sources, properties andapplications ofmicrobial ...€¦ · vol 2,july 2003, pp 334-341 sources,...

Indian Journal of BiotechnologyVol 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 digestingfood proteins. Enzymes accelerate and control all biochemical processes in the body and in a single second severalmillions of enzyme mediated chemical reactions occur in a human body. Each enzyme is programmed to carry outone special task. The immense number of enzymes acts as a perfectly matched orchestra to ensure that enormouslycomplex life mechanisms and processes occur in a right direction. Sufficient amount and optimal function of enzymespresent 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 topicis very meagre and thus the present review is an attempt to compile information on the sources, properties andapplications of important therapeutic enzymes.

Keywords: therapeutic enzymes, glutaminase, asparaginase, enzyme therapy, tumour, biodrug

IntroductionEnzymes are proteinaceous in character. Each

enzyme is programmed to carry out one special task.Like a key in a lock each enzyme fits together withone specific substrate modifying it in one proper way.The manufacture or processing of enzymes for use asdrugs is an important facet of today's pharmaceuticalindustry (Cassileth, 1998). Attempts to capitalize onthe advantages of enzymes as drugs are now beingmade at virtually every pharmaceutical researchcentre in the world. Since the later years of the 19thcentury, crude proteolytic enzymes have been usedfor gastrointestinal disorders, e.g., pepsin fordyspepsia. In fact, other than as digestion aids,enzymes were largely ignored as drugs until a groupof researchers observed that an extra cellular secretionof Bacillus pyocyaneus was capable of killing anthraxbacilli, and of protecting mice from otherwise lethalinocula of the bacterium. They deduced that thesecretion in question was a nuclease, i.e. it was actingby enzymatically degrading nucleic acids. Thismilestone study gradually opened up the way for theuse of parenteral enzymes first in the treatment ofinfections, then of cancer, and finally of a diversespectrum of diseases. Enzyme supplements areavailable in pills, capsules and powders. Supplementsoften consist of combinations of several differentenzymes. John Beard, an English scientist, was first to

*Tel: 91-471-2515339; Fax: 91-471-2491712E-mail: [email protected]

use pancreatic enzymes to treat cancer in 1902(Gonzalez & Isaacs, 1999). He proposed in 1906 thatpancreatic proteolytic enzymes, in addition to theirwell- known digestive function, represent the body'smain defence against cancer. He further proposed thatpancreatic enzymes would most likely be useful asanticancer agents. During the first two decades of thiscentury, a number of physicians, both in Europe andthe USA, used injectable pancreatic enzymes to treatadvanced human cancer, often with great success.There are several studies from the 1960s showing, inan animal model, that orally ingested pancreaticenzymes have an anticancer effect, and might workthrough immune modulation. German researcherslater used enzyme therapy to treat patients withmultiple sclerosis, cancer, and viral infections. DrEdward Howell introduced the term enzyme therapyto the United States in the 1920s. He believed that byeating raw meat, people created an enzyme surplus,which resulted in better health and increasedresistance to diseases (Cassileth, 1998).Therapeutic enzymes have a broad variety of

specific uses: as oncolytics, thrombolytics oranticoagulants, and as replacements for metabolicdeficiencies. Additionally, there is a growing group ofmiscellaneous enzymes of diverse function.Proteolytic enzymes have been widely used as anti-inflammatory agents. Reduction of inflammation andedema is ascribed to the dissolution of soft fibrin andto the clearance of proteinaceous debris found ininflammatory exudates.

SABU: MICROBIAL THERAPEUTIC ENZYMES

Information on the utilization of microbial enzymesfor therapeutic purposes is scarce and the availablereports are largely on some anticancer enzymes andothers, which are active against cystic fibrosis.Development of medical applications for enzymes hasbeen at least as extensive as those for industrialapplications, reflecting the magnitude of potentialrewards. The variety of enzymes and their potentialtherapeutic applications are considerable. A selectionof those enzymes, which have realised this potentialto become important therapeutic agents, is given inTable 1. At present, the most successful applicationsare extra cellular, purely topical uses, the removal ofcytotoxic substances and the treatment of life-threatening disorders within the blood circulation(Sabu, 2003).Since the enzymes are specific biological catalysts,

they should make the most desirable therapeuticagents for the treatment of metabolic diseases.Unfortunately, a number of factors severely reducethis potential utility; enzymes are too large to bedistributed simply within the body's cells. This is themajor reason why enzymes have not yet beensuccessfully applied to the large number of humangenetic diseases. A number of methods are beingdeveloped in order to overcome this by targetingenzymes; for example, enzymes with covalentlyattached external ~-galactose residues are targeted athepatocytes and enzymes covalently coupled totarget-specific monoclonal antibodies are being usedto avoid non-specific side-reactions.In contrast· to the industrial use of enzymes,

therapeutically useful enzymes are required with veryhigh degree of purity. The favoured kinetic propertiesof these enzymes are low Km and high Vmax in order tobe maximally efficient even at very low enzyme andsubstrate concentrations. Thus, the sources of suchenzymes are chosen with care to avoid any possibilityof unwanted contamination by incompatible materialand to enable ready purification. Therapeutic enzymepreparations are generally offered for sale aslyophilised pure preparations with only biocompatiblebuffering salts and mannitol diluents added. The costsof such enzymes may be quite high but stillcomparable to those of competing therapeutic agentsor treatments.A major potential therapeutic application of

enzymes is in the treatment of cancer. Asparaginasehas proved to be particularly promising for thetreatment of acute lymphocytic leukaemia. Its actiondepends upon the fact that tumour cells are deficient

335

Table l--Some important therapeutic enzymes and theirapplications

Enzyme Application

L-AsparaginaseL-GlutarninaseSuperoxide dismutaseSerratio peptidasePenicillin acylaseCollagenaseLipaseStreptokinaseUrokinaseLaccaseL-arginaseL-TyrosinaseGlucosidaseGalactosidase

13-lactamaseRibonuclease

AntitumourAntitumourAnti-oxidant, anti-inflammatoryAnti-inflammatorySynthetic antibiotic productionTo treat skin ulcersDigests lipidsAnticoagulantAnticoagulantDetoxifierAntitumourAntitumourAntitumourAntitumourPenicillin allergyAntiviral

in aspartate-ammonia ligase activity, which restrictstheir ability to synthesise the normally non-essentialamino acid, L-asparagine. Therefore, they are forcedto extract it from body fluids. The action ofasparaginase does not affect the functioning of normalcells, which are able to synthesise enough for theirown requirements, but reduce the free exogenousconcentration, and so induce a state of fatal starvationin the susceptible tumour cells. A 60% incidence ofcomplete remission has been reported in a study ofalmost 6,000 cases of acute lymphocytic leukaemia.This enzyme is administered intravenously.

Microbial Therapeutic EnzymesMicrobial enzymes are preferred over plant or

animal sources due to their economic production,consistency, ease of process modification andoptimization. They are relatively more stable thancorresponding enzymes derived from plants oranimals. Further, they provide a greater diversity ofcatalytic activities. The majority of enzymes currentlyused in industry are of microbial origin. But once weenter into the therapeutic applications of microbialenzymes, a number of factors severely reduce theirpotential utility. One of the major problems is thelarge molecular size of biological catalysts, whichprevents their distribution within the somatic cells.Investigations are on to overcome these problems bythe technique of drug targeting. Another importantproblem related to enzyme therapy is the elicitation of

336 INDIAN J BIOTECHNOL, JULY 2003

immune response in the host cells after injecting theforeign enzyme protein. Modern medical sciencecould overcome this problem also by disguising theenzyme as an apparently non-proteinaceous moleculeby covalent modification. L-glutarninase modified bycovalent attachment of polyethylene glycol, has beenshown to retain its antitumour effect whilst possessingno immunogenicity. Other methods like entrapment ofthe enzyme within artificial liposomes, syntheticmicro spheres and red blood cell ghosts have also beenfound useful. These inherent problems necessitate therequirement of therapeutic enzymes with a very highdegree of purity and specificity (Sabu, 2003).

Salt Tolerance and the Role of MarineMicroorganismsUse of salt tolerant enzymes from marine bacteria

provides an interesting alternative for therapeuticpurpose. The marine biosphere is one of the richest ofthe earth's innumerable habitats, yet one of the leaststudied and characterized fauna. Currently, marinemicroorganisms are considered as untapped sourcesof metabolites and products, which may possess novelproperties. Marine microorganisms have a diverserange of enzymatic activity and are capable ofcatalyzing various biochemical reactions with novelenzymes. Thus, there is enormous scope for theinvestigations exploring the probabilities of derivingnew products of economic importance from potentialmarine microorganisms. Considering the fact thatmarine environment, particularly seawater, which issaline in nature and chemically closer to human bloodplasma, it could provide microbial products, inparticular the enzymes that could be safer having noor less toxicity or side effects when used for humantherapeutic application. Yet another fact, which isleading an increasing interest on exploring andexploitation of marine microorganisms for industrialapplications, is their high levels of salt toleranceability. Hence, there is an increasing interest in themarine microorganisms for therapeutic purposes(Sabu et al, 2000).

SourcesTherapeutic enzymes are widely distributed in plant

and animal tissues and microorganisms includingbacteria, yeast and fungi. Although microorganismsare potential sources of therapeutic enzymes,utilization of such enzymes for therapeutic purposes islimited because of their incompatibility with thehuman body. But there is an increased focus on

utilization of microbial enzymes because of economicfeasibility. Microbial sources of some therapeuticenzymes are given in Table 2.

ProductionThere are different methods of fermentation by

which we can produce these important enzymes. Oncommercial scale, liquid cultures in huge bioreactorsare preferred for the bulk production of therapeuticenzymes. Other processes like solid state fermentation(SSF), immobilization and fermentation on inert solidsupports are also widely used for the production oftherapeutic enzymes. Recombinant E. coli strains witha foreign gene are generally cultivated in liquid media(submerged fermentation) for expressing the foreignprotein. Submerged fermentation (SmF) is thecultivation of microbial cells in liquid media undercontrolled conditions in bioreactors for the productionof desirable metabolites. SmF offers advantages likeeasy online monitoring of process parameters andprocess automation. SSF is the culturing ofmicroorganisms on moist solid substrates in theabsence or near absence of free water. It is alsodescribed as a fermentation process that takes placeon solid or semisolid substrate or that occurs on anutritionally inelt solid support, which provides someadvantages to the microorganisms with respect toaccess to nutrients and the product derived will bewith high purity. Immobilization of cells can bedefined 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 rouxiiPseudomonas acidovorans,Acinetobacter sp.Citrobacter freundii, Serratiamarcescens, Klebsiella pneumoniaeSerratia marcescensCandida lipolytica, C. rugosa,Aspergillus oryzaePseudomonas aeruginosaAspergillus nigerBacillus polymyxa, BeauveriabassianaMycobacterium sp, Nocadia sp.Aspergillus nigerAspergillus oryzae, Bacillus sp.Serratia marcescensPenicillium sp.Trametes versicolor

L-asparaginase

~-Lactamase

Serratia peptidaseLipase

Alginate lyaseL-arabinofuranosidaseProtease

Superoxide dismutaseGlucosidaseAmylaseSerrapeptasePenicillin acylaseLaccase


a distinct solid phase that permits exchange ofsubstrates, products, inhibitors, etc., but at the sametime separates the catalytic cell biomass from the bulkphase containing substrates and products.

rDNA Technology for the Production ofTherapeutic EnzymesAdvent of rDNA technology allows production of

large quantities of pharmaceutical proteins, whichwere previously difficult and costly to produce.Protein activity is often modified by rDNAtechnology and can be overcome by shufflingfunctional domains and site directed mutagenesis.This is done to modify activities, regulation and avoidunwanted side effects. The principle behind rDNAtechnology can be simply represented as:

Clone cDNA for proteint

Insert into-expression vectort

Transform E. colit

Over expresst

Purify

So far four hundred human proteins have beenproduced by rDNA technology for therapeutic use.Commercial value of these therapeutic products isenormous (Tables 3 & 4). Present global market fortherapeutic recombinant proteins is around $200.billion. Major market is shared by pharmaceuticalgiants like Genentech ($250 million) for growthhormone, Eli Lilly ($277 million) for insulin andAmgen ($2.15 billion) for erythroprotein. Since thereis a need for large quantity of therapeutic enzymes forclinical trials and for sales once approved, the gene-expression process must be optimized.

General ApplicationsEnzymes are being used to treat many diseases like

cancer, cardiac problems, cystic fibrosis, dermalulcers, inflammation, digestive disorders, etc.Collagenase, an enzyme unique that hydrolyses nativecollagen and spares hydrolysis of other proteins, hasbeen used in the debridement of dermal ulcers andbums. Another protease, papain, has been shown toproduce marked reduction of obstetrical inflammationand the edema following dental surgery. Deoxyribo-nuclease, an enzyme that degrades nucleic acids, has

337

Table 3--Human proteins produced by rDNA technology

Recombinant protein Application

AntitrypsinCell growth factorsEpidermal growth factorErythroproteinFactor VIII and Factor IXGrowth hormoneInsulin

For treating EmphysemaFor immunological disordersTo treat burnsAnemia, kidney disorders, etc.HemophiliaGrowth defectsDiabetes

Table 4--Commercially available FDA-approved recombinanthuman proteins

Recombinant Manufacturer Applicationprotein

DNase I Genentech Cystic fibrosisErythroprotei n Amgen Anaemia

Growth hormone Genentech Growth hormonedeficiency

Insulin Eli Lilly DiabetesIFN-a2a Hoffmann-La Roche LeukemiaInterleukin-2 Chiron Renal carcinoma

recently been investigated as a mucolytic agent foruse in patients with chronic bronchitis. The enzyme,lysozyme hydrolyzes the chitins and mucopeptides ofbacterial cell walls. Accordingly, it has been used asan antibacterial agent usually in combination withstandard antibiotics. The proteolytic enzymes, trypsinand chymotrypsin have been successfully used in thetreatment of post-operative hand trauma, athleticinjuries and sciatica. Hyaluronidase exerts action bydestroying the intracellular ground substancehyaluronic acid, thus allowing diffusion of vitalmolecules through this normally impermeableconnective tissue barrier. In 1959, improvements ofthe electrocardiograms of patients with acutemyocardial infarction were demonstrated followingtreatment. Lysostaphin whose lytic effects oncoagulase-positive Staphylococcus aureus arepresently under considerable study. It is a proteasethat lyses susceptible cells in a highly efficientmanner probably by peptidase-like cleavage of theglycoprotein of the bacterial wall. At present,lysostaphin has been administered in humans onlytopically for reduction of staphylococcal carrier ratein the nose and throat where it has been found to beeffective and non-toxic. Ultimately, the potential drugapplications are twofold. Since lysostaphin is uniqueamong antistaphylococcal agents in that it destroys


bacteria, whether they are active or resting, and is thuscapable of killing large numbers of organisms; it maybe useful in instances of endocarditis and otherconditions where an initial and rapid reduction inbacterial count is necessary. The in vivo effectivenessof this enzyme against methicillin-resistant strains ofS. aureus has been demonstrated; lysostaphin mightprove useful in the treatment of methicillin-resistantstaphylococcal infections, of which many have begunto appear in Europe as well as in the USA.Protease, the enzyme that digests proteins, has a

very different and powerful function when taken onan empty stomach. It is a powerful all natural bloodenhancer, able to break down protein invaders in theblood supply, so that the body's natural immunesystem can destroy them. Parasites, fungal forms andbacteria are made up of proteins. Viruses are made upof nucleic acids covered by a protein film. Sinceprotease can break down undigested protein, cellulardebris, and toxins in the blood, it frees up the immunesystem for the more important work of destroying theunnatural invaders like bacteria. Cancer cells are moresensitive to enzymes than normal cells becauseenzymes dissolve the fibrous coating on cancer cells,allowing the immune system to work. The enzymescan also diminish the ability for cancer cells to attachto healthy organs or tissue.The oncolytic enzymes fall into two major classes:

those that degrade small molecules for whichneoplastic tissues have a requirement, and those thatdegrade macromolecules such as membranepolysaccharides, structural and functional protein, ornucleic acids. At present, tumour-cell specificity isobserved only in the former category. An example isthe typical oncolytic enzyme, L-asparaginase. Certaintumour cells are deficient in their ability to synthesizethe non-essential amino acid, L-asparagine, and areforced to extract it from body fluids; by contrast, mostnormal cells can produce their own L-asparagine.Asparaginase given parenterally acts in this way inmany susceptible tumours. Only acute lymphocyticleukemia ordinarily responds to chemotherapy withthe enzyme. Nevertheless, the response of this onetumour type is promising-60% incidence of completeremissions in 6,000 cases. The search is beingextended to other enzymes that degrade smallmolecules. A bi-functional amidohydrolase, L-glutaminase, L-asparaginase is undergoing clinicaltrials in the United Kingdom and shows activity inother diseases. L-methioninase, which effectivelydismantles L-methionine to yield methanethiol,

ammonia and a ketobutyric acid, is effective againstseveral murine tumours, but no clinical trials havebeen undertaken. The same is true for L-phenylalanine ammonialyase, which deaminates bothL-phenylalanine and L-tyrosine yielding trans-cinnamic and trans-coumaric acids, respectively. Inthe case of both these enzymes, mammalian cells areincapable of reconstructing the substrate from theproducts, so the reaction is effectively irreversible invivo. Other amino acid degrading enzymes withoncolytic activity in experimental tumours include: L-arginase, L-tyrosinase, L-serine dehydratase, L-threonine deaminase and indolyl-3-alkanehydroxylase, which decompose L-tryptophan. Thislist is expanding at a notable rate since the techniqueof enrichment of bacterial culture increased yields ofmicrobial enzymes capable of decomposing aminoacids in novel ways. Diphtheria toxin, a different typeof oncolytic enzyme still in the experimental stage,catalyzes transfer of the adenosine diphosphate ribose(ADP-ribose) moiety of nicotinamide adeninedinucleotide (NAD) to elongation factor 2. Thisenzyme stops the process of protein synthesis. Mostimportant from a chemotherapeutic standpoint is theobservation that protein synthesis in tumour cells isone hundred to ten thousand times more sensitive tothis toxin than the analogous process in normal cells.Among the oncolytic enzymes that degrademacromolecules, neuraminidase, ribonuclease, and adiverse group of proteases are the most prominentexamples. Neuraminidase removes sialic acid residuesfrom the surface of neoplastic cells, thereby alteringtheir immunogenicity, and rendering them sensitive toimmune response. To date this effect has been studiedmainly in experimental trials. In addition, severalribonucleases have shown modest activity againstexperimental murine neoplasms, but their use is besetby the problem of forcing these molecules into thecytoplasm where the substrate ribonucleic acid (RNA)is present. Pepsin, given intralesionally, was one ofthe first enzymes used for the chemotherapy ofcancer, but its clinical use was surrounded bycontroversy and has ceased. On the other hand, amixture of vitamins and proteolytic enzymes, markedunder the name Wobe Mugos, is widely prescribed forthe control of cancer in Europe and appears to be ofsome use in the palliation of the disease. Thecarboxypeptidases are catalysts that cleave thecarboxyl-terminal residue of many peptides; certain ofthese enzymes also are capable of hydrolyzing the L-glutamyl moiety of folic acid. In doing so, they


achieve a state of folic acid deficiency deleterious tothe tumour cell. Use of this approach has, so far, beenrestricted to test animals, but human trials arebeginning with a preparation designatedcarboxypeptidase G 1. Because carboxypeptidase G 1can decompose the drug methotrexate--a folic acidanalogue and antagonist, the enzyme is also envisagedas an antidote to overdose of methotrexate.

Therapeutic Enzymes for the Treatment of CysticFibrosisCystic fibrosis is the most common fatal hereditary

disease among Caucasians. This dreaded diseaseaffects c. 30,000 people in USA. Affected persons aresusceptible to bacterial infections in lungs and theinfecting bacteria cause accumulation of thick mucus.Bacterial DNA and polysaccharides induce thesecretion of mucus. Now, enzymes are available forthe treatment of cystic fibrosis. Genentech producesrecombinant human DNase I under the trade namePulmozyme®. Cloned and over-expressed DNase I isdelivered to patients as an aerosol, which digestsDNA in mucus and hence reduces viscosity of mucus.This enzyme has been approved by the Food andDrug Administration (FDA) of the United States.Mucus also contains the polysaccharide alginate,

which is produced by seaweeds and soil and marinebacteria. Pseudomonas aeruginosa is one among themand is the main infectious agent in cystic fibrosisaffected lungs. Alginate lyase in combination withDNase is used to degrade alginate as well as DNA.Alginate lyase gene from the soil bacterium,Flavobacterium was isolated and the alginatedegradation domain was amplified. This was thencloned in to an expression vector.An innovative use of enzymes as therapeutic agents

entails their administration to tumour-bearing subjectsalong with a prodrug conjugated to a functional groupthat is susceptible to attack by an enzyme. To achievethe requisite selectivity advantage is taken of twofeatures: the acidic intracellular environment of manyneoplasms as compared to normal tissues, and anenzyme with an acidic pH-activity optimum. Using acombination of L-arabinofuranosidase fromAspergillus niger and Peltatin-L-arabinofuranoside,scientists have successfully used this technique todepress thymidine incorporation by mammaryadenocarcinomas.Most organisms are exposed to oxygen for their

lives. However, oxygen can be converted to formextremely reactive radicals that bind to DNA, proteins

339

and lipids and cause permanent loss of structure tosuch molecules. Superoxide radicals are the mostdangerous. To protect from such danger, the cellshave superoxide dismutase (SOD) and catalaseenzymes. Hydrogen peroxide is itself dangerous andmust be destroyed by catalase. A number of tumourcells have been found to be deficient in SOD. Initialplan was to treat this as a target for reactive radicals.But then it was discovered that re-expression of SODgene cancels immortality. It seems that an essentialstep in becoming immortal is switch off SOD gene ormay be a cluster of genes that include SOD. Absenceof SOD activity seems to support cancer. Phagocytesdestroy cells by pumping superoxide radicals intocells and tissues, and other systems such as Ab-Agcomplexes can trigger phagocytes to dump superoxideseems to be a general alert signal to attract wbc, etc.to the scene causes swelling, etc. SOD mops up thesuperoxide. SOD is also an effective defence weap?n;and Mycobacteria and Nocardia have SOD, whichenables them to resist the injection of superoxide byphagocytes. When these organisms cause seriousdisease, it takes the body a very long time to win, ~ddepending on the strength of the patient the bactenamay win. The extent of SOD in bacteria in un~o,:n,but it may be that the next generation of antibioticsrequired will be inhibitors of SOD. .Serrapeptase is a proteolytic enzyme sornetimes

known as or serratiopeptidase. For over 30 yearsserrapeptase has been gaining wide acceptance inEurope and Asia as a potent analgesic and anti-inflammatory drug (Yamasaki et al, 1967; Mazzone etal, 1990). It has been used to promote wound healingand surgical recovery. Recent Japanese patents evensuggest that oral serrapeptase may help tre~~ orprevent viral diseases such as AIDS an? h~patI~IS.Band C. But its most spectacular application IS inreversing cardiovascular disease. Serrapeptase iseffective in unblocking carotid arteries. Themechanism behind the action of this enzyme is theability of the enzyme to cut or cleave a protein tar~etinto two or more pieces, usually at very specificcleavage sites. The same mechanism makes itpossible for peptidases to inactivate HI~, the A~S-associated virus, by pruning the VIral proteinsnecessary for infectivity (Tang et al, 1991).Serrapeptase is commercially obtained from Serratiamarcescens cultures.

Enzyme Replacement TherapyThe treatment of enzyme

represents an obvious use ofdeficiencyenzymes.

stateMore


intriguing is the treatment of inborn errors ofmetabolism in which deficiency of a single enzymeleads to accumulation of abnormal amounts ofsubstrate. With the recognition that many of theseerrors are owing to inadequacies of lysosomalenzymatic catabolism, it was reasoned thatexogenously administered enzyme might react withand dispose of such accumulations. The infusion ofcrude glucosidase from Aspergillus niger into patientswith type II glycogenolysis, a condition attributed to adeficiency of this enzyme, was reported in the mid1960s.

f3-Lactamases and their Role in AntibioticResistanceMany members of the enterobacteriaceae including

Enterobacter cloacae, E. aerogenes, Citrobacterfreundii, Serratia marcescens, Klebsiella pneumoniae,etc. are generally resistant to amoxycillin and earlygeneration cephalosporins and have variableresistance profiles to second generationcephalosporins. These bacterial species produce achromosomally encoded f3-lactamase belonging toambler class C (amb C) gene (Bush, 1988). 13-lactamases hydrolyze the cyclic amide bonds in the 13-lactum ring of penicillins, cephalosporins and relatedcompounds. A combined administration of f3-lactumsand f3-lactamase inhibitors may lead to a discovery ofnew effective antibacterial compounds.

The Biodrug ConceptThe biodrug concept involves the use of orally

administered recombinant microorganisms as a newdrug delivery route to prevent or treat diseases. Thetools used for genetic engineering that have beendeveloped to date have led to the emergence of novelapplications using genetically modifiedmicroorganisms to produce drugs in large-scalebioprocesses (Primrose, 1986). An innovativeextension of these approaches is drug productiondirectly in the digestive environment by ingestedliving recombinant microorganisms. For this purpose,recombinant bacteria, mainly lactic acid bacteria,have been studied (Chang & Prakash, 1998). Yeast isa convenient host and a good alternative for theproduction of biodrug. The most common yeasts,Saccharomyces cerevisiae and S. boulardii, have agenerally regarded as safe (GRAS) status and haverecently been used both in animals and humans, andin some human digestive pathologies, such asantibiotic-associated diarrhoea and Clostridium

difficile-disease. In the past few decades, S. cerevisiaehas become an attractive host for the production ofrecombinant proteins and bioconversion owing to itshigh productivity and ease of genetic engineering.The biodrug concept was validated (Alric, 2000)using a recombinant model S. cerevisiae expressingthe plant P450 73Al. This enzyme provides a relevantmodel of bioconversion for potential therapeuticapplications, such as 'biodetoxication' in the digestiveenvironment. The yeasts have been studied in anartificial digestive system, which simulates humandigestion.The potential medical applications of these new

generation of biodrugs are numerous, for example, thecorrection of enzyme deficiencies, the control of theactivation of pro-drug to drug or the production oftherapeutic proteins, such as vaccines, directly in thedigestive tract. In particular, by increasing the body'sprotection against environmental xenobiotics, thesebiodrugs can offer an innovative way to prevent ortreat diseases that escape traditional drug action, suchas cancer or other widespread multifactorial diseases.

ConclusionsEnzyme industry is one among the major industries

of the world and there exists a great market forenzymes in general. Pharmaceutical industry is beingrecognized as an important consumer for commercialenzymes. Enzymes are in great demand for use astherapeutic agents against many dreaded diseases.Accelerated and in-depth studies to utilize the vastmicrobial resources--both terrestrial and marine--asso'urces of novel therapeutic enzymes are highlysignificant. Microbial enzymes offer potential to treatmany important diseases, which are resurging afteracquiring resistance to antibiotics.

ReferencesAlric M et al, 2000. Microorganisms actifs dans l'environnement

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their implications on future. Rev Infect Dis, 10,681-690.Cassileth B, 1998. The Alternative Medicine Handbook. W W

Norton & Co, New York, USA.Chang T M S & Prakash S, 1998. Therapeutic uses of

microencapsulated genetically engineered cells. Mol MedToday, 4, 221-227.

Gonzalez N J & Isaacs L L, 1999. Evaluation of pancreaticproteolytic enzyme treatment of adenocarcinoma of thepancreas, with nutrition and detoxification support. NutrCancer, 33, 117-124.

Mazzone A et al, 1990. Evaluation of Serratia peptidase in acuteor chronic inflammation of otorhinolaryngology pathology:A multi centre; double-blind, randomized trial versus placebo.


J lnt Med Res, 18, 379-388.Primrose S B, 1986. The application of genetically engineered

microorganisms in the production of drugs. 1 Appl Bacteriol,61,8211-8216.

Sabu A et al, 2000. Biopotential of microbial glutaminases.Chemistry Today, 11/12, 21-25.

Sabu A, 2003. Microbial glutaminase. in Concise Encyclopedia of

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Bioresource Technology (in press).Tang S & Levy J A, 1991. Inactivation of HIV-l by trypsin and

its use in demonstrating specific virus infection of cells. 1Virol Methods, 33, 39-46.

Yamasaki H et al, 1967. Anti-inflammatory action of a protease,TSP, produced by Serratia. Folia Pharmacol lPN, 63, 302-314.

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