Indian Journal of BiotechnologyVol 2, July 2003, pp 370-377

Microbial Transformation of Rifamycin: A Novel Approach to RifamycinDerivatives

A H Jobanputra, G D Patil, R Z Sayyed, A B Chaudhari and S B Chincholkar=School of Life Sciences, North Maharashtra University, P B 80, Jalgaon 425 001, India

Received 10 December 2002; accepted 8 April 2003

Biotransformation of an ansamycin group of antibiotic, rifamycin B (clinically less active) to a more potentantibiotic rifamycin S, involves the action of rifamycin oxidase. The extracellular rifamycin oxidase (RO) fromCurvularia lunata offers ease in the process and increased biotransformation potential and yield. Extracellular ROfrom C. lunata deserves a great future since it shows higher activity and biotransformation potential vis-a-visintracellular RO from Monocillium and Humicola. Rifamycin S, especiallyamong the derivatives, has been of greatcommercial significance as a key intermediate for the synthesis of several hundreds of semisynthetic rifamycins.Besides these, rifamycin S has broad-spectrum activity against Brucella, Chlamydia, Haemophilus, Helicobacterpylori, Legionella and Staphylococcus. It has also emerged as an antituberculosis and antileprosy drug. Chemicalconversion of rifamycin B against microbial transformation has been discussed with special emphasis onbiotransforming systems, characteristics of rifamycin oxidase and factors influencing the production of rifamycinoxidase and biotransformation of rifamycin B to S.

Keywords: biotransformation, rifamycin s, rifamycin oxidase,Curvularia lunata, Monocillium and Humicola

IntroductionBiotransformation of organic compounds to active

derivatives of medicinal importance is a commerciallyattractive area. Microbial transformation of organiccompounds offers a vast repertoire of reactions, a fewof which have successfully emerged as industrialprocesses. The industrialist's choice oscillatesbetween organic synthesis and biotransformation formanufacturing organic chemicals. The final decisionis purely based upon cost effectiveness of the process.

During biotransformation an organic compound ismodified into a recoverable product by simple andchemically defined reaction catalysed by enzymescontained in the cell. Numerous microbial speciesprovide enzymes for biotransformation. Theseenzymes are able to adapt to an artificial environmentimposed as per the technical and economicalrequirements. Along with microbes, animal cellcultures and organ perfusion preparations may beuseful in transforming organic compounds; but theseare complex to operate even at laboratory scale, ascompared to microbial transformation. Biotrans-

=Author for correspondence:Tel: + 91-257-2252193; Fax: + 91-257-2252183E-mail: chincholkar_sb@indiatimes.com

formation involves reactions like oxidation,hydroxylation, hydrolysis, isomerization, resolution ofracemic mixtures, amination, enolization etc. (Patil,1998).

Antibiotic BiotransformationBiotransformation of antibiotics is far advantageous

with respect to duration, safety, energy inputs,pollution and economics. Biochemical transformationof naturally produced antibiotics can potentially beused to alter their antimicrobial activity, spectrum ofaction, oral absorption, toxicity and allergicresponses. A commercially used example of antibioticbiotransformation is the conversion of benzylpenicillins to 6-aminopencillanic acid (6-APA) whichis precursor for the synthesis of semisyntheticpenicillins (Banerjee et al, 1992). The reactioninvolves the selective hydrolysis of amide bond in thepenicillin side chain catalysed by penicillin acylase.Similarly transformation of rifamycin B to Y or B toS has also been reported. Transformation of tylosin torelomycin has been reported with washed cells ofStreptomyces hygroscopicus, S. griseospiralis andNocardia coralina (Kieslich, 1976). Biotransforma-tion of cephalosporin to 7-amino cephalosporanic acid(7ACA) was catalysed by D-amino acid oxidase

JOBANPUTRA et al: MICROBIAL TRANSFORMA nON OF RIFAMYCIN

(DAO) (Deshpande et al, 1996). Antibiotics areknown to exhibit enhanced activity aftermodification(s) in the parent structure. The product ofantibiotic biotransformation can be used as startingmaterial for the synthesis of another superior drugderivative with respect to their pharmacologicalaction (Banerjee, 1994a). Antibiotic biotrans-formation is achieved with the help of cell-freeenzymes (biocatalysts) or microbes secreting suchbiocatalysts.

RifamycinsThe rifamycin group of antibiotics was isolated by

the Lepetit Laboratories (Sensi et al, 1960; Sensi &Thiemann, 1967). Rifamycins belong to ansamycingroup of antibiotics. Their derivatives have been usedsuccessfully over the years for the treatment oftuberculosis and leprosy (Fox, 1985; Shepard, 1981).Nocardia mediterranei has been known to produce abattery of rifamycins (Rifamycin A, B, C, and D)some of which though produced in lower quantitieshave greater antibiotic activity (Lancini, 1986; Vohra,1992). The clinically useful precursor is rifamycin B.Rifamycins are basically yellow to orange compoundssoluble in polar solvents and insoluble in non-polarsolvents with acidic properties (Patil & Chincholkar,1997). In the presence of sodium barbiturate, producermicrobe exclusively secretes only rifamycin B withtraces of other components. This observation haspaved the way for the development of the rifamycin Bfermentation process. Streptomyces tolypophorus isalso recognized as potential producer of rifamycin(Kishi et al, 1972).

Chemical Properties of RifamycinsRifamycins (MW. 755.80) are yellow to orange

compounds with a specific rotation of (0 + 400 to +7000. UV-Visible spectrum of rifamycin at pH 7.3, 1%, 1 em is 555 (223 nm-E), 275 (304 nrn-E) and 220(425 nm-E). A pH-mediated change in the spectra is acharacteristic feature of rifamycin. Rifamycin B issoluble in polar solvents but insoluble in nonpolarsolvents with acidic properties. It does not melt under30°C, but decomposes at 160 -164°C. Rifamycin Bitself has no antibiotic property. Its apparent propertyis due to transformation in aqueous solution toanother active compound, rifamycin S, which is aprecursor far many rifamycin derivatives. From allthe chemical and physical studies done on rifamycins,it has been concluded that the structural features

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necessary for activity are: i) presence of naphthalenering carrying oxygen atoms at C (1) and C (8) eitherin the quinone or hydroquinone form; ii) hydroxylgroups at position C (21) and C (23) of ansa chain;and iii) a well defined spatial arrangement of oxygenatoms at C (21) and C (23) (Arora &Main, 1984).

Rifamycin SV shows good in-vivo activity,tolerability and solubility. Rifamycins have beenreported to exhibit inhibition of DNA-dependentRNA synthesis (Umezawa et al, 1968) Thus, it wasused in the treatment of Gram negative bacteria, butshowed poor activity against Mycobacteriumtuberculosis. Hundreds of compounds have beensynthesized from B, 0, S with a view to get superioractivity of rifamycin SV against tuberculosis, leprosyand several other infectious diseases (Sensi &Thiemann, 1967). Rifamycin was developed as one ofthe antituberculosis drugs in 1966 and has been usedfor almost 30 years (Hidaka, 1999). Rifamycin SV,rifamide and rifampicin are antibiotics, which are nowproduced industrially. Rifamycin S is of considerableeconomic importance because it is the precursor formajority of semisynthetic rifamycins and is highlyactive against Gram positive bacteria likeMycobacteria, Staphylococci and Streptococci) andsome Gram negative bacteria, viz. Brucella,Chlamydia, Hemophilus, Legionella and Neisseria sp.Inhibitory activity against HIV -1 reverse transcriptasehas been reported in open ansa chain rifamycin Sderivative (Bartolucci et al, 1995). Caruso (1997)observed that intra-auricular rifamycin was effectiveagainst active synovitis and could be used with anybasic thera~y with slow acting antirheumatic drugs.Important activities of novel semisynthetic derivativesof rifamycin B are given in Table 1.

Chemical Conversion of RifamycinSince the discovery of rifamycins, synthetic

processes for rifamycin derivatives have relied uponchemical means. Existing conventional chemicalconversion process of rifamycin B to S has warranteduse of strong oxidizing agents like Mn02,tetrahydrofuran and H2S04 for oxidation andsubsequent acid hydrolysis. Such stringent chemicalssignificantly reduced the yield (50-60%). Conse-quently, the process generates large amounts ofhazardous acidic water. Overall, the process sufferssevere set back due to requirement for low pH, acidresistant equipment, vigorous foaming during reactionand low yield. Biological alternatives to such process

372 INDIAN J BIOTECHNOL, JULY 2003

Rifamycin Chemistry

Table l---Chemical properties and some important activities of novel semisynthetic derivatives of rifamycin B

Important activities

Rifamycin B with amines, hydrazine and alcohol,yields amides, hydrazides and esters,respectivelywith aromatic amines and hydrazineyield quinonimine derivativeswith 0 - aminophenols producephenazine

Rifamycin 0

Rifamycin S

Rifampin (Rifadinelrimactane)

obtained from rifaldehyde

Rifabutin(Mycobutin)Rifapentine Piperazinyl hydrazone of 3 - formyl

rifamycin SVKRM 1648

Derivatives obtained

Rifamycin 0 I S I SV andRifamide

Biologically inactive. Derivativesactive against bacteria, lower toxicity.

Rifamycin AG Broad-spectrum antimicrobial.

Rifazine, rifamycin SVRifaldehydeRifampin

Broad-spectrum antimicrobial,Inhibits polymerase of RNA tumourvirusAntibacterial, used in tuberculosisdrug regime, leprosy treatment,nontuberculosis infection and S.aureus and S. epidermis, inhibits viralreverse transcriptaseAntiMAC complex and TB bacilli,bactericide, inhibits RNA polymeraseActivity similar to rifampin, longerhalf lifeActive against M. tuberculosis &MAC complex, high tissuedistribution

were thought to replace the conventional chemicaltransformation. Seong and Han (1982) found 85 %efficiency with biological system vis-a-vis chemicalprocess (60 % yield). They also noticed that chemicalmodification of rifamycins has largely beenconcentrated on the naphthalene ring moiety becausemodification of the ansa chain moiety reduces thebiological activity, ii) the presence of both hydroxylgroups, at C21 and C23 position, were particularlyrequired for biological activity, iii) increased activityof rifamycin B in aqueous solution on aging is due toits spontaneous oxidation to S.

Biotransformation of Rifamycin BRecent increase in tuberculosis and leprosy cases

due to emergence of multiple drug resistance strains(MDR) has prompted rigorous research on analternative regime. The circumstances paved the wayto recommend rifamycin regime and its derivativesfor abatement of MDR strains. Due to the criticallimitation, biological system has ventured to convertrifamycin B to S under mild conditions without anyenvironmental pollution, Biotransformation was firstreported by Lancini et al (1967) who noticed thatwashed mycelia of N. mediterranei could convertrifamycin B to Y. Biotransformation of rifamycin B toS was reported in 1969. Han and colleagues (1983)reported bioconversion of rifamycin B to S with anintracellular rifamycin oxidase from Humicola sp.

ATCC 20620 and Monocillium sp. ATCC 20621.Subsequently, an extracellular rifamycin oxidase fromCurvularia lunata var. aeria was also reported byVohra et al (1989). Further investigations haverevealed that rifamycin and its derivatives areeffective against certain RNA viruses and MAIS(Mycobacterium aviumlintracellulare/scrofulaceum)complex (MAC), which is associated with the AIDScomplex (Muralikrishna et al, 1999). At elevatedconcentration, rifamycin has been reported to showantitumour and antiviral activities. Recently, intra-auricular rifamycin appeared effective against activesynovitis. Such therapeutic value recognizedrifamycin as a 'wonder drug' and its therapeuticimportance tremendously raised the demand for theproduction of rifamycin and its derivativesworldwide. This review updates some of the recentefforts on the production of various rifamycinderivatives. Due to the industrial secrecy policy, it isimpossible to encompass all information on processdevelopment.

Rifamycin Biotransforming EnzymeHan and colleagues (1983) reported bioconversion

of rifamycin B to S with an intracellular rifamycinoxidase from Humicola sp. ATCC 20620 andMonocillium sp. ATCC 20621. Subsequently, anextracellular rifamycin oxidase from Curvularialunata var. aeria was also reported by Vohra et al,

JOBANPUTRA et al: MICROBIAL TRANSFORMATION OF RIFAMYCIN

(1989). The enzyme system reported for thebiotransformation belongs to dioxygenase type. It hasbeen predicted that the enzyme might be requiring co-factors like NADH, flavoproteins and cytochromeP450 (Patil, 1998). The key steps associated withtransformation of rifamycin B to S catalyzed byrifamycin oxidase are represented by the followingscheme.

02 HZ02 H20 OHCHzCOOHRifamycin B ~ Rifamycin 0 ~RiramYCin S

Oxidation Hydrolysis

Characteristics of Rifamycin OxidaseRifamycin oxidase from Monocillium sp. has been

found to have the properties quite different from otherpolyphenol oxidases. The substrate specificity of theenzyme was different from that of laccase obtainedfrom other organisms. Rifamycin oxidase rapidlyoxidized rifamycin Band hydroquinone but not theresorcinol and the Mannich derivative of rifamycinSV [3-diethyl arninomethyl rifamycin SV]. Theenzyme was reported to contain no flavins, heme,non-heme iron nor other metal ions as cofactors, northe SH group in the active site (Han et al, 1983).

Substrate SpecificityThe catalytic properties of rifamycin oxidase

produced intracellularly by Humicola sp. werereported by Seong et al in 1985. The enzyme wasmost specific for rifamycin B (Banerjee et al, 1992).The substrate specificity of the enzyme wasinvestigated using various substrate analogues,including rifamycin B derivatives and polyolcompounds. p-hydroxyphenoxyacetic acid and p-hydroquinone, the corresponding structural analoguesin the quinonoid moiety, caused a significant decreasein the enzyme activity compared to rifamycin B andrifamycin SV respectively (Seong et al, 1985).

Optimum pH and TemperatureReports on the characteristics of rifamycin oxidase

from C. lunata showed that a neutral pH was mostfavourable for growth as well as enzyme production.The maximum specific enzyme activity was also highat neutral pH of the medium. Without pH controlgrowth and enzyme production were limited atglucose concentrations above 1.5% (w/v). Thetemperature optimum of the enzyme was 50°C, at40°C and 45°C the enzyme exhibited 75% and 85% ofits maximum activity respectively (Vohra et al, 1989).

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While rifamycin oxidase from Monocillium sp.showed pH and temperature optima of 7.8 and 40°C,respectively.

Production of Rifamycin OxidaseHumicola sp. ATCC 20620 was found to produce

an intracellular enzyme, which was most specific forrifamycin B. Three preparations of rifamycin oxidasefrom Humicola sp.-whole cells, acetone dried wholecells and ammonium sulphate precipitate were studiedby Seong et al in 1985. To increase intracellularproduction of this enzyme by Humicola sp.biochemical engineering studies were conducted. Themaximum specific enzyme activity in fermentations'without pH control were reported to be 9.5 IU/g drycells and 15 IU/g dry cells for cells cultured atcontrolled pH, 8.0 (Kim et al, 1984).

Production of extracellular rifamycin oxidase wasreported from Curvularia lunata var. aeria grown inYPD medium at 28°C. Mycelial form of growth wasfound to produce higher enzyme activity than thepellet form (Banerjee & Srivastava, 1993). Studies onthe influence of different factors on rifamycin oxidaseproduction revealed that lOgL-I carboxymethylcellulose (CMC) was effective in producing mycelialgrowth, which yielded maximum enzyme activity(Banerjee, 1993b). The organism was found to formblack pigment when the glucose level was almost zeroand the medium became alkaline. Formation of blackpigment was considered as an indication of reducedenzyme activity (Banerjee, 1993a). While thecontrolled pH at 7.0 favored both growth as well asenzyme production. Effect of metal ions on rifamycinoxidase production showed that the metal ions (10-75mgl," like Mg2+' C02+, Zn2+ and Mn2+ had nostimulatory effect where as Fe3+, Ca2+ and Cu2+stimulated rifamycin oxidase production. Method ofsubstrate preparation in different solvents gave theidea that ethanol is best suited for maximum secretionof enzyme (Patil & Chincholkar, 1997). Rifamycinoxidase of C. lunata being an extracellular enzymeoffered ease in downstreaming (Banerjee et al, 1992).

Biotransformation with Immobilized SystemsImmobilized system offers distinct advantages of

greater stability, reusability and productivity and isreplacing free enzyme or cell systems. Exclusion ofpossible allergenic effects of the enzyme is anadditional advantage of immobilization inpharmaceutical industry (Vohra & Vyas, 1992). Toreduce the diffusion resistance in the membrane

374 INDIAN J BIOTECHNOL, JULY 2003

defattation with acetone has been practiced. Theapparent Km values of rifamycin oxidase for free andimmobilized acetone defatted cells are 0.3 and 0.6mM, respectively (Lee et al, 1984).

Rifamycin oxidase of C. lunata immobilized onnylon fibres by using gluteraldehyde as a crosslinking agent showed better results, the enzymeactivity of 18 Ug-l of nylon fibres and operationalstability of seven days with a protection againstthermal inactivation was reported. Immobilization onalginate gel has been reported to protect the enzymefrom heat shocks even at elevated temperature[500°C] (Vohra & Vyas, 1992). Enzyme immobilizedon kappa carrageenan also showed excellentcatalyzing and operational stability and reusability forseveral times vis-a-vis free enzyme (Banerjee, 1993c).

Biotransformation by using whole cells ofHumicola sp. ATCC 20620 immobilized bycopolymerisation with acrylarnide has also been tried.Compared to free enzyme the whole cell immobilizedpreparations were found to be less sensitive totemperature and pH (Lee et al, 1984).

Factors Influencing Rifamycin B BiotransfonnationDespite of a few reports on rifamycin oxidase, in

rifamycin B biotransformation, series of attemptshave been made to improve the activity and yield ofenzyme in culture filtrate by varying differentphysicochemical parameters.

Nutritional FactorsSeveral investigators have found that only C3

precursors such as glycerol or sugars like glucose orribose are used for glycolic acid moiety in rifamycinBand L but not C2 precursors like glycolate,glyoxylate, glycine or ethanol (Ghisalba et al, 1984).Kim and co-investigators (1984) observed that•rifamycin oxidase secretion in Humicola sp. was notrepressed by glucose concentration up to 60 gL-1 ofculture broth. While Ghisalba and co-workers (1984)claimed carboxymethylcellulose [CMC] as aneffective carbon source for rifamycin oxidasesynthesis. Since CMC enhances the viscosity ofmedium and thereby promotes better mycelial growththan pellet form, such conditions are conducive forsecretion of enzyme. Apart from CMC, peptone andpeanut meal as a carbon source were also proved.comparatively effective. While, ammonium sulphatewas the most effective nitrogen source to secretmaximum enzyme. But compounds like ammonium

acetate and ammonium oxalate were found inhibitoryto enzyme synthesis. C. lunata could neither grow norsecrete the enzyme in synthetic medium withoutpeptone and yeast extract (Banerjee, 1994a).

Co-factors and Metal IonsThe effect of various electron acceptors or

cofactors such as NAD+, NADP+ and FAD on theenzymatic oxidation of rifamycin B was examined inHumicola sp. Apparent inhibition of enzyme activityin presence of cysteine and mercaptoethanol wasaccompanied with the reduction of rifamycin 0 torifamycin B. These experimental findings indicatedthat the enzyme could be classified as an oxidase andnot dehydrogenase since there was no requirement ofelectron acceptor other than O2 (Han, 1983). Variouscations showed no or negligible effect on enzymeactivity. Enzyme activity was significantly inhibitedby metal ions such as Fe++, Ag", Zn'", Mn++, andHg++ (10-20 rnM), whereas Mg++and Fe+++(25 rnM)restored 83-89 % biotransformation. Catalytic activityof enzyme is not affected by the presence of metalchelating agent like EDTA (Seong et al, 1985).Contrary to this, increased enzyme activity was seenwith these cations only in C. lunata (Banerjee, 1994).

Temperature and pHThe temperature and pH requirement for optimum

rifamycin oxidase varies among the strains. Optimaltemperature range for growth and enzyme activity inMonocillium sp was found between 28-30°C in the pHrange of 7.5-8.5 (Seong et al, 1983), while Humicolasp showed optimal enzyme activity at 45°C in the pHrange of 7.0-8.0. But in case of C. lunata var. aeria,temperature optima for rifamycin oxidase was 50°C,(pH 6.5), at 40°C and 45°C, it exhibited only 75-85%activity (Seong et al, 1985). The rate of enzymaticreaction declines sharply to 80% at pH above 7.0 asgiven in Table 2 (Vohra et al, 1989; Seong et at,1985).

BiomassSeveral investigators have reported that the specific

activity of enzyme increased with increase in cellbiomass. In C. lunata, 5% inoculum size was optimalfor growth and enzyme secretion (Banerjee, 1994).Growing cells of 24 and 48 hrs-old mycelium wereable of transform rifamycin B but not 72 and 96 hrs-old cells. Whereas, resting cells of same age, wererepeatedly used for effective transformation(Banerjee, 1994b).

JOBANPUTRA et al: MICROBIAL TRANSFORMA nON OF RIFAMYCIN 375

Table 2--List of microorganisms exhibiting potential to transform rifamycin B to its derivatives

Organism Rifamycin Biotransfor Type of pH Tempe- Km Vmax Referenceoxidase -mation reaction rature (mM) (/-LMIhI

(0C) ml)

Nocardia Intracellular Rifamycin Hydroxylation Margalith &mediterranei B toY Pagani, 1961

Margalith &Beratta, 1960

Monocillium sp. Intracellular Rifamycin Oxidation 7.5- 8.5 28 -30 0.5 6.5-7.1 Han et al,BtoS/O Oxidative 1983

cyclizationHumicola sp. Intrace IIular Rifamycin Oxidation 7.8 45 0.3-0.6 19.2 Seong et al,

B to S 1983C. lunata Extracellular Rifamycin Oxidation 6.5 50 0.67 11 Banerjee &

Immobilized B toS 7.0 30 2.0 Srivastava,1993

Amycolatopsis Muralikrishnamediterranei et al, 1999

Krishna etal,1998

Substrate Specificity and KineticsThe enzyme oxidizes hydroquinone, rifamycin B,

rifamycin SV, 3-formyl rifamycin SV rapidly, whileMannich derivative of rifamycin SV (3-diethyl-methyl rifamycin SV) was not attacked at all.Rifamycin B becomes self inhibitory above thecritical concentration of 3.4 gL-I. The enzyme exhibitsa Km value of 0.67 mM and Vmax of 11 mM hr-I

ml-I. The substrate inhibition is apparent at substrateconcentration higher than 2 mM (Banerjee, 1993a).Kinetic studies with enzyme have shown uniformenzyme activity up to 9 days at 30°C. The activationand deactivation energies of partially purifiedenzymes, are 5.80 and 35.10 Kcal mol", respectively(Banerjee, 1993a; Banerjee & Patnaik, 1996).

Among the strains, Monocillium and Humicola sp.were first recognized for the production ofintracellular rifamycin oxidase (Seong et al, 1985).However, both species exhibited less specificactivities. While C. lunata showed the production ofextracellular rifamycin oxidase with high specificactivity and therefore offers ease in its purification.Culture filtrate of C. lunata was directly applied toammonium sulphate precipitation followed by columnchromatography using sephacyl S-200 and 0.05 Mphosphate buffer, pH 7.0 (Banerjee, 1993a).

Current Status of the Development of RifamycinDerivatives

A large number of rifamycin derivatives have beensynthesized/developed over the years. For

semisynthetic rifamycins, naphthalene ringmodification is preferred since ansa chainmodification reduces the biological activity ofrifamycins. Rifamycin B, 0 and S have served asstarting materials for the preparation of numerousclasses of derivatives. Several of the semisyntheticderivatives are more active, have broader spectrum ofbiological activity and therapeutically more usefulthan their parent antibiotics.

Rifamycin S, especially among the derivatives, hasbeen of great commercial significance as a keyintermediate for the synthesis of several hundreds ofsemisynthetic rifamycins. Besides these, rifamycin Shas broad-spectrum activity against Brucella,Chlamydia, Haemophilus, Helicobacter pylori,Legionella and Staphylococcus.

Rifampin, a physiological derivative of rifamycinS, has been used clinically for the treatment oftuberculosis and leprosy. Now, rifabutin is approvedas a preventive drug for MAC infection in AIDSpatients in the USA and European countries. It isnoteworthy that rifapentine was approved as anantituberculosis drug by FDA in the USA during199&.

A newly synthesised rifamycin derivative, rifalazil(KRM - 1648) possesses a promising potentantituberculosis and anti-MAC activity. It is nowunder clinical trial for the treatment of tuberculosis.The derivative, KRM 1648 possesses many usefulattributes like i) potent activity, ii) high tissuedistribution capability, iii) long shelf-life, iv)

376 INDIAN J BIOTECHNOL, JULY 2003

suitability for intermittent therapy and v) conversionto yet metabolically active versions like 30-hydroxyKRM and 25-D acetyl KRM.

KRM 1648 did not induce liver P450 whilerifabutin and rifapentine induce liver P450 in animalsand human beings. These facts accelerate themetabolism of concomitant drugs (protease inhibitors)in HIV patients thereby lowering their blood levels.For the future novel antituberculosis drugs andtherapy for tuberculosis, it is therefore prerequisiteand imperative to develop new rifamycin derivativeswith i) a preferable antimicrobial activity; ii)shortened treatment period and iii) activity againstMDR bacilli.(http://www.aegis.com/pubslaidslineI1999/jul/ A9971 077 .html

Future Perspectives for RifamycinBiotransfonnationThe free enzymes are now being replaced by

immobilized enzymes. Immobilized enzymes offerdistinct advantages in terms of greater stability,reusability and productivity. Moreover, it excludes thepossible allergenic effects of the enzyme in finishedpharmaceutical preparations. Extracellular rifamycinoxidase from C. lunata deserves future significancesince it shows highest activity vis-a-vis intracellularenzyme from Monocillium and Humicola sp. Theapplication of immobilized enzyme as biocatalyst willbe useful tool for the future industrial production ofrifamycin derivatives. Few reports have indicated theimmobilization of rifamycin oxidase from C. lunata.The enzyme immobilized on nylon fibres showed anoperational stability of 7 days, protected from thermalinactivation with apparent Km of 2.0 mM. Someinvestigators immobilized whole cells of Humicolasp. with rifamycin oxidase activity by copolymeriza-tion with acrylarnide. The biocatalyst demonstrated noappreciable loss of activity for one month when storedat 4°C, pH 7.8. The operational stability of 8 days wasrecorded at 40°C and pH 8.0 with immobilized cellsof Humicola sp. Preliminary experiments on solidstate cultivation of C. lunata, a local isolate, forbiotransformation of rifamycin in our laboratory alsooffered another possibility in this regard (Rasalkar etal, 2002). Due to lack of reports on bacterial strainssearch for novel bacterial sp. capable ofbiotransforming rifamycin B is underway and willoffer promising results in the near future (Jobanputraand Chincholkar, unpublished data).

The basic requisite in above section thereforewarrants surmounting the stability of rifamycin

oxidase. The exploration of new commercially viablealternative for biotransformation of rifamycin hingesupon several factors. With some success at laboratoryscale, researchers can be optimistic that furtherexploration of microbial transformations of rifamycinwill lead to new, highly potent rifamycin derivativeswith therapeutic value.

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