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Screening of stereoselective lipase and its application in resolution of menthol

立体选择性脂肪酶的筛选及其在薄荷醇拆分中的应用



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ChineseJournalofBioprocessEngineering
Vol.11No.1
Jan.2013
doi:10.3969/j.issn.1672-3678.2013.01.001
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Screeningofstereoselectivelipaseanditsapplication
inresolutionofmenthol
XUGang,LIMu,SUNMiaomiao,WUJianping,YANGLirong
(DepartmentofChemicalandBiologicalEngineering,ZhejiangUniversity,Hangzhou310027,China)
Abstract:AstrainwithstereoselectivehydrolyingLmenthylpropionatewasscreenedbydoubleplate
screeningmethod.ItwasidentifiedasPseudomonasalcaligenesby16sDNA sequencing.Thehigh
enantiomericexcess(e.e.p>99%)anddiastereomericexcess(d.e.p>90%)wereachievedwhenthe
conversionofLmenthylpropionatereached50% at35℃.Theresultsshowedthattheefective
biocatalysthadapotentialindustrialapplication.
Keywords:lipase;Lmenthol;screening;Pseudomonasalcaligenes;stereoselectivity
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[1] ZhengGaowei,YuHuilei,ZhangJiandong,etal.Enzymatic
productionoflmentholbyahighsubstrateconcentrationtolerable
esterasefromnewlyisolatedBacilussubtilisECU0554[J].Adv
SynCatal,2009,351:405414.
[2] TaniK,YamagatT,AkutagawaS.Highlyenantioselective
isomerization ofprochiralalylamines catalyzed by chiral
diphosphinerhodium(I)complexes:preparationofopticaly
activeenamines[J].JAmChemSoc,1984,106:52085217.
[3] YuLijuan,XuYan.Highlyenantioselectivehydrolysisofdl
menthylacetate to lmentholby wholecel lipase from
BurkholderiacepaciaATCC25416[J].JMolCatalB:Enzymatic,
2007,47(3):149154
[4] VorlovaS,BornscheuerUT,GatfieldI,etal.Enantioselective
hydrolysisofD,L(±)menthylbenzoatetoL(-)mentholby
recombinantCandidarugosalipaseLIP1[J].AdvSynCatal,
2002,344(10):11521155.
[5] GaoRenjun,FengYan.Cloning,purificationandpropertiesofa
hyperthermophilicesterasefromarchaeonAeropyrumpernixK1
[J].JMolCatalB:Enzymatic,2003,24:18.
[6] BradyaD,ReddyaS.Biocatalyticenantiomericresolutionofl
mentholfrom an eightisomeric mentholmixture through
transesterification[J].JMolCatalB:Enzymatic,2012,75:110.
[7] LorenzP,LiebetonK,NiehausF.Screeningfornovelenzymesfor
biocatalyticprocesses:accessingthemetagenomeasaresourceof
novelfunctionalsequencespace[J].CurOpinBiotechnol,2002,
13:572577.
[8] BaharumSN,SalehAB,RazakCNA.Organicsolventtolerant
lipasebyPseudomonassp.strainS5:stabilityofenzymeinorganic
solventandphysicalfactorsafectingitsproduction[J].Ann
Microbiol,2003,53:7583.
[9] BozogluF,Swaisgood H E,AdamsD M.Isolation and
characterizationofanextracelularheatstablelipaseproducedby
PseudomonasfluorescensMC50[J].AgricFoodChem,1984,32:
26.

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