海栖热袍菌α-葡萄糖苷酶基因aglA的克隆、表达及酶学性质  

Expression and Characterization of a Thermostable α-Glucosidase from Hyperthermophile,Thermotoga maritima

作　　者：王月宏[1] 段作营[1] 邵蔚蓝[2] 李华钟[1] 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214222 [2]南京师范大学生命科学学院,江苏南京210097

出　　处：《食品与生物技术学报》2011年第6期940-944,共5页Journal of Food Science and Biotechnology

基　　金：江苏省自然科学基金项目(BK2006220)

摘　　要：主要研究了在大肠杆菌中克隆和表达海栖热袍菌(Thermotoga maritima)的一个α-葡萄糖苷酶(TM1834)。通过PCR方法克隆编码T.maritima的一个α-葡萄糖苷酶基因aglA,构建重组质粒pHsh-AglA,电击转化Escherichia coli JM109,通过热激诱导高效表达。SDS-PAGE检测出蛋白相对分子质量约55 000,经热处理,阴离子交换层析和疏水层析纯化后的α-葡萄糖苷酶最适反应温度为90℃,最适反应pH为7.5,在pH 6.5～8.5,温度65～100℃之间酶活仍达到50%以上。在辅助因子NAD+,Mn2+和还原剂DTT的存在条件下达到最高酶活。A thermostable α-glucosidase gene aglA（TM1834） from a hyperthermophile bacterium,Thermotoga maritima,was expressed in Escherichia coli JM109 using the new heat-shock expression vector pHsh.The recombinant α-glucosidase aglA was purified by heat treatment,ion exchange chromatography,and hydrophobic chromatography,and had a molecular weight of 55 kDa as indicated by SDS-PAGE analysis.The optimum pH and temperature for this enzyme was pH 7.5 and 90 ℃,respectively.More than 50% was detected at pH values between 6.5 and 8.5 and temperatures between 65 ℃ and 100 ℃.The maximal aglA activity was observed in the presence of cofactor NAD＋,Mn2＋,and DTT.

关 键 词：Α-葡萄糖苷酶 海栖热袍菌 NAD+ 酶学性质 

分 类 号：Q78[生物学—分子生物学]