Cloning, sequencing, and overexpression in Escherichia coli of the Enterobacter sp. Px6-4 gene for ferulic acid decarboxylase

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1、ApplMicrobiolBiotechnol(2011)89:1797–1805DOI10.1007/s00253-010-2978-4BIOTECHNOLOGICALLYRELEVANTENZYMESANDPROTEINSCloning,sequencing,andoverexpressioninEscherichiacolioftheEnterobactersp.Px6-4geneforferulicaciddecarboxylaseWenGu&XuemeiLi&JingwenHuang&YanqingDuan&ZhaohuiMeng&

2、Ke-QinZhang&JinkuiYangReceived:28September2010/Revised:17October2010/Accepted:18October2010/Publishedonline:18November2010#Springer-Verlag2010AbstractFerulicaciddecarboxylase(FADase)cancata-recombinantFADasewaspurifiedasaproteinofca.lyzethetransformationofferulicacidinto4-v

3、inylguaiacol23kDawithanoptimalactivityatpH4.0and28°C.Theviadecarboxylationinmicroorganisms.Inthisstudy,apurifiedFADasecouldconvertferulicacidto4-vinylgeneencodingFADasewasfirstisolatedfromthebacteri-guaiacoleffectively,anditshydrolyticactivitycouldbe2+2+umEnterobactersp.Px6

4、-4usingdegenerateprimersandainhibitedbyCu(99%)andHg(99.5%).Aphylogeneticgenomewalkingtechnique.TheputativeencodinggeneanalysisoftheFADaseproteinfrombacteriarevealed(fad)ofFADaseconsistsof507-bpnucleotides,codingaseveraldifferentclades.Ourresultprovidedabasisforpolypeptideof

5、168aminoacidresidues.Inaddition,afurtherstudiesoftheferulicacidtransformationpathwayputativegeneencodingthetranscriptionalregulatorwasandforenhancedproductionofvanillininthefuture.identifiedfromtheupstreamofthefadgene.ThededucedpeptidesequenceoftheFADasefromEnterobactersp.K

6、eywordsEnterobactersp.Px6-4.FerulicacidPx6-4showeda51.2–53.3%sequenceidentitytodecar-decarboxylase.Cloning.Expression.Enzymeanalysisboxylasesfromotherbacteria.Thegenefadwassuccess-fullyexpressedinEscherichiacoliBL21,andtheIntroductionW.Gu:J.Huang:Z.Meng:K.-Q.Zhang:J.Yang(*)

7、FerulicacidisanextremelyabundantcinnamicacidderivativeLaboratoryforConservationandUtilizationofBio-resources,YunnanUniversity,foundinthecellwallofwoods,grasses,andcornhullsKunming650091,People’sRepublicofChina(Rosazzaetal.1995;Ishii1997;Oosterveldetal.2000).Itise-mail:jinku

8、iyun@yahoo.com.cncovalentlylinkedtovariouscarbohydratesasaglycosidicW.Gu:J.Huang:Z