Research Journal of Microbiology 1 (5): 433-437, 200 ISSN 1816-4935 (© 2005 Academie Journals Ine, USA Production of L-phenyl Acetyl Carbinol (L-PAC) by Different Novel Strains of ‘Yeasts in Molasses and Sugar Cane Juice as Production Medium M, Ravi Kumar, M, Adharvana Chari and M, Lakshmi Narasu Centre for Biotechnology, Institute of Seience and Technology. JIN-T. University, Kuketpally, Hyderabed-500072, India "Abstract: Isolation and serning of novel stains of yeass from natural sourees like fits tnd sugareane juice for biotransformation studies was investigated, These stains were identified at the Institut of Microbial ‘Technology, Chandigarh, basing on sequencing of yD, domain of 265 rRNA gene ane assigned MTCC numbers. These three strains were designated as Candida preudotntermedia MTCC No, 6225 (GY), Issatchenkia artentials MTCC No, 6351 (DY), Candida pseudointermedia MTCC No. 6352 (SCY), respectively Production of phony! acetyl eabinal(L-PAC) though biowansformation of benzaldehyde by free ccs of the yeast of efferent strains has been attempted. In our experiments the L-PAC product obtained from benzaldehyde tough biotransformation by four difetent yeasts like Baker's yeas, Candida pseudotntermedta (6225), Candida pseudotntermedia (6352) and Jesrchenkia orsentals (6351), Key words Benzaldshyde, novel stains, molasses, sugareane juice, biotransformation, PAC Introduction Biotechnology employs microorganism as wells higher ells and thir active principles with the aim of achieving desirable conversions of various substrates (Tripathi etal, 1997). L-phengl acetyl ‘arbinol is the starting material for chemical synthesis of L-Ephedkine hydro ehlorde und pseudo epheine pharmaceutical compounds used as decongestant, antasthmsties (Shin and Rogers, 1995) ‘and recenly reported used in obesity control (Astrup etal, 1992). Aromatic substrate benzaldehyde will give L-PAC by biotransformation method. Certain yeast stains possess pyruvate decaroxylase (PDO) and alchol dehydrogenase (ADHD enzymes that produce L-PAC and benzyl alechol, a by product, respectively from berwaldehyde (Nikelova and Ward, 1991), Biotransformation potentials ‘ofthe growing els free harvested cells immobilized cells and isclated crude as well as purified enzyme have bee nextensively studied (Liew ea, 1995, Shin and Rogers, 19960, b). ‘The role of novel strains in the bioconversion isan important aspect. L-PAC production was studied by fiee and immobilized cells of saccharomyces cerevisie under Vasious growth and Diotransformation conditions. But ve have studied L-PAC production from benzaldehyde by using various novel stains under varios growth and biottansformaton redalites witha view to monitor ‘the ideal conditions permiting maximum product yield at constant substrate eoneenttation and cell density. L-PAC productions given in Scheme 1 : = Oftaamas Sra, “abe emgceti Conta Lphedsine Scheme 1: ‘Corresponding Astor: NE Aira Cr. Cente fr Brecnolog state o Science Tinos, INT: Unive, Kus, dead S007 IndiaRes. J. Microbiol, 1 (5): 433-437, 2006 Materials and Methods Production of -phensl Acetsl Carbinol with Normal Cells of Secharemuces Cerevisiae (BE) Production of L-Pac (a key ilermediate for many drugs) ffom benzaldehytle by yeast is the potential route inthe Fermentation Industry for the production of Ephedrine and other drugs. The present study was ccnductod by the author in Sultan-Ul -Uloom college of Pharmacy, located in Hyderabad India in the yeat 2004.The stock culture of bakers yeast was freshly subeultuzed (Elaish and Krishna, 1987) on fish sterile YEMA medium slants and incubated at room temperature (about 28°C) for 36 h. Thus the 36 h culture was used for harvesting. The culture of Bakers yeast was harvested by shaking in 5 ml. sterile water. Harvested microbial suspension was transferred into Inoculum medium The composition of inoculum medium is as fellows Molasses Urea. In Sterile water Tomake up to 100 mL. pH 5s ‘The flask was incubated at 28°C on a rotary shaker (180 RPM) for 24, ‘The microbial count was mad by using Neubauer counting chamber. The Microbial suspension ‘was diluted so ut each mL of suspension contained 200<10* calls, Ten nalliliter of inoculum From TMI was transferred to 100 mL of inoculation mediusn- who's composition is indicated below: Molasses 10% Urea 02s InSterle water Tomake up to 100 mL pH 3s ‘The flasks were incubated ona rotary shaker (180 RPM) for 161. ‘One hundked milter of production media were prepared, In most of the study, molzsses medium, used as @ production medium and for comperisan study sugarcane juice medium vith urea wes used 8 a production medium, Ten milter of inoculum fiom IMI was wansfrred to production media ‘whose compositions same as IMI and incubated for 9h on rotary shaker, At 8, nuitient (20 mL of 50% molasses) was added to molasses production meskum and nutrient (20 mL of 50% sugar cane juice) was added to sugar cane production medium respectively and incubated on rotary shaker. From 10 h onwards 0.6% of distilled benzaldehyde was added in 6 divided doses of half an hour intervals to prosuetion medium, ‘Then asks were incubated cn the rotary shaker for 24 h. the asks containing 130 mL broth 2,100 mi production medivan+-10 mL inceulum+20 mL sutient medium) was treated with 130 mL, ‘erzene (solvent) and shaken for 1S min in separating finaels. Then the organic layer was separated and filtered through absorbent cotion, Filly the solvent benzene was distilled of 10 get L-PAC product, Production of L-phensl Acetsl Carbinol with New Isolates of Yeasts: Procediwes for Bioconversion: In comparative studies the novel cultures were used to estimate and compare their biotransformation potential with tukers yeast. The stock cultures of Candia pseudointermedia MTCC No. 6225, Candida pseudountermedia MTCC No, 6352 an Issatchenkia orientalis MTCC No. 6351 were subeultured aseptically on Stile YEMA slants with stslized tansTersng loop in the sterile area (Laminar air ow). a4Res. J. Microbiol, 1 (5): 433-437, 2006 Previously mentioned procedure (Elaiah and Krishna, 1987) was repeated forthe novel strains Also, In mos ofthe study molasses medium was used as a production madium and for comparative study sugarcane juice medium with urea (Kaur and Kocher, 2002) was used as a production medium. Results and Discussion L-Phenyl acetyl carbo isa yellow colour liquid. ts spectie gravity reported is 0.99 at room temperature, L-PAC produced by isolates ineucing Bakers yeasts (S.cerevisia) showed the same specific gravity value. pH value of L-PAC (1:1 Ratio of L-PAC sample and water is reported as 3.84 In this experiments the L-PAC product obtained through biotransformation by four diffrent yeasts like baker's yeast, Candida preudomtermedia (6225), Candida pseudointermedia (6352) and “ssatchenkiaorientals (6351) showed the same pH values. R value of L-PAC wus reported (Groger ‘and Erge, 1965) in chloroform as motile phase on siliea gl odin vapons were used for the detection of spats. In developing chromatograms chloroform as a front solvent was compared with solvent mixture (30% ethyl acetate 70% hexane) as mobile phase The later solvent ent shoved better seperation than chloroform. So we used 30% cthylacctate and 709% hexane as solvent front in all our experiments, L-PAC isreaetive in UV light, odine vapours and f-Methoxynaplihalene charring also, The RE value of standard L-PAC is around 0.33. L-PAC produet of different isolates showed the same RE value ‘The met Ketone present in L-PAC undergoes the Idofinm reaction (Smith and Hendlin, 1954), Itinially undergoes halogenation and the cleavage in the presence of alkali like NaOH to give rise to Idoform. This reaction is very spocifie for L-PAC and does net oecur with by products, L-PAC produced by different isolates gave rise to Idoferm By polarimetric and colerimeti estimations, dhe percentage bioconversion wus estimated in different yeast isolates. Other fermentation peoduets (2. benzyl alcohol, benzoic acid as well as ‘unconverted benzaldehyde were assayed by High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). The chemical stueture of -PAC was identified and conformed to NMR and UV spectal data Soveral workers (Ellaiah and Krishna, 1987) conducted fermentaticn in molasses as preaucton| _meddum, in addition to that in indkastry use molasses medium for bioconversion reaction inthe L-PAC produetion In the present investigation we wied the suger cane juice as produetion medium in L-PAC production. Sugar cane juice with 0.25% wea was used as production modium which yielded more oduct of L-PAC than molasses. Inaction to that product extraction from sugarcane juice medium ‘was very convenient than from the molasses mediuun, The bicconversion obtained with different ‘yeas isolates is shown in Table 1 Biotransformation potential of fice cells of Bakers yeast (Scerevisiae) was staied in both ‘molasses ao sugarcane juice as production mediuan, In molasses medium 25% bioconversion was ‘observed where asin sygareane juice medium 28% bioconversion was cbserved, Name of the cere Melimn vset -L-PAC concentration (gL!) Binconenson 89) SixcharomzercoreMsion Moles 158 S00 Cds psuckinormeda MTCCNo, 6028 Molases tar aS Canta psoubonmeda MTCCNS, 882 Molasses 210 aur seis renal MTICCN. S1—‘Molaees a sue Saccharomyces crevsae Sugeane ue tan 2200 Candi pieudanermeda MTCC Ne 228 Sugarcane lee Las Bs Canta pseukinermedda MTCC Ne, 52 Suane ice 297 nae seats orinale MECCNe 6831 Sugarane lee iso east 425Res. J. Microbiol, 1 (5): 433-437, 2006 Biotransformation potential of free cells of Candida pseutointermedia MTCC No. 6225 was studied in both molasses and sugarcane juice as production mediuan In molases 23.43% bioconversion was observed whereas in sugarcane juice medium 23.75% bioconversion was observed. Biotransformation potential of free cells af Candida pseudointermedia MTCC No. 6332 was studied in both molasses and sugarcane juice as production medium. In molasses 33.47% bioconversion ‘was observed where as in sugarcane juice medium 48 76% bioconversion was observed. Biowransformation potanal of fee cals of ssatchenkia orientalis MICC No, 6351 was studied inboth molasses and sugateane juice as production medium, In molasses 37.16% biowanversion was ‘observed where asin sugareane juice medium 60.61% bioconversion was observed. Conclusion In conclusion the present procedure forthe usage of novel strains of yeasts from natural sources for biotransformation studies were investigated and used for the bioconversion of benzaldehyde to L- PAC. Three strains were isolated from different natural sources ike biackgrapes, date fruit and sugarcane juice and Were identified at the Insite of Microbial Technology, Chandigath. These three sins were designated as Candida pseudointermesdia MTCC No, 6225 (EGY), Isateenkia orentials MTCC No. 6351 (DY), Candida pseudotermedia MTCC No. 6352 (SCY)Which will be an ‘important addition tothe present existing procedures. The most significant Findings of the present research ate use of3 new strains of yeasts forthe production of L-PAC and the use of Sugarean juice 4s production medium for the production of L-PAC. We are success in both attempt and there is considerable nerease inthe parcontage yield of L-L-PAC when saarcane juice was ised as production ‘medium. Though sugarear juice is expensive compared to Molasses, te extraction procedure was proved to be much easier with sugarcane juice compared to Motases. Further research modalities lke different factors, immobilization studies, mutation studies et., could help in establishing the cost effective methods forthe production of L-PAC by using sugarcane juice asa production medium. This is the first report on using Candida pseudointermedia and Esatehenkia ortentalts for bioconversion studies of benzaldehyde to L-PAC. Use of sugarcane juice as production medium in biotznsformation studies of benzaldehyde to L-PAC is also the first eport ofits kind. Moreover sugarcane juice showed ‘increased bioconversion potential than molasses medium, The novel stains ve ean explore fie the «different chemical reactions, Further studies in this diction axe in peogtess Acknowledgments “The authors are thank o IMTECH, Chandigarh for identifying the nove strains and assigning the MTCC numbers, References ‘Astrup, A., Breum, S. Toubro, P. Hein and P. Quaade, 1992. Gastroplasty for obesity: Long-term ‘Weight loss improved by vagotomy. In J. Obesity, 16: 269-277 [llaah,P. und KT. Krishna, 1987, Studies onthe Production of Phenyl Acetyl Cutbinol (PAC) from benzaldehyde by S.oetevisue. Ind. Drugs, 24: 192-195. Ellsiah,P. and K-T. Krishna, 1988, Effect of Acration and Alterning current on the production of Phenyl Acsty! Carbine by Saccharomyces cerevisiae. In. J Technol , 26: $09-S10. roger, D. and D. Erge, 1905. Analysis for PAC; Die Pharmazie, 20: 92, Kaur, M, and GS. 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