes, synthetic are preferred for the bulk production of therapeutic
micro spheres and red blood cell ghosts have also been enzymes. Other processes like solid state fermentation
found useful. These inherent problems necessitate the (SSF), immobilization and fermentation on inert solid
requirement of therapeutic enzymes with a very high supports are also widely used for the production of
degree of purity and specificity (Sabu, 2003). therapeutic enzymes. Recombinant E. coli strains with
a foreign gene are generally cultivated in liquid media
Salt Tolerance and the Role of Marine (submerged fermentation) for expressing the foreign
Microorganisms protein. Submerged fermentation (SmF) is the
Use of salt tolerant enzymes from marine bacteria cultivation of microbial cells in liquid media under
provides an interesting alternative for therapeutic controlled conditions in bioreactors for the production
purpose. The marine biosphere is one of the richest of of desirable metabolites. SmF offers advantages like
the earth's innumerable habitats, yet one of the least easy online monitoring of process parameters and
studied and characterized fauna. Currently, marine process automation. SSF is the culturing of
microorganisms are considered as untapped sources microorganisms on moist solid substrates in the
of metabolites and products, which may possess novel absence or near absence of free water. It is also
properties. Marine microorganisms have a diverse described as a fermentation process that takes place
range of enzymatic activity and are capable of on solid or semisolid substrate or that occurs on a
catalyzing various biochemical reactions with novel nutritionally inelt solid support, which provides some
enzymes. Thus, there is enormous scope for the advantages to the microorganisms with respect to
investigations exploring the probabilities of deriving access to nutrients and the product derived will be
new products of economic importance from potential with high purity. Immobilization of cells can be
marine microorganisms. Considering the fact that defined as the attachment of cells or their inclusion in
marine environment, particularly seawater, which is Table 2-Microbial sources of therapeutic enzymes
saline in nature and chemically closer to human blood
plasma, it could provide microbial products, in Enzyme Source
particular the enzymes that could be safer having no
L-glutarninase Beauveria bassiana, Vibrio costicola,
or less toxicity or side effects when used for human Zygosaccharomyces rouxii
therapeutic application. Yet another fact, which is L-asparaginase Pseudomonas acidovorans,
leading an increasing interest on exploring and Acinetobacter sp.
exploitation of marine microorganisms for industrial ~-Lactamase Citrobacter freundii, Serratia
applications, is their high levels of salt tolerance marcescens, Klebsiella pneumoniae
ability. Hence, there is an increasing interest in the Serratia peptidase Serratia marcescens
marine microorganisms for therapeutic purposes Lipase Candida lipolytica, C. rugosa,
(Sabu et al, 2000). Aspergillus oryzae
Alginate lyase Pseudomonas aeruginosa
L-arabinofuranosidase Aspergillus niger
Sources Protease Bacillus polymyxa, Beauveria
Therapeutic enzymes are widely distributed in plant bassiana
and animal tissues and microorganisms including Superoxide dismutase Mycobacterium sp, Nocadia sp.
bacteria, yeast and fungi. Although microorganisms Glucosidase Aspergillus niger
are potential sources of therapeutic enzymes, Amylase Aspergillus oryzae, Bacillus sp.
utilization of such enzymes for therapeutic purposes is Serrapeptase Serratia marcescens
limited because of their incompatibility with the Penicillin acylase Penicillium sp.
human body. But there is an increased focus on Laccase Trametes versicolor
 SABU: MICROBIAL THERAPEUTIC ENZYMES 337

a distinct solid phase that permits exchange of Table 3--Human proteins produced by rDNA technology
substrates, products, inhibitors, etc., but at the same
time separates the catalytic cell biomass from the bulk Recombinant protein Application
phase containing substrates and products.
Antitrypsin For treating Emphysema
Cell growth factors For immunological disorders
rDNA Technology for the Production of Epidermal growth factor To treat burns
Therapeutic Enzymes Erythroprotein Anemia, kidney disorders, etc.
Advent of rDNA technology allows production of Factor VIII and Factor IX Hemophilia
large quantities of pharmaceutical proteins, which Growth hormone Growth defects
were previously difficult and costly to produce. Insulin Diabetes
Protein activity is often modified by rDNA
technology and can be overcome by shuffling Table 4--Commercially available FDA-approved recombinant
functional domains and site directed mutagenesis. human proteins
This is done to modify activities, regulation and avoid
Recombinant Manufacturer Application
unwanted side effects. The principle behind rDNA protein
technology can be simply represented as:
DNase I Genentech Cystic fibrosis
Clone cDNA for protein Erythroprotei n Amgen Anaemia

t Growth hormone Genentech Growth hormone

Insert into-expression vector deficiency
Insulin Eli Lilly Diabetes
t
Transform E. coli IFN-a2a Hoffmann-La Roche Leukemia
Interleukin-2 Chiron Renal carcinoma
t
Over express
t recently been investigated as a mucolytic agent for
Purify use in patients with chronic bronchitis. The enzyme,
lysozyme hydrolyzes the chitins and mucopeptides of
So far four hundred human proteins have been bacterial cell walls. Accordingly, it has been used as
produced by rDNA technology for therapeutic use. an antibacterial agent usually in combination with
Commercial value of these therapeutic products is standard antibiotics. The proteolytic enzymes, trypsin
enormous (Tables 3 & 4). Present global market for and chymotrypsin have been successfully used in the
therapeutic recombinant proteins is around $200 treatment of post-operative hand trauma, athletic
.billion. Major market is shared by pharmaceutical injuries and sciatica. Hyaluronidase exerts action by
giants like Genentech ($250 million) for growth destroying the intracellular ground substance
hormone, Eli Lilly ($277 million) for insulin and hyaluronic acid, thus allowing diffusion of vital
Amgen ($2.15 billion) for erythroprotein. Since there molecules through this normally impermeable
is a need for large quantity of therapeutic enzymes for connective tissue barrier. In 1959, improvements of
clinical trials and for sales once approved, the gene- the electrocardiograms of patients with acute
expression process must be optimized. myocardial infarction were demonstrated following
treatment. Lysostaphin whose lytic effects on
General Applications coagulase-positive Staphylococcus aureus are
Enzymes are being used to treat many diseases like presently under considerable study. It is a protease
cancer, cardiac problems, cystic fibrosis, dermal that lyses susceptible cells in a highly efficient
ulcers, inflammation, digestive disorders, etc. manner probably by peptidase-like cleavage of the
Collagenase, an enzyme unique that hydrolyses native glycoprotein of the bacterial wall. At present,
collagen and spares hydrolysis of other proteins, has lysostaphin has been administered in humans only
been used in the debridement of dermal ulcers and topically for reduction of staphylococcal carrier rate
bums. Another protease, papain, has been shown to in the nose and throat where it has been found to be
produce marked reduction of obstetrical inflammation effective and non-toxic. Ultimately, the potential drug
and the edema following dental surgery. Deoxyribo- applications are twofold. Since lysostaphin is unique
nuclease, an enzyme that degrades nucleic acids, has among antistaphylococcal agents in that it destroys
338 INDIAN J BIOTECHNOL,
bacteria, whether they are active or resting, and is thus
capable of killing large numbers of organisms; it may
be useful in instances of endocarditis and other
conditions where an initial and rapid reduction in
bacterial count is necessary. The in vivo effectiveness
of this enzyme against methicillin-resistant strains of
S. aureus has been demonstrated; lysostaphin might
prove useful in the treatment of methicillin-resistant
staphylococcal infections, of which many have begun
to appear in Europe as well as in the USA.
Protease, the enzyme that digests proteins, has a
very different and powerful function when taken on
an empty stomach. It is a powerful all natural blood
enhancer, able to break down protein invaders in the
blood supply, so that the body's natural immune
system can destroy them. Parasites, fungal forms and
bacteria are made up of proteins. Viruses are made up
of nucleic acids covered by a protein film. Since
protease can break down undigested protein, cellular
debris, and toxins in the blood, it frees up the immune
system for the more important work of destroying the
unnatural invaders like bacteria. Cancer cells are more
sensitive to enzymes than normal cells because
enzymes dissolve the fibrous coating on cancer cells,
allowing the immune system to work. The enzymes
can also diminish the ability for cancer cells to attach
to healthy organs or tissue.
The oncolytic enzymes fall into two major classes:
those that degrade small molecules for which
neoplastic tissues have a requirement, and those that
degrade macromolecules such as membrane
polysaccharides, structural and functional protein, or
nucleic acids. At present, tumour-cell specificity is
observed only in the former category. An example is
the typical oncolytic enzyme, L-asparaginase. Certain
tumour cells are deficient in their ability to synthesize
the non-essential amino acid, L-asparagine, and are
forced to extract it from body fluids; by contrast, most
normal cells can produce their own L-asparagine.
Asparaginase given parenterally acts in this way in
many susceptible tumours. Only acute lymphocytic
leukemia ordinarily responds to chemotherapy with
the enzyme. Nevertheless, the response of this one
tumour type is promising-60% incidence of complete
remissions in 6,000 cases. The search is being
extended to other enzymes that degrade small
molecules. A bi-functional amidohydrolase, L-
glutaminase, L-asparaginase is undergoing clinical
trials in the United Kingdom and shows activity in
other diseases. L-methioninase, which effectively
dismantles L-methionine to yield methanethiol,
JULY 2003
ammonia and a ketobutyric acid, is effective against
several murine tumours, but no clinical trials have
been undertaken. The same is true for L-
phenylalanine ammonialyase, which deaminates both
L-phenylalanine and L-tyrosine yielding trans-
cinnamic and trans-coumaric acids, respectively. In
the case of both these enzymes, mammalian cells are
incapable of reconstructing the substrate from the
products, so the reaction is effectively irreversible in
vivo. Other amino acid degrading enzymes with
oncolytic activity in experimental tumours include: L-
arginase, L-tyrosinase, L-serine dehydratase, L-
threonine deaminase and indolyl-3-alkane
hydroxylase, which decompose L-tryptophan. This
list is expanding at a notable rate since the technique
of enrichment of bacterial culture increased yields of
microbial enzymes capable of decomposing amino
acids in novel ways. Diphtheria toxin, a different type
of oncolytic enzyme still in the experimental stage,
catalyzes transfer of the adenosine diphosphate ribose
(ADP-ribose) moiety of nicotinamide adenine
dinucleotide (NAD) to elongation factor 2. This
enzyme stops the process of protein synthesis. Most
important from a chemotherapeutic standpoint is the
observation that protein synthesis in tumour cells is
one hundred to ten thousand times more sensitive to
this toxin than the analogous process in normal cells.
Among the oncolytic enzymes that degrade
macromolecules, neuraminidase, ribonuclease, and a
diverse group of proteases are the most prominent
examples. Neuraminidase removes sialic acid residues
from the surface of neoplastic cells, thereby altering
their immunogenicity, and rendering them sensitive to
immune response. To date this effect has been studied
mainly in experimental trials. In addition, several
ribonucleases have shown modest activity against
experimental murine neoplasms, but their use is beset
by the problem of forcing these molecules into the
cytoplasm where the substrate ribonucleic acid (RNA)
is present. Pepsin, given intralesionally, was one of
the first enzymes used for the chemotherapy of
cancer, but its clinical use was surrounded by
controversy and has ceased. On the other hand, a
mixture of vitamins and proteolytic enzymes, marked
under the name Wobe Mugos, is widely prescribed for
the control of cancer in Europe and appears to be of
some use in the palliation of the disease. The
carboxypeptidases are catalysts that cleave the
carboxyl-terminal residue of many peptides; certain of
these enzymes also are capable of hydrolyzing the L-
glutamyl moiety of folic acid. In doing so, they
 SABU: MICROBIAL THERAPEUTIC ENZYMES 339

achieve a state of folic acid deficiency deleterious to and lipids and cause permanent loss of structure to
the tumour cell. Use of this approach has, so far, been such molecules. Superoxide radicals are the most
restricted to test animals, but human trials are dangerous. To protect from such danger, the cells
beginning with a preparation designated have superoxide dismutase (SOD) and catalase
carboxypeptidase G 1. Because carboxypeptidase G 1 enzymes. Hydrogen peroxide is itself dangerous and
can decompose the drug methotrexate--a folic acid must be destroyed by catalase. A number of tumour
analogue and antagonist, the enzyme is also envisaged cells have been found to be deficient in SOD. Initial
as an antidote to overdose of methotrexate. plan was to treat this as a target for reactive radicals.
But then it was discovered that re-expression of SOD
gene cancels immortality. It seems that an essential
Therapeutic Enzymes for the Treatment of Cystic
step in becoming immortal is switch off SOD gene or
Fibrosis
may be a cluster of genes that include SOD. Absence
Cystic fibrosis is the most common fatal hereditary
of SOD activity seems to support cancer. Phagocytes
disease among Caucasians. This dreaded disease
destroy cells by pumping superoxide radicals into
affects c. 30,000 people in USA. Affected persons are
cells and tissues, and other systems such as Ab-Ag
susceptible to bacterial infections in lungs and the
complexes can trigger phagocytes to dump superoxide
infecting bacteria cause accumulation of thick mucus.
seems to be a general alert signal to attract wbc, etc.
Bacterial DNA and polysaccharides induce the
to the scene causes swelling, etc. SOD mops up the
secretion of mucus. Now, enzymes are available for
superoxide. SOD is also an effective defence weap?n;
the treatment of cystic fibrosis. Genentech produces
and Mycobacteria and Nocardia have SOD, which
recombinant human DNase I under the trade name
enables them to resist the injection of superoxide by
Pulmozyme®. Cloned and over-expressed DNase I is
phagocytes. When these organisms cause serious
delivered to patients as an aerosol, which digests
disease, it takes the body a very long time to win, ~d
DNA in mucus and hence reduces viscosity of mucus.
depending on the strength of the patient the bactena
This enzyme has been approved by the Food and
may win. The extent of SOD in bacteria in un~o,:n,
Drug Administration (FDA) of the United States.
but it may be that the next generation of antibiotics
Mucus also contains the polysaccharide alginate,
required will be inhibitors of SOD. .
which is produced by seaweeds and soil and marine
Serrapeptase is a proteolytic enzyme sornetimes
bacteria. Pseudomonas aeruginosa is one among them
known as or serratiopeptidase. For over 30 years
and is the main infectious agent in cystic fibrosis
serrapeptase has been gaining wide acceptance in
affected lungs. Alginate lyase in combination with
Europe and Asia as a potent analgesic and anti-
DNase is used to degrade alginate as well as DNA.
inflammatory drug (Yamasaki et al, 1967; Mazzone et
Alginate lyase gene from the soil bacterium,
al, 1990). It has been used to promote wound healing
Flavobacterium was isolated and the alginate
and surgical recovery. Recent Japanese patents even
degradation domain was amplified. This was then
suggest that oral serrapeptase may help tre~~ or
cloned in to an expression vector.
prevent viral diseases such as AIDS an? h~patI~IS.B
An innovative use of enzymes as therapeutic agents
and C. But its most spectacular application IS in
entails their administration to tumour-bearing subjects
reversing cardiovascular disease. Serrapeptase is
along with a prodrug conjugated to a functional group
effective in unblocking carotid arteries. The
that is susceptible to attack by an enzyme. To achieve
mechanism behind the action of this enzyme is the
the requisite selectivity advantage is taken of two
ability of the enzyme to cut or cleave a protein tar~et
features: the acidic intracellular environment of many
into two or more pieces, usually at very specific
neoplasms as compared to normal tissues, and an
cleavage sites. The same mechanism makes it
enzyme with an acidic pH-activity optimum. Using a
possible for peptidases to inactivate HI~, the A~S-
combination of L-arabinofuranosidase from
associated virus, by pruning the VIral proteins
Aspergillus niger and Peltatin-L-arabinofuranoside,
necessary for infectivity (Tang et al, 1991).
scientists have successfully used this technique to
Serrapeptase is commercially obtained from Serratia
depress thymidine incorporation by mammary
marcescens cultures.
adenocarcinomas.
Most organisms are exposed to oxygen for their Enzyme Replacement Therapy
lives. However, oxygen can be converted to form The treatment of enzyme deficiency state
extremely reactive radicals that bind to DNA, proteins represents an obvious use of enzymes. More
340 INDIAN J BIOTECHNOL,
intriguing is the treatment of inborn errors of
metabolism in which deficiency of a single enzyme
leads to accumulation of abnormal amounts of
substrate. With the recognition that many of these
errors are owing to inadequacies of lysosomal
enzymatic catabolism, it was reasoned that
exogenously administered enzyme might react with
and dispose of such accumulations. The infusion of
crude glucosidase from Aspergillus niger into patients
with type II glycogenolysis, a condition attributed to a
deficiency of this enzyme, was reported in the mid
1960s.
f3-Lactamases and their Role in Antibiotic
Resistance
Many members of the enterobacteriaceae including
Enterobacter cloacae, E. aerogenes, Citrobacter
freundii, Serratia marcescens, Klebsiella pneumoniae,
etc. are generally resistant to amoxycillin and early
generation cephalosporins and have variable
resistance profiles to second generation
cephalosporins. These bacterial species produce a
chromosomally encoded f3-lactamase belonging to
ambler class C (amb C) gene (Bush, 1988). 13-
lactamases hydrolyze the cyclic amide bonds in the 13-
lactum ring of penicillins, cephalosporins and related
compounds. A combined administration of f3-lactums
and f3-lactamase inhibitors may lead to a discovery of
new effective antibacterial compounds.
The Biodrug Concept
The biodrug concept involves the use of orally
administered recombinant microorganisms as a new
drug delivery route to prevent or treat diseases. The
tools used for genetic engineering that have been
developed to date have led to the emergence of novel
applications using genetically modified
microorganisms to produce drugs in large-scale
bioprocesses (Primrose, 1986). An innovative
extension of these approaches is drug production
directly in the digestive environment by ingested
living recombinant microorganisms. For this purpose,
recombinant bacteria, mainly lactic acid bacteria,
have been studied (Chang & Prakash, 1998). Yeast is
a convenient host and a good alternative for the
production of biodrug. The most common yeasts,
Saccharomyces cerevisiae and S. boulardii, have a
generally regarded as safe (GRAS) status and have
recently been used both in animals and humans, and
in some human digestive pathologies, such as
antibiotic-associated diarrhoea and Clostridium
JULY 2003
difficile-disease. In the past few decades, S. cerevisiae
has become an attractive host for the production of
recombinant proteins and bioconversion owing to its
high productivity and ease of genetic engineering.
The biodrug concept was validated (Alric, 2000)
using a recombinant model S. cerevisiae expressing
the plant P450 73Al. This enzyme provides a relevant
model of bioconversion for potential therapeutic
applications, such as 'biodetoxication' in the digestive
environment. The yeasts have been studied in an
artificial digestive system, which simulates human
digestion.
The potential medical applications of these new
generation of biodrugs are numerous, for example, the
correction of enzyme deficiencies, the control of the
activation of pro-drug to drug or the production of
therapeutic proteins, such as vaccines, directly in the
digestive tract. In particular, by increasing the body's
protection against environmental xenobiotics, these
biodrugs can offer an innovative way to prevent or
treat diseases that escape traditional drug action, such
as cancer or other widespread multifactorial diseases.
Conclusions
Enzyme industry is one among the major industries
of the world and there exists a great market for
enzymes in general. Pharmaceutical industry is being
recognized as an important consumer for commercial
enzymes. Enzymes are in great demand for use as
therapeutic agents against many dreaded diseases.
Accelerated and in-depth studies to utilize the vast
microbial resources--both terrestrial and marine--as
so'urces of novel therapeutic enzymes are highly
significant. Microbial enzymes offer potential to treat
many important diseases, which are res urging after
acquiring resistance to antibiotics.
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