谷氨酸棒杆菌海藻糖合成酶基因的分子进化及其酶学性质的研究

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1、广西大学硕士学位论文谷氨酸棒杆菌海藻糖合成酶基因的分子进化及其酶学性质的研究姓名：朱绮霞申请学位级别：硕士专业：微生物学指导教师：黄日波20080701单位，而F244L和F244W均为6．5，均下降了近1．5个单位；突变酶的热稳定性均有不同程度的提高，其中F244Y、F244W和A288G的Tm值比野生型的提高约1℃；F244L提高约2。C，而F244C热稳定性提高了近4。C。通过分析和比较这些突变酶的性质变化，可为今后研究海藻糖合成酶的结构和功能的关系提供有用的数据和信息，为进一步对海藻糖合成酶的分子改造提供理论依

2、据。关键词：谷氨酸棒杆菌海藻糖合成酶分子进化ⅡSTUDIES0NMOLECULEEVOLUTIONANDTHEENZYMEPRoPERTIESoFTHETREHALOSESYNTHASEFRoMcoRYNEBACTERlUMGLUTAMlCUMABSTRACTTrehalosesynthaseisthekeyenzymeinenzymaticprocesstoproducetrehalose．Itcancatalyzemaltosebondedinan0【，伐一1，4·glycosidiclinkagetoproduc

3、etrehalosewitha，0【-1，4-glycosidiclinkage．Recently,Trehalosehasbeenwidelyattendedbecauseofit’Snon—specialprotectingfunctiononbigmoleculeofbiology．Thescientistsfrommanycountrieshavebeenengaginginstudyingonthetechnologyofproductionandapplicationtoreduceit’Scostofpr

4、oduction．Theexistingtrehalosesynthasehavesomedisadvantages：thetransformefficiencyisnotenoughhigh；Thethermostabilityislow；ThereactiontimeislongandSOon．Therefore，itisnecessarytoperformmutagenesisontrehalosesynthasetoimprovetheindustrialcapability．Inthispaper’treha

5、losesynthasewasevolvedbyhalf-rationaldesign．Thethree．dimensionalmodelofTreSfromCorynebacteriumglutamicumwasconstructedbasedonthecrystalstructureoftrehalulosesynthaseMutBfromPseudomonasmesoacidophilaMX-45ontheSWISS—MODELIntemet．Theenergyofprimalstructurewasoptimi

6、zed．Combinedwithanalyzingtheconservedregionoftrehalosesynthaseamiosequencefromdifferentorigin．TheconservedamioacidR245andE289werepredictedlikelyasactivesiteandweremutated．Furthermore，thepositionF244、D247andA288nearthetwosites(R245andE289)wereperformedsite—mutage

7、nesis．TheresultsindicatedIIIthattheactivitywaslostafterR245andE289wereconvertedtoother19kindsofamioacidsandthemutantsD247EandD247Nwerealsoshownnoactivities．Thereasonofactivitylostprobablywasthesepositionswereimportanttomaintainthestructurestabilityoftheprotein．a

8、ndtheypossiblywerecrucialresiduesoractivesitesoftheenzyme．ThespecificactivityofthemutantsF244C，F244L，F244W，F244YandA288Gweredecreasedlargelyandreducedto38％，24％，62％，64