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
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
use pancreatic enzymes to treat cancer in 1902
(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 antiinflammatory 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
Information on the utilization of microbial enzymes
for therapeutic purposes is scarce and the available
reports are largely on some anticancer enzymes and
others, which are active against cystic fibrosis.
Development of medical applications for enzymes has
been at least as extensive as those for industrial
applications, reflecting the magnitude of potential
rewards. The variety of enzymes and their potential
therapeutic applications are considerable. A selection
of those enzymes, which have realised this potential
to become important therapeutic agents, is given in
Table 1. At present, the most successful applications
are extra cellular, purely topical uses, the removal of
cytotoxic substances and the treatment of lifethreatening disorders within the blood circulation
(Sabu, 2003).
Since the enzymes are specific biological catalysts,
they should make the most desirable therapeutic
agents for the treatment of metabolic diseases.
Unfortunately, a number of factors severely reduce
this potential utility; enzymes are too large to be
distributed simply within the body's cells. This is the
major reason why enzymes have not yet been
successfully applied to the large number of human
genetic diseases. A number of methods are being
developed in order to overcome this by targeting
enzymes; for example, enzymes with covalently
attached external ~-galactose residues are targeted at
hepatocytes and enzymes covalently coupled to
target-specific monoclonal antibodies are being used
to avoid non-specific side-reactions.
In contrast· to the industrial use of enzymes,
therapeutically useful enzymes are required with very
high degree of purity. The favoured kinetic properties
of these enzymes are low Km and high V max in order to
be maximally efficient even at very low enzyme and
substrate concentrations. Thus, the sources of such
enzymes are chosen with care to avoid any possibility
of unwanted contamination by incompatible material
and to enable ready purification. Therapeutic enzyme
preparations
are generally offered for sale as
lyophilised pure preparations with only biocompatible
buffering salts and mannitol diluents added. The costs
of such enzymes may be quite high but still
comparable to those of competing therapeutic agents
or treatments.
A major potential therapeutic application of
enzymes is in the treatment of cancer. Asparaginase
has proved to be particularly promising for the
treatment of acute lymphocytic leukaemia. Its action
depends upon the fact that tumour cells are deficient
ENZYMES
Table l--Some
335
important therapeutic enzymes and their
applications
Enzyme
Application
L-Asparaginase
L-Glutarninase
Antitumour
Superoxide dismutase
Anti-oxidant, anti-inflammatory
Serratio peptidase
Anti-inflammatory
Antitumour
Penicillin acylase
Synthetic antibiotic production
Collagenase
To treat skin ulcers
Lipase
Digests lipids
Streptokinase
Anticoagulant
Urokinase
Anticoagulant
Laccase
Detoxifier
L-arginase
Antitumour
L- Tyrosinase
Antitumour
Glucosidase
Antitumour
Antitumour
Galactosidase
13-lactamase
Ribonuclease
Penicillin allergy
Antiviral
in aspartate-ammonia ligase activity, which restricts
their ability to synthesise the normally non-essential
amino acid, L-asparagine. Therefore, they are forced
to extract it from body fluids. The action of
asparaginase does not affect the functioning of normal
cells, which are able to synthesise enough for their
own requirements, but reduce the free exogenous
concentration, and so induce a state of fatal starvation
in the susceptible tumour cells. A 60% incidence of
complete remission has been reported in a study of
almost 6,000 cases of acute lymphocytic leukaemia.
This enzyme is administered intravenously.
Microbial Therapeutic Enzymes
Microbial enzymes are preferred over plant or
animal sources due to their economic production,
consistency, ease of process modification
and
optimization. They are relatively more stable than
corresponding enzymes derived from plants or
animals. Further, they provide a greater diversity of
catalytic activities. The majority of enzymes currently
used in industry are of microbial origin. But once we
enter into the therapeutic applications of microbial
enzymes, a number of factors severely reduce their
potential utility. One of the major problems is the
large molecular size of biological catalysts, which
prevents their distribution within the somatic cells.
Investigations are on to overcome these problems by
the technique of drug targeting. Another important
problem related to enzyme therapy is the elicitation of
336
INDIAN J BIOTECHNOL,
immune response in the host cells after injecting the
foreign enzyme protein. Modern medical science
could overcome this problem also by disguising the
enzyme as an apparently non-proteinaceous molecule
by covalent modification. L-glutarninase modified by
covalent attachment of polyethylene glycol, has been
shown to retain its antitumour effect whilst possessing
no immunogenicity. Other methods like entrapment of
the enzyme within artificial liposomes, synthetic
micro spheres and red blood cell ghosts have also been
found useful. These inherent problems necessitate the
requirement of therapeutic enzymes with a very high
degree of purity and specificity (Sabu, 2003).
Salt Tolerance and the Role of Marine
Microorganisms
Use of salt tolerant enzymes from marine bacteria
provides an interesting alternative for therapeutic
purpose. The marine biosphere is one of the richest of
the earth's innumerable habitats, yet one of the least
studied and characterized fauna. Currently, marine
microorganisms are considered as untapped sources
of metabolites and products, which may possess novel
properties. Marine microorganisms have a diverse
range of enzymatic activity and are capable of
catalyzing various biochemical reactions with novel
enzymes. Thus, there is enormous scope for the
investigations exploring the probabilities of deriving
new products of economic importance from potential
marine microorganisms. Considering the fact that
marine environment, particularly seawater, which is
saline in nature and chemically closer to human blood
plasma, it could provide microbial products, in
particular the enzymes that could be safer having no
or less toxicity or side effects when used for human
therapeutic application. Yet another fact, which is
leading an increasing interest on exploring and
exploitation of marine microorganisms for industrial
applications, is their high levels of salt tolerance
ability. Hence, there is an increasing interest in the
marine microorganisms
for therapeutic purposes
(Sabu et al, 2000).
Sources
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. But there is an increased focus on
JULY 2003
utilization of microbial enzymes because of economic
feasibility. Microbial sources of some therapeutic
enzymes are given in Table 2.
Production
There are different methods of fermentation by
which we can produce these important enzymes. On
commercial scale, liquid cultures in huge bioreactors
are preferred for the bulk production of therapeutic
enzymes. Other processes like solid state fermentation
(SSF), immobilization and fermentation on inert solid
supports are also widely used for the production of
therapeutic enzymes. Recombinant E. coli strains with
a foreign gene are generally cultivated in liquid media
(submerged fermentation) for expressing the foreign
protein. Submerged fermentation
(SmF) is the
cultivation of microbial cells in liquid media under
controlled conditions in bioreactors for the production
of desirable metabolites. SmF offers advantages like
easy online monitoring of process parameters and
process automation.
SSF is the culturing of
microorganisms on moist solid substrates in the
absence or near absence of free water. It is also
described as a fermentation process that takes place
on solid or semisolid substrate or that occurs on a
nutritionally inelt solid support, which provides some
advantages to the microorganisms with respect to
access to nutrients and the product derived will be
with high purity. Immobilization of cells can be
defined as the attachment of cells or their inclusion in
Table 2-Microbial
sources of therapeutic enzymes
Enzyme
Source
L-glutarninase
Beauveria bassiana, Vibrio costicola,
Zygosaccharomyces rouxii
L-asparaginase
Pseudomonas acidovorans,
Acinetobacter sp.
~-Lactamase
Citrobacter freundii, Serratia
marcescens, Klebsiella pneumoniae
Serratia peptidase
Serratia marcescens
Lipase
Candida lipolytica, C. rugosa,
Aspergillus oryzae
Alginate lyase
Pseudomonas aeruginosa
L-arabinofuranosidase
Aspergillus niger
Protease
Bacillus polymyxa, Beauveria
bassiana
Mycobacterium sp, Nocadia sp.
Superoxide dismutase
Glucosidase
Amylase
Aspergillus niger
Serrapeptase
Serratia marcescens
Penicillin acylase
Penicillium sp.
Laccase
Trametes versicolor
Aspergillus oryzae, Bacillus sp.
SABU: MICROBIAL THERAPEUTIC
a distinct solid phase that permits exchange of
substrates, products, inhibitors, etc., but at the same
time separates the catalytic cell biomass from the bulk
phase containing substrates and products.
rDNA Technology for the Production of
Therapeutic Enzymes
Advent of rDNA technology allows production of
large quantities of pharmaceutical proteins, which
were previously difficult and costly to produce.
Protein activity is often modified by rDNA
technology and can be overcome by shuffling
functional domains and site directed mutagenesis.
This is done to modify activities, regulation and avoid
unwanted side effects. The principle behind rDNA
technology can be simply represented as:
Clone cDNA for protein
t
337
ENZYMES
Table 3--Human
proteins produced by rDNA technology
Application
Recombinant protein
Antitrypsin
For treating Emphysema
Cell growth factors
For immunological disorders
Epidermal growth factor
To treat burns
Erythroprotein
Anemia, kidney disorders, etc.
Factor VIII and Factor IX
Hemophilia
Growth hormone
Growth defects
Insulin
Diabetes
Table 4--Commercially
available FDA-approved recombinant
human proteins
Recombinant
protein
Manufacturer
Application
DNase I
Erythroprotei n
Genentech
Cystic fibrosis
Anaemia
Growth hormone
Genentech
Insulin
Eli Lilly
Diabetes
IFN-a2a
Hoffmann-La Roche
Leukemia
Interleukin-2
Chiron
Renal carcinoma
Amgen
Insert into-expression vector
t
Transform E. coli
t
Growth hormone
deficiency
Over express
t
Purify
So far four hundred human proteins have been
produced by rDNA technology for therapeutic use.
Commercial value of these therapeutic products is
enormous (Tables 3 & 4). Present global market for
therapeutic recombinant proteins is around $200
.billion. Major market is shared by pharmaceutical
giants like Genentech ($250 million) for growth
hormone, Eli Lilly ($277 million) for insulin and
Amgen ($2.15 billion) for erythroprotein. Since there
is a need for large quantity of therapeutic enzymes for
clinical trials and for sales once approved, the geneexpression process must be optimized.
General Applications
Enzymes are being used to treat many diseases like
cancer, cardiac problems, cystic fibrosis, dermal
ulcers,
inflammation,
digestive
disorders,
etc.
Collagenase, an enzyme unique that hydrolyses native
collagen and spares hydrolysis of other proteins, has
been used in the debridement of dermal ulcers and
bums. Another protease, papain, has been shown to
produce marked reduction of obstetrical inflammation
and the edema following dental surgery. Deoxyribonuclease, an enzyme that degrades nucleic acids, has
recently been investigated as a mucolytic agent for
use in patients with chronic bronchitis. The enzyme,
lysozyme hydrolyzes the chitins and mucopeptides of
bacterial cell walls. Accordingly, it has been used as
an antibacterial agent usually in combination with
standard antibiotics. The proteolytic enzymes, trypsin
and chymotrypsin have been successfully used in the
treatment of post-operative hand trauma, athletic
injuries and sciatica. Hyaluronidase exerts action by
destroying
the intracellular
ground
substance
hyaluronic acid, thus allowing diffusion of vital
molecules
through this normally
impermeable
connective tissue barrier. In 1959, improvements of
the electrocardiograms
of patients with acute
myocardial infarction were demonstrated following
treatment. Lysostaphin
whose lytic effects on
coagulase-positive
Staphylococcus
aureus
are
presently under considerable study. It is a protease
that lyses susceptible cells in a highly efficient
manner probably by peptidase-like cleavage of the
glycoprotein of the bacterial wall. At present,
lysostaphin has been administered in humans only
topically for reduction of staphylococcal carrier rate
in the nose and throat where it has been found to be
effective and non-toxic. Ultimately, the potential drug
applications are twofold. Since lysostaphin is unique
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,
Lglutaminase, 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 Lphenylalanine ammonialyase, which deaminates both
L-phenylalanine
and L-tyrosine yielding transcinnamic 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: Larginase, L-tyrosinase, L-serine dehydratase, Lthreonine
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 Lglutamyl moiety of folic acid. In doing so, they
SABU: MICROBIAL THERAPEUTIC
achieve a state of folic acid deficiency deleterious to
the tumour cell. Use of this approach has, so far, been
restricted to test animals, but human trials are
beginning
with
a
preparation
designated
carboxypeptidase G 1. Because carboxypeptidase G 1
can decompose the drug methotrexate--a folic acid
analogue and antagonist, the enzyme is also envisaged
as an antidote to overdose of methotrexate.
Therapeutic Enzymes for the Treatment of Cystic
Fibrosis
Cystic fibrosis is the most common fatal hereditary
disease among Caucasians. This dreaded disease
affects c. 30,000 people in USA. Affected persons are
susceptible to bacterial infections in lungs and the
infecting bacteria cause accumulation of thick mucus.
Bacterial DNA and polysaccharides
induce the
secretion of mucus. Now, enzymes are available for
the treatment of cystic fibrosis. Genentech produces
recombinant human DNase I under the trade name
Pulmozyme®. Cloned and over-expressed DNase I is
delivered to patients as an aerosol, which digests
DNA in mucus and hence reduces viscosity of mucus.
This enzyme has been approved by the Food and
Drug Administration (FDA) of the United States.
Mucus also contains the polysaccharide alginate,
which is produced by seaweeds and soil and marine
bacteria. Pseudomonas aeruginosa is one among them
and is the main infectious agent in cystic fibrosis
affected lungs. Alginate lyase in combination with
DNase is used to degrade alginate as well as DNA.
Alginate lyase gene from the soil bacterium,
Flavobacterium
was isolated and the alginate
degradation domain was amplified. This was then
cloned in to an expression vector.
An innovative use of enzymes as therapeutic agents
entails their administration to tumour-bearing subjects
along with a prodrug conjugated to a functional group
that is susceptible to attack by an enzyme. To achieve
the requisite selectivity advantage is taken of two
features: the acidic intracellular environment of many
neoplasms as compared to normal tissues, and an
enzyme with an acidic pH-activity optimum. Using a
combination
of
L-arabinofuranosidase
from
Aspergillus niger and Peltatin-L-arabinofuranoside,
scientists have successfully used this technique to
depress thymidine
incorporation
by mammary
adenocarcinomas.
Most organisms are exposed to oxygen for their
lives. However, oxygen can be converted to form
extremely reactive radicals that bind to DNA, proteins
ENZYMES
339
and lipids and cause permanent loss of structure to
such molecules. Superoxide radicals are the most
dangerous. To protect from such danger, the cells
have superoxide dismutase (SOD) and catalase
enzymes. Hydrogen peroxide is itself dangerous and
must be destroyed by catalase. A number of tumour
cells have been found to be deficient in SOD. Initial
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
step in becoming immortal is switch off SOD gene or
may be a cluster of genes that include SOD. Absence
of SOD activity seems to support cancer. Phagocytes
destroy cells by pumping superoxide radicals into
cells and tissues, and other systems such as Ab-Ag
complexes can trigger phagocytes to dump superoxide
seems to be a general alert signal to attract wbc, etc.
to the scene causes swelling, etc. SOD mops up the
superoxide. SOD is also an effective defence weap?n;
and Mycobacteria and Nocardia have SOD, which
enables them to resist the injection of superoxide by
phagocytes. When these organisms cause serious
disease, it takes the body a very long time to win, ~d
depending on the strength of the patient the bactena
may win. The extent of SOD in bacteria in un~o,:n,
but it may be that the next generation of antibiotics
required will be inhibitors of SOD.
.
Serrapeptase is a proteolytic enzyme sornetimes
known as or serratiopeptidase. For over 30 years
serrapeptase has been gaining wide acceptance in
Europe and Asia as a potent analgesic and antiinflammatory drug (Yamasaki et al, 1967; Mazzone et
al, 1990). It has been used to promote wound healing
and surgical recovery. Recent Japanese patents even
suggest that oral serrapeptase may help tre~~ or
prevent viral diseases such as AIDS an? h~patI~IS.B
and C. But its most spectacular application IS in
reversing cardiovascular disease. Serrapeptase is
effective in unblocking
carotid arteries. The
mechanism behind the action of this enzyme is the
ability of the enzyme to cut or cleave a protein tar~et
into two or more pieces, usually at very specific
cleavage sites. The same mechanism makes it
possible for peptidases to inactivate HI~, the A~Sassociated virus, by pruning the VIral proteins
necessary for infectivity (Tang et al, 1991).
Serrapeptase is commercially obtained from Serratia
marcescens cultures.
Enzyme Replacement Therapy
The treatment
of enzyme deficiency
represents an obvious use of enzymes.
state
More
INDIAN J BIOTECHNOL,
340
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
Resistance
and
their
Role
in
Antibiotic
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). 13lactamases hydrolyze the cyclic amide bonds in the 13lactum 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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