Abdul Razak, Che Nyonya

Basri, Mahiran

Salleh, Abu Bakar

Universiti Putra Malaysia Institutional Repository

Applied Sciences and TechnologiesM. Basri, C.NA Razak and A.B. 5allehEnzyme Catalyzed Stereo selective Reactions to Produce Useful Products*Faculty of Science and Environmental StudiesUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaE-mailofCorrespondingAuthor:mahiran@fsas.upm.edu.myKey words: enzyme, lipase, stereo selective, reactions, products.IntroductionThe increasing understanding of themechanism of drugs, pesticides andhormones interaction on a molecularlevel has led to the increasing aware-ness of the significance of chirality's asthe key to the efficacy and safety ofmany of these products. It is nowknown that in many cases only onestereo isomer (enantiomer) of thesesubstances is required for efficacy andsafety, and the other stereo isomer iseither inactive or may be toxic. Enan-tiomers can be prepared by separationof the racemes compounds by chemicalmethods. However, the laborious stepsand the expensive and sometimes toxicchemicals needed to produce enantio-mers meant much higher productioncosts. In this project, we are developingan alternative method, which is usingenzymes as biocatalysts to produceenantiomers via asymmetric synthesis.Besides being known for their favor-able properties, such as their mild andenvironmentally friendly reactions,enzymes are also known for their highsubstrate, region- and stereo-specificities. In this work, specificchital compounds such as 2-(4-chlorophenoxy) propanoic acid esters,the R-enantiomer is an important her-bicide, were synthesized by using vari-ous lipases. The enzyme was designedto acquire high enantioselectivity bychemical modification and immobili-zation. Changing the parameters suchas organic solvents and water activityoptimized the reaction systems.Materials and MethodsEight kinds of lipases from yeast, fungiand bacteria were used in the screeningstudies. (RS)-2-(4-chlorophenoxy)propanoic acids were obtained fromAldrich, Milwaukee, Wis., USA. Tet-radecanol was obtained from SigmaChemical Co., St. Louis, Missouri, andUSA. All other reagents were of ana-lytical grade. The organic solvents andsubstrates were dried over a molecularsieve 3°A before use (unless otherwisestated). The etherification reaction wascarried out as described by Basri et al.(1998). The remaining acid and alco-hol, and the ester formed, were isolatedby column chromatography (silica gel60, 70-230 mesh) as described by Basriet al., (In press). The optical purity ofremaining (Sj-acid was determinedfrom optical rotation readings using anAtago Polax-D polarimeter (Japan) at25°C. The amounts of remaining alco-hol and ester produced were deter-mined by gas chromatography (Shima-dzu GC-14A, Japan) with a flame-ionization detector using a 30 m capil-lary column type Restek (AT-IOOO).Lipase was modified with rnonometh-oxy-polyethylene glycol as describedby Basri etal. (1991,1995). Reductivealkylation of lipase was as described byBasri et al. (1997). Amid nation oflipase was carried out as describe byBasri et al. (1992). Lipase was immo-bilized to various polymer organicbeads as described by Basri et al.(1996a, 1996b, 1999).Results and DiscussionIn the stereoselective screening, 8 li-pases exhibited some stereobias with(Rl-acid. They are lipases from Can-dida rugosa. Rhizopus nivieus, Pseu-domonas roqueforti and Aspergillusniger, Rhizopus arrhizus and Mucusjavanicus as well as myceliallipasesfrom mesophilic Rhizopus oryzae(MroML) and Rhizopus rhizopodifor-mis (RrML). However, lipase fromCandida rugosa, which showed thehighest stereobias, was selected, as theenzyme to be used for further study asit also exhibited high activity as well asit is readily available. In the modifica-tion of lipase with various modifiers,the stereoselective activities of all themodified lipase preparations werehigher than the native enzyme. In-creasing the degree of modificationincreased the stereo selective activityas shown by the increase in the enanti-omeric excess (% ee). Immobilizationprocedure using various supports ex-hibited different enantioselective ac-tivities. The native lipases from differ-ent sources and the Candida rugosa-immobilized lipases were most activein solvents of log P values greater than2. However, certain lipases ie. thosefrom Pseudomonas roqueforti andMucus javanicus are active even inaceton. Generally, the enantioselectiveactivity of the lipases is relativelyhigher in non-polar solvents (log Pvalues equal or greater than 2). Hydro-phobic organic solvents appeared to bemore effective in inducing the esterifi-cation of the R-2-(4-chlorophenoxy)propanoic acid with tetradecanol com-pared to the S-2-(4-chlorophenoxy)propanoic acid. The initial water activ-ity (Aw) of the reaction systems mark-edly influenced the enantioselectivityof the enzyme. Both percentages ofester conversion and enantioselectivityshowed dependence on Aw in the re-action mixtures in organic solvents ofdifferent polarity. Optimum Aw for theenantioselective esterification was at0.529 for all solvents used.ConclusionsLipase from candida rugosa is the bestenzyme tested for enantioselectivesynthesis. Enzyme can be designed toacquire high stereo selectivity bychemical modification and/or immobi-lization. The enantioselective reactionwas enhanced when organic solvents ofhigher log P and optimum Aw wereused. These improved enzymes andreaction systems were used in theresolution of important race mates foruse in the chemical and agro industries.Benefits from the studyDesigner enzymes with improvedproperties were produced. This enzymeis of potential interest in the field of193UPM R~arch Repoft 1997·2000. 5«tion 2-Ex1~ndM1 Abstractsapplied enlymulogi\1. They flOdnu-mcruU\ applt~illion' in the field ormolecular separauon and cillilly,i,. Onepani~ular inlerC,1 i. in the reM,IUII"n"ran Importanl race male. a herbi~idc: roruse in the cbernical and agro indu\lrie\An optimized reacuon 'Y'lem wa.\dc:vclupo;d.Literature cited In the text1I ... n,M .• Amp"n. K .• W .... Yunu ••WMI' •• KUlik. CNA. and Sillkh. A.II.1'JlJ~. Synltle ••• of fillly ~.k''' by1'01~lhykM ,ly~ol·m .. lllfi~t1 111"''''' J.CIu-",. , .."hno. illllruhn"I"K_Y. M: 10·16" ...ri. M. Amp"n. K. Wiln Yunu ••WMI' •• KlIlilk. CNA. and Salkh. A.Il.11J'l2. Amllllnallun of lira'" IO-lIhhyd"'phublc 1IlIIft(lC',k'f'.J A",. OilCh ..",. S<Jc b'J(t.): ~79·~KlProject Publications InRefereed JournalsIh\fl. M .• Ampun. K .. Wlin Yunus,W.M.Z_. Kil/ak. C.N.A. And Salleh,A.IUIJ'J7. EIlIYlTllllic .ynltle,i. of fillly~1k'1\hy alk y lated hpa",. J. Mol",'. CIII.B: f:n:,-nlllll,·. 3: 171·176.lIalri. M .• Amp"n. K..Wan Yunu ••W.M.Z .. KUlik. CN.A .• ntI Salleh, A.II.IW6a. Entymlc 'ynltle.i. of fUlly~.I~"by hyd",phobic lira'" dcrivuliveli,nllll"'. hzed un orgilnic pul ymer heud,_ J.Am_ Oil (,h ..m. S<Jt'. 72(4): 407-411.Ihlri. M .. Ampun. K ..Wan Yunul.W.M.Z_. KUlik. C.N.A.And Salleh. A.O.IW{"'. ImlTll"'ili/llli"n of hpil'" fromCllntl..... ruau ... on ,ynlhelic polymerhelld. fur UIC in orgilnic .ynlhc,i. "f fUlly. ellen. J. Ch,.",. Tt'I'hlllJl. BilllrdlnlJl. 66:ItN·I73.II ... ". M .. Sallch. A_Il .. Ampon. K..WunYunu •• W.M.Z_ and KU/-lik. CN.A. 19'11.Mudlficlilion uf llra-e by pulyclhylenea:lycol. ilill(-IIltIly,IJ. 4: 313·317.194lIu,ri. M., Th'ng, II.L .• RUlUk. CN.A. lindSulleh. A.Il. IWK. Elfecl of reductiveIIlkylalion of Candida rugu,a lipase un illenanuoselecuve esterification reaction.Ann. New York Acad. Sd.II64: 192·1')11.1I11'ri. M .• Wona:. C_C.• Ahmad, M.B ..Itallik. CN.A and SlIlIch. A.B. 11J'I9.Immuhililalion of Iipa!<Con poly (n-vmyl-2·pyrrolidone.co.2.hydroxyclhylmethacrylate) hydrogel for lhe 'ynlhe.i.of butyl oleate, J. Am. 011 Chem. 511('.76(~): ~71·~77Project Publications InConference ProceedingsNonc.Graduate ResearchlIillila JU'lTllIni. 2(K)2. Hioorgllnic Synlhe,il(Ph.D.(. Univenili Pulra MlIluy,ill.Ai/ll lIamn.199M. Biourgunic Synlhe.i.IM.S_). Univenili Putra Malay.iD.Resellt'ch funded hy Ihe Inlen~ificalion of Researchin Priority ure", (IRPM' Supported by IKPA KM·7(I Wo·2()OO), Cycle IWo. Granl(1'}·()2.04·(K)2S.

Enzyme catalyzed stereo selective reactions to produce useful products

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Enzyme catalyzed stereo selective reactions to produce useful products

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