Chemical Papers 67 (6) 667–671 (2013)DOI: 10.2478/s11696-013-0323-y

SHORT COMMUNICATION

In situ bioconversion of compactin to pravastatin by ����������

species in fermentation broth of ������ �� ������

aAjaz Ahmad, bMohd Mujeeb, bRohit Kapoor, bBibhu Prasad Panda*

aDepartment of Clinical Pharmacy, College of Pharmacy, King Saud University, Riyadh-11451; Saudi Arabia

bMicrobial & Pharmaceutical Biotechnology Laboratory, Centre for Advanced Research in Pharmaceutical Science,

Faculty of Pharmacy, Jamia Hamdard, Hamdard Nagar, New Delhi-110062, India

Received 20 August 2012; Revised 28 October 2012; Accepted 9 November 2012

The biocatalytic production of pravastatin from compactin by hydroxylation has found manyapplications in health care and pharmaceuticals. Actinomadura macra, Actinomadura madurae, andActinomadura livida can efficiently bioconvert compactin to pravastatin. The fermentation broth(Penicillium citrinum fermented media) harvested on the eighth day contained 388.90 mg L−1 ofcompactin and an undetectable level of mycotoxin (citrinin). Bioconversion by A. macra was highest(87 %) in the yeast extract-amended medium. The anti-actinomadura effects of citrinin reducethe bioconversion capacity of Actinomadura. The in situ hydroxylation of compactin produced byP. citrinum represents a preferable alternative for the use of purified compactin, as a way to reducecost and time processing.c© 2013 Institute of Chemistry, Slovak Academy of Sciences

Keywords: Actinomadura sp., P. citrinum, compactin, pravasatin, in situ bioconversion

Pravastatin suppresses cholesterol biosynthesis byinhibiting a 3-hydroxy-3-methyl glutaryl CoA reduc-tase. Initially, pravastatin was isolated as a metaboliteproduct of compactin from canine urine. Subsequently,pravastatin was developed as a new therapeutic agentfor the treatment of hypercholesterolemia (Yamashitaet al., 1985; Shepherd et al., 1995; Barrios-González &Miranda, 2010). Pravastatin is produced by chemicalsynthesis from compactin. However, due to the highcost and the occurrence of stereoisomers, pravastatinproduction by microbial hydroxylation is preferred.Pravastatin can be produced by a two-step process,in which the first step is the production of compactinby an appropriate microbial strain such as Penicilliumcitrinum; subsequently, hydroxylation of compactin atthe C-6 position can be performed either by a chemi-cal method or by a fermentation process (Chen et al.,2006).An extensive survey of the literature shows the use

of purified compactin for the production of pravastatinby actinomycetes such as Streptomyces carbophillus

SANK 62585, Streptomyces sp. Y-110, catalysed bythe cytochrome P450 monooxygenase system, or byActinomadura sp. ATCC 55678, catalysed by the hy-droxylase enzyme (Hosobuchi et al., 1993; Peng & De-main, 2000; Park et al., 2003). To reduce the costand time of processing, the present research proposesuse of the entire fermented broth containing com-pactin rather than the purified compactin. To this end,a fermented broth containing spent medium, com-pactin, mycotoxin (citrinin), and Penicillium citrinumcell fragments were used for testing the bioconversionof compactin to pravastatin by three Actinomaduraspecies, namely Actinomadura livida, Actinomaduramacra, and Actinomadura madurae.Cultures of Penicillium citrinum MTCC 1256,

Actinomadura madurae MTCC 1120, Actinomaduramacra MTCC 2559, and Actinomadura livida MTCC1382 were obtained from MTCC, IMTECH, India.P. citrinum was maintained on PDA slants. A. madu-rae and A. livida were maintained in a growthmedium, A. macra was maintained in a growth

*Corresponding author, e-mail: bibhu panda31@rediffmail.com

668 A. Ahmad et al./Chemical Papers 67 (6) 667–671 (2013)

medium supplemented with 2 g L−1 CaCO3. All mi-crobial cultures were stored at 4◦C and sub-culturedevery 30 days (Peng & Demain, 2000; Chakravarti &Sahai, 2002a).Spores of P. citrinum (4 × 106 spores per mL) were

suspended in a glycerol–water solution (15 g L−1). Theseed culture was prepared according to the proceduregiven by Chakravarti and Sahai (2002b). Fermenta-tive production of compactin was carried out as perthe method detailed by Ahmad et al. (2011a). Cell sus-pensions of A. madurae, A. livida, and A. macra wereprepared in a basal medium (glucose 4 g L−1, maltextract 10 g L−1, and yeast extract 4 g L−1) from ac-tively growing slants; the medium pH was adjusted to7.2 with 0.1 M KOH, or 0.1 M HCl. A. macra andA. livida were incubated at 28◦C for two days, andA. madurae was incubated at 37◦C for seven days.The fermented medium containing compactin and

fungal cells (8, 10, 12, and 14 day-old culture) was ul-trasonicated by a Vibra Cell VCX 130 (Sonics, USA)at 20 kHz for 5 min and autoclaved at 121◦C for20 min. Subsequently, the fermentation broth con-taining depleted medium, compactin, mycotoxin (cit-rinin), and non-viable Penicillium citrinum cell frag-ments was used for the bioconversion of compactinto pravastatin by A. livida, A. macra, and A. madu-rae. The spent medium containing different concen-trations of compactin, citrinin and cell fragments wassupplemented with Actinomadura growth-promotingmedium components (dextrose 4 g L−1, malt extract10 g L−1, CaCO3 2 g L−1, yeast extract 4 g L−1, andurea 4 g L−1, at pH 7.2). Seed cultures of A. macra,A. livida, and A. madurae were added (5 vol. %) intothe medium separately. All the bioconversion reactionswere carried out in 250 mL flasks by A. macra andA. livida at 28◦C and by A. madurae at 37◦C for fourdays, shaking at 210 min−1.Citrinin was extracted from the fermentation broth

following the procedure detailed by Jackson andCiegler (1978) and quantified by HPTLC (Ahmad etal., 2010). Compactin and pravastatin were extractedfrom the fermentation broth following the proceduredetailed by Chakarvarti and Sahai (2002b) and anal-ysed by HPTLC (Ahmad et al., 2011b). Liquid chro-matographic and mass spectrophotometric (LC-MS)analysis of compactin and pravastatin was carriedout by using an ultra-pressure liquid chromatogra-phy system (UPLC, Waters, USA) using an AQUITYUPLC� BEH, C-18, (2.1 mm × 100 mm, 1.7 µm) ana-lytical column tempered maintained at temperature to40◦C. Acetonitrile–water (ϕr = 65 : 35) acidified with0.1 vol. % formic acid at a flow-rate of 0.75 mL min−1

was used as the mobile phase. Detection was carriedout using PDA photodiode array (235 nm) and massdetectors.The cultures of A. madurae, A. macra, and

A. livida were grown in a growth medium. A. madu-rae and A. macra were incubated at 37◦C for three

days, and A. livida was incubated at 28◦C for sevendays in an orbital shaker at 110 min−1. Each acti-nomycetes culture (5 mL) with 6 × 107 CFU (colony-forming unit) per mL was treated for 12 h with 1 mL ofP. citrinum fermentation broth (14th day). The CFUof A. madurae, A. macra, and A. livida, as developedin the growth media, were measured after incubationfor 3 d for A. madurae and A. macra and 7 d forA. livida.Compactin production in a previously optimised

medium started from the second day onwards withan initial concentration of 67.59 mg L−1, reachinga maximum level of 541.41 mg L−1 on the 15thday and remaining almost constant thereafter. Cit-rinin biosynthesis in the optimised production me-dia started from the 9th day with a concentrationof 113.6 mg L−1. The fermentation media (8th day,10th day, 12th day, 14th day) containing compactin(388.90 mg L−1, 419.26 mg L−1, 458.75 mg L−1,and 522.50 mg L−1) and citrinin (0.00 mg L−1,126.03 mg L−1, 165.92 mg L−1, and 252.36 mg L−1)were used for bioconversion. The amount of pravas-tatin produced after bioconversion by either A. livida,A.macra, or A. madurae in a medium containing com-pactin 388.90 mg L−1 and citrinin 0.00 mg L−1 sup-plemented with yeast extract (4 g L−1) is representedin (Fig. 1a) with bioconversion of 87 %, 85 %, and86 % by A. macra, A. livida, and A. madurae, respec-tively. The bioconversion percentages of compactin topravastatin by A. livida, A. macra, and A. madu-rae in a medium containing 419.26 mg L−1 of com-pactin, and 126.03 mg L−1 of citrinin were 78.9 %,81.12 %, and 81.13 %, respectively (Fig. 1b). The bio-conversion was highest at 96th h of fermentation inthe yeast extract-supplemented medium. A sharp de-crease in the percentage of the bioconversion of com-pactin to pravastatin was observed in the P. citrinumfermentation medium containing a higher amount ofcitrinin, 165.92 mg L−1 harvested on the 12th day offermentation with bioconversion of 74 %, 72 %, and75 % by A. macra, A. livida, and A. madurae, re-spectively (Fig. 1c). In the media containing citrinin,252.36 mg L−1 (14th day of fermentation), the bio-conversion was 67 %, 70 %, and 68 % by A. macra,A. livida, and A. madurae, respectively (Fig. 1d).The pravastatin produced was analysed by UPLC-

MS. The retention time (Rt) of the pravastatin pro-duced and unused compactin exhibited a coincidencewith the Rt of standard pravastatin (0.71 min) andstandard compactin (2.65 min) in Fig. S1 (see sup-plementary data) with the m/z value of compactinand pravastatin is 391 and 424 respectively (Fig S2).The chromatograms of components in the fermenta-tion broths of A. madurae, A. macra, and A. lividashowed a very similar pattern, confirming that all theActinomadura sp. tested were capable of bioconvertingcompactin to pravastatin. The percentage of pravas-tatin production in the medium supplemented with

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Fig. 1. Pravastatin production by A. madurae ( ), A. macra ( ), and A. livida ( ) in medium supplemented with yeast extractcontaining fermentation broth obtained on 8th day (a), 10th day (b), 12th day (c), and 14th (d) day of P. citrinumcultivation.

urea (4 g L−1) was lower than in the medium supple-mented with yeast extract, with differences of 6 % forA. macra, 7 % for A. madurae, and 5 % for A. livida,respectively.On the basis of the antibacterial effect of citrinin

(Chan, 2008), the anti-actinomadura activity of theP. citrinum fermentation broth (14th day) containingcitrinin (252.36 mg L−1) was analysed in terms of thenumber of colony-forming units (i.e. CFU) per mL.There was a decrease from 6 × 107 CFU mL−1 to7 × 102 CFU mL−1, 2 × 103 CFU mL−1, and 1 × 103CFU mL−1 for A. madurae, A. livida, and A. macra,respectively. The inhibitory effect of citrinin was great-est against A. madurae. The percentage of bioconver-sion of compactin to pravastatin was lowest in the cul-ture broth containing maximum citrinin, i.e. in the14th-day P. citrinum fermentation media.Pravastatin is a natural HMG Co-A reductase in-

hibitor produced in two-step fermentation processes.Initially, compactin (mevastatin) is produced by fer-mentation using P. citrinum, then the purified com-pactin is bioconverted to pravastatin by hydroxyla-tion at the C-6 position. Biosynthesis of compactin byP. citrinum and mycelium formation (glucosamine)showed a parallel response which is revealed by thefermentation kinetics. During the log phase growth,

citrinin biosynthesis was found to be almost zero andits production started during the second growth phaseof fungus until the idiophase was reached. The produc-tion of compactin and citrinin is independent of eachother; this may be due to different biosynthetic geneclusters and/or regulatory genes for compactin andcitrinin.In situ bioconversion of the compactin-containing

fermentation broth was carried out, instead of the useof purified compactin, which would represent a possi-bility to reduce cost and time required for processing.Actinomadura species such as Actinomadura livida,Actinomadura macra, and Actinomadura maduraewere selected for the bioconversion of compactin topravastatin. Bioconversion reaction was previouslyachieved by a number of microorganisms, includ-ing Amycolata autotrophica, Mucor hiemalis, Nor-cardiaceae, Actinomycesa madurae, Streptomyces car-bopilus, Streptomyces exfoliates, andMicromonospora.However, these microbes proved to be highly sensi-tive towards compactin concentration. The pro-drug(compactin) of pravastatin is highly toxic to these mi-croorganisms (Peng & Demain, 2000). However, bio-conversion of pure compactin is 68 % after 6 days offermentation by Pseudonocardia carboxydivorans (Linet al., 2011).

670 A. Ahmad et al./Chemical Papers 67 (6) 667–671 (2013)

In the present work, the P. citrinum 8th-day fer-mentation broth containing 388.90 mg L−1 of com-pactin, an undetectable level of mycotoxin (citrinin),non-viable fragmented mycelium, supplemented withActinomadura growth-promoting medium components(dextrose 4 g L−1, malt extract 10 g L−1, CaCO32 g L−1, yeast extract 4 g L−1), led to a maximum87.02 % of bioconversion of compactin to pravastatin,with A. macra MTCC 2559 in 96 h.The growth rates of the Actinomadura species

(A. livida, A. macra, and A. madurae) selected forbioconversion studies were inhibited by the high levelof antibacterial mycotoxins (citrinin) produced dur-ing mevastatin production by P. citrinum MTCC1256. The nitrogen source influences the produc-tion of pravastatin. Bioconversion was higher in themedium supplemented with yeast extract than in themedium supplemented with urea. This suggests thatthe medium constituents, micronutrients, and growthfactors present in yeast extract are essential for bio-conversion. The hydroxylase enzyme of Actinomadurasp. (Watanabe et al., 1995; Chen et al., 2006) car-ries out the hydroxylation of compactin to pravas-tatin. In the cell-free extract, Actinomadura hydrox-ylase required NADPH and ATP, ascorbic acid, andMg2+ as co-factors for maximum activity (Peng & De-main, 1998). This suggests that a medium composed ofMg2+ ions and ATP will contribute to a higher biocon-version reaction yield and better hydroxylase enzymeactivity. The yeast extracts, unused Mg2+ ions andATP released from the cell-free extract of P. citrinumwould help in achieving higher bioconversion.The in situ bioconversion of compactin (by means

of microbial hydroxylation reaction) may be consid-ered for the production of pravastatin by Actino-madura macra instead of using purified compactin.However, the concentration of mycotoxin (citrinin) re-quires to be controlled, since it regulates the biocon-version. In situ bioconversion may prove to be a betteralternative in order to reduce cost and time requiredfor processing, and this production model may be use-ful in the development of other two-step fermentationprocesses.

Acknowledgements. We wish to express our thanks to UGC,Government of India for providing financial support.

Supplementary data

Supplementary data associated with this articlecan be found in the online version of this paper (DOI:10.2478/s11696-013-0323-y).

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