蜡状芽孢杆菌环糊精糖基转移酶的基因克隆表达·酶学特性及定点突变研究  

Cloning, Expression, Enzymatic Properties and Site-directed Mutation of Cyclodextrin Glycosyltransferase from Bacillus cereus

作　　者：花敬涵 杨静文 胡雪芹[1] 张洪斌 HUA Jing-han;YANG Jing-wen;HU Xue-qin(School of Food and Biological Engineering, Hefei University of Technology, Hefei,Anhui 230009)

机构地区：[1]合肥工业大学食品与生物工程学院,安徽合肥230009

出　　处：《安徽农业科学》2019年第5期110-115,126,共7页Journal of Anhui Agricultural Sciences

摘　　要：研究克隆、表达了蜡状芽孢杆菌来源的环糊精糖基转移酶(cyclodextrin glycosyltransferase,CGTase,EC 2.4.1.19)基因,研究了其酶学性质,并在此基础上构建突变菌株以探究关键氨基酸位点与产物特异性的关系。结果表明,重组CGTase的最大比活力达5 292 U/mL,分子量约为68 kDa,最适温度和pH分别为55℃和8.5;以淀粉为底物催化合成的主要产物是β-环糊精(β-CD);通过序列对比选取了第47位氨基酸(Ala)进行定点突变,构建了A47R、A47M、A47S、A47Y。研究发现活性区域中第47位氨基酸对于产物特异性和产量具有一定的影响,其中亲水性氨基酸更利于CDs(尤其β-CD)的合成,这为酶法合成β-CD的工业应用提供了方法。In this study, the cloning, expression and enzymatic properties of the cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus cereus were studied. Based on this, four mutant strains A47R, A47M, A47S, A47Y were constructed to explore the relationship between key amino acid sites and product specificity. The results showed that the maximum specific activity of recombinant CGTase was 5 292 U/mL, the molecular weight was about 68 kDa. The optimum temperature and pH were 55 ℃ and 8.5, respectively. The main product of starch-based catalytic synthesis was β-cyclodextrin (β-CD). Through sequence alignment, the 47 th amino acid (Ala) was selected for sequence-directed mutagenesis, and A47R, A47M, A47S, A47Y were constructed. It was found that the active-site residue 47 had certain influence on product specificity and yield, among which hydrophilic amino acids were more conducive to the synthesis of CDs (especially β-CD). This study provided a method for the industrial application of enzymatic synthesis of β-CD.

关 键 词：β-环糊精糖基转移酶 克隆 表达 定点突变 

分 类 号：S188[农业科学—农业基础科学]