γ-CGTase酶学性质及产物特异性影响因素  

Expression,characterization and product specificity of gamma-CGTasefrom Bacillus sp.

作　　者：郑丹妮 柏玉香[1,2] 纪杭燕 李晓晓 王禹 蔣彤 金征宇[1,2] ZHENG Danni;BAI Yuxiang;JI Hangyan;LI Xiaoxiao;WANG Yu;JIANG Tong;JIN Zhengyu(School of Food Science and Technology,Jiangnan University,Wuxi 214122,China;State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]食品科学与技术国家重点实验室(江南大学),江苏无锡214122

出　　处：《食品与发酵工业》2020年第5期38-45,共8页Food and Fermentation Industries

基　　金：国家自然科学基金(31701644);江苏省自然科学基金(BK20170184);现代农业重点及面上项目(BE2018304)。

摘　　要：将γ-环糊精葡萄糖基转移酶(γ-CGTase)的特有区域作为判断依据,在美国国家生物信息中心(National Center for Biotechnology Information,NCBI)数据库中,通过序列比对及生物信息学分析,筛选出一种新型基因,并将其克隆表达得到一种酶。利用高效液相色谱-质谱联用进行产物鉴定,表明该酶为γ-CGTase;酶分子质量大约为80 kDa,最适温度50℃,最适pH 10.0;无α-环糊精生成;甲醇、乙醇、环己烷、十二醇激活γ-CGTase活力,异丙醇和正丁醇抑制γ-CGTase活力;反应时间、淀粉质量浓度及乙醇体积分数均会影响γ-CGTase的产物特异性。HPLC结果显示,在10%的乙醇最终体积分数条件下,γ-CGTase催化产物中γ-环糊精的比例由40.11%增加至78.20%,专一性提高了94.96%。该研究提供了一种新型的γ-CGTase,为提高γ-CGTase产物特异性提供研究基础。Based on the specific region ofγ-cyclodextrin glycosyltransferase(γ-CGTase),a gene was explored from the NCBI database by sequence alignment and biological information analysis.The gene was cloned and expressed,and a recombinant enzyme was obtained.The reaction product was identified by high performance liquid chromatography-mass spectrometry(HPLC-MS),which indicated that the enzyme was aγ-CGTase.The molecular weight of the recombinant enzyme was about 80 kDa,the optimum temperature was 50℃,and the optimum pH was 10.0.There was no detectableα-cyclodextrin production.The enzyme activity was activated by methanol,ethanol,cyclohexane and dodecanol,but was inhibited by isopropanol and butyl alcohol.The product specificity of the enzyme was affected by the reaction time,the concentration of starch and the volume fraction of ethanol.As indicated by the HPLC analysis result,the proportion ofγ-cyclodextrin was increased from 40.11%to 78.20%and the specificity increased by 94.96%in the presence of 10%ethanol.This research could not only provide a novelγ-CGTase but also provide a research basis for changing the product specificity ofγ-CGTase.

关 键 词：γ-环糊精葡萄糖基转移酶 酶学性质 乙醇 产物特异性 

分 类 号：Q55[生物学—生物化学]