海栖热袍菌木聚糖酶b的克隆和表达

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1、维普资讯http://www.cqvip.com应用与环境生物学报2002，8(3)：280～284ChinJApplEnvironBiol=ISSN1006-687X2oo2-25极端嗜热茵海栖热袍菌木聚糖酶B的克隆和表达木江正强一李里特林清MOHAMMADMainulAhsan(中国农业大学食品学院北京100083；食品综合研究所酵素利用研究室筑波305日本)摘要极端嗜热菌海栖热袍菌MSB8(ThermotogamaritimaMSB8)能够利用木聚糖代谢，并具有两个产木聚糖酶的基因．本研究首次克隆和在大肠杆菌中表达海栖热袍菌MSB8的第2个木聚糖酶基因即xynB基因．以基因组DNA为

2、模板，根据xynB基因的全序列设计了两对引物，在合适的PCR反应条件下有效地扩增出了xynB基因片段．选用pET28a(+)表达载体，Ncol—HindⅢ酶切位点并编码了羧基端6×His标签的阳性克隆表达成可溶且具有生物活性的蛋白质．xynB结构基因由1O44对碱基组成，编码347个氨基酸．根据已知木聚糖酶蛋白质氨基酸的同源性分析，XynB与sp．strainFjSS3一B．1的XynA的同源性最大，为85％，与Tneapolitana的XynB同源性次之，为82％，与其它木聚糖酶的同源性小于43％．另外，XynB属于F／IO族木聚糖酶，且含有单一功能区．表3图3参12关键词海栖热袍菌；木

3、聚糖酶B基因；克隆；表达CLCQ78：0939．1lCLONINGANDEXPRESSIoNoFANxynBGENEINj5『．C(L，FRoMTHEEXTREME—THERMoPHILIC日EGIAMAR1JIANGZhengqiang一，LILite，HAYASHIKyoshi，MOHAMMADMainulAhsan(FoodCollege，ChinaAgriculturalUniversity，Beijing100083，China)(EnzymeApplicationsLaboratoryofNationalFoodResearchInstitute，Tsukuba305，Japa

4、n)AbstractThehyperthermophilicbacterium，Thermotogamaritima，isabletometabolizexylan，andpos—sessestwodifferentxylanasegeneswhichencodexylanaseAandB，respectively．ThexynBgenewasisola—tedfromagenomeDNAofmaritimaMSB8．clonedandexpressedinEscherichia．coliforthefirsttimeinthispaper．Twoprimersweredesignedon

5、thebasisofthenucleotidesequenceofxynBgene．xynBgenefragmentsweresuccessfullyamplifiedbyPCRreactionsunderthesuitableconditions．E．coliBL21compe—tentcellsweretransformedbyelectroporationwiththeligatedxynB—pET28containingNcoI—HindllIsite．Thetranslatedproteinwassolublewithxylanaseactivity，andtheC—termin

6、alHis—tagwasintroducedintherecombinantenzyme．ThexynBgeneconsistsof1044bp，encodingaproteinwith347aminoacidresidues．ThehomologyanalysisofthededucedaminoacidsequencesshowedthattheXynBwasmostclosetoXynAfromspstrainFjss3一B．1(85％identity)，andshowedthesecondhighestdegreeofsimilarity(82％i—dentity)withXynB

7、fromneapolitana．Theotherxylanasesshowedtheidentitylessthan43％．These—quenceofthegeneshowedthatitencodedafamily10xylanasewithasingledomain．Tab3，Fig3，Ref12KeywordsThermotogamaritima；xynBgene；cloning；expressionCLCQ78