大黄欧文氏菌蔗糖异构酶控制产物特异性基序的定点突变  

Site-directed Mutation in a Product-Specificity-Controlling Motif of Sucrose Isomerase from Erwinia rhapontici

作　　者：周兴[1,2] 韦星明[2] 杨祥开[2] 郑元涛[2] 庞浩[3] 许黎明[2] 黄日波[1,3] 

机构地区：[1]广西大学生命科学与技术学院,南宁530005 [2]广西科学院生物研究所,南宁530007 [3]生物质能源酶解技术国家重点实验室,广西科学院,南宁530007

出　　处：《基因组学与应用生物学》2011年第5期556-563,共8页Genomics and Applied Biology

基　　金：国家科技支撑项目(2007BAD75D06);广西科技攻关项目(桂科攻0895003-4-1)共同资助

摘　　要：大黄欧文氏菌(Erwinia rhapontici)蔗糖异构酶催化蔗糖异构为异麦芽酮糖和海藻酮糖,具有一个可能控制产物特异性的325RLDRD329基序。本研究以定点突变方法对该基序的带电荷氨基酸进行突变,共构建R325D、R328A、R328D、R328Q和D329N5个突变体。通过对突变体的酶学特性及突变体转化蔗糖的产物组成分析,结果显示所构建突变体的Km值上升约2～5倍,比活力下降至野生型SI比活力的11.8%～25.3%。HPLC分析显示Arg325和Arg328分别突变为Asp,导致产物中异麦芽酮糖/海藻酮糖的比例从6.93分别降至0.96和2.92,并伴随一个未知寡糖出现。Arg328突变为Ala和Gln同样导致反应产物中海藻酮糖比例上升,异麦芽酮糖比例下降。但是突变体D329N反应产物比例没有变化。以上结果表明325RLDRD329基序对大黄欧文氏菌蔗糖异构酶的酶活具有重要作用,并对酶的产物特异性产生影响。本研究结果将为该酶的作用机理研究奠定基础。Sucrose isomerase（SI） from Erwinia rhapontici isomerizes surcose to produce isomaltulose and trehalulose.It has a motif（325RLDRD329） which may control the product specificity.In this study,the charged amino acid residues of the motif have been site-directedly mutated,and 5 mutants（R325D,R328A,R328D,R328Q and D329N） were obtained.By analyzing the enzymatic characteristics of the mutants and the product composition of sucrose conversion catalysed by the mutants,we found that the Km of these mutants increased by about 2~5 folds and the specific activity decreased to 11.8%~25.3% that of wild-type enzyme.The replacement of residues Arg325 and Arg328 respectively to residue Asp resulted in a decrease of the isomaltulose/trehalulose ratio from 6.93 to 0.96 and 2.92 respectively in the reaction products and the emergence of an undetermined oligosaccharide by HPLC assay.Mutation of residue Arg328 with Ala and Gln respectively also increased the percentage of trehalulose and reduced the percentage of isomaltulose in the sucrose converted product.However,the ratio of isomaltulose/trehalulose formation by D329N mutant was similar to that of wild-type enzyme.Based on our results,we believe that the 325RLDRD329 motif is essential for the activity of SI from E.rhapontici and also affects the product specificity of this enzyme.Our results will provide a foundation for understanding the mechanism of SI.

关 键 词：蔗糖异构酶 定点突变 产物特异性 大黄欧文氏菌 

分 类 号：Q78[生物学—分子生物学]