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

DOI：

中文关键词: α-葡萄糖苷酶 海栖热袍菌 NAD+ 酶学性质

英文关键词: α-glucosidase Thermotoga maritima NAD+ characterization

基金项目:江苏省自然科学基金项目(BK2006220)

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中文摘要:

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。

英文摘要:

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.

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