环糊精葡萄糖基转移酶my20结构域删除突变体的表达和酶学性能  

Expression and Enzymatic Properties of Domain-Deleted Mutants of Cyclodextrin Glucosyltransferase my20

作　　者：孙腾腾 王伟[2] 孙晶晶[2] 江承程 郝建华 SUN Tengteng;WANG Wei;SUN Jingjing;JIANG Chengcheng;HAO Jianhua(Jiangsu Key Laboratory of Marine Biological Resources and Environment,Jiangsu Ocean University,Lianyungang 222005,China;Key Laboratory for Sustainable Utilization of Polar Fisheries,Ministry of Agriculture and Rural Affairs,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China)

机构地区：[1]江苏海洋大学江苏省海洋生物资源与环境重点实验室,江苏连云港222005 [2]农业农村部极地渔业可持续利用重点实验室,中国水产科学研究院黄海水产研究所,山东青岛266071

出　　处：《食品科学》2024年第20期154-160,共7页Food Science

基　　金：江苏省海洋生物资源与环境重点实验室开放课题基金项目(SH20211201);中国水产科学研究院基本科研业务费项目(2023TD71)。

摘　　要：本实验以海洋来源的环糊精葡萄糖基转移酶(cyclodextrin glycosyltransferase,CGTase)my20为研究对象,构建了D、E以及DE结构域截短突变体,通过pET-24a载体和宿主菌E.coli BL21(DE3)进行异源表达,并利用镍亲和层析柱纯化得到纯酶。以my20环化比活力为100%计,my20ΔD、my20ΔE、my20ΔDE相对环化活力分别约为197%、22%、17%,表明E结构域是CGTase催化环化反应的重要结构域,D结构域的存在可能降低了底物分子进入催化结构域的能力。my20为耐热酶,3种截短突变酶相较之下耐热性均有不同程度的降低,说明D、E结构域在CGTase耐热性方面具有重要的作用。my20在缺失了DE结构域后,催化产物中α-环糊精占比相较其他截短酶提升约8%,表明缺失DE结构域影响其产物的特异性。本实验结果可为CGTase通过结构域改造在工业生产方面发挥作用提供一定的理论基础。In this study,we constructed domain D,E and DE truncated mutants of a marine-derived cyclodextrin glucosyltransferase(CGTase)my20 and heterologously expressed these mutants using the pET-24a vector and E.coli BL21(DE3)as the host,and purified the expressed enzyme by nickel affinity column chromatography.The specific cyclization activities of my20ΔD,my20ΔE,and my20ΔDE were approximately 197%,22%,and 17%of that of my20,respectively,indicating that domain E is important for the catalytic activity of CGTase,and the presence of domain D might hinder substrate molecules from entering the catalytic domains.my20 was a heat-resistant enzyme,and the three truncated mutants showed different degrees of reduction in heat resistance,suggesting that domains D and E played an important role in the heat resistance of CGTase.The domain DE-deleted mutant showed an 8%increase in the percentage ofα-cyclodextrin(CD)in the catalytic product compared with the other truncated mutants,which suggested that the loss of domain DE affected the product specificity.The results of this study provide a theoretical basis for CGTase domain modification for its application in industrial production.

关 键 词：环糊精葡萄糖基转移酶 结构域 环化活力 酶学性质 

分 类 号：Q71[生物学—分子生物学]