Thermococcus sp.B1001环糊精酶的重组表达及在麦芽七糖制备中的应用  

Recombinant Expression of Cyclodextrinase from Thermococcus sp.B1001 and Application in Preparation of Maltoheptaose

作　　者：武权 王蕾[1,2,3] 吴敬 陈晟[1,2,3] Wu Quan;Wang Lei;Wu Jing;Chen Sheng(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi,214122;Key Lab of Ministry of Education for Industrial Biotechnology,School of Biotechnology,Jiangnan University,Wuxi,214122;International Joint Laboratory on Food Safety,Jiangnan University,Wuxi,214122)

机构地区：[1]江南大学,食品科学与技术国家重点实验室,无锡214122 [2]江南大学生物工程学院,工业生物技术教育部重点实验室,无锡214122 [3]江南大学,食品安全国际合作联合实验室,无锡214122

出　　处：《基因组学与应用生物学》2021年第2期671-677,共7页Genomics and Applied Biology

基　　金：国家重点研发项目(2019YFA0706900);国家自然科学基金项目(31571776);国家杰出青年科学基金项目(31425020)共同资助。

摘　　要：成功构建pET-24a-tscd质粒,实现Thermococcus sp.Strain B1001来源的环糊精酶(TsCDase)在Escherichia coli BL21(DE3)中表达。通过热处理和镍柱分离对重组TsCDase进行纯化。酶学性质研究表明,重组Ts CDase的比活为1208.04 U/mg,最适温度为90℃、最适pH值为5.5。重组酶TsCDase在85℃、90℃、95℃条件下的半衰期分别为180、120、30 min。酶转化研究表明,以80 g/Lβ-环糊精为底物,当酶转化温度为90℃、反应pH值为5.5~6.0,加酶量为25 U/g,反应时间为4 h时,麦芽七糖产率为81.19%和85.95%,七糖占产物麦芽寡糖的比例为95.24%和92.92%。本研究结果为工业化制备麦芽七糖奠定良好基础。The cyclodextrinase from Thermococcus sp.Strain B1001(TsCDase)was expressed in Escherichia coli BL21(DE3),which carried the plasmid pET-24 a-tscd.The recombinant TsCDase was purified by Ni2+-NTA affinitychromatoragraphyafter heat treatment.Then the recombinant TsCDase was characterized in detail.The specific activity of recombinant enzyme was 1208.04 U/mg.The optimal temperature was 90℃and the optimal pH was value 5.5.The half-life of recombinant TsCDase at 85℃,90℃,95℃was 180,120,30 minutes,respectively.Furthermore,the condition for maltoheptaose preparation by the recombinant TsCDase was investigated.The yield of maltoheptaose was 81.19%and 85.95%,accounting for 95.24%and 92.92%of all maltooligosaccharides,when the following conditions were adopted:a dosage of 80 g/Lβ-cyclodextrin(β-CD),pH 5.5 and 6.0,90℃,25 U TsCDase per gramβ-CD,incubated for 4 hours.Our research provides a basisfor the industrial production ofmaltoheptaose.

关 键 词：热球菌 环糊精酶 Β-环糊精 麦芽七糖 麦芽寡糖 

分 类 号：TQ929[轻工技术与工程—发酵工程]