耐酸性高温α-淀粉酶突变基因在大肠杆菌中的表达及酶学性质研究

Expression of the Acid-resistant and Heat-stable α-amylase Mutation Gene in Escherichia coli and Study on Characterization of the Enzyme

利用表达载体pET-30a，实现了去信号肽的耐酸性高温α-淀粉酶突变基因amyd及未突变的高温”淀粉酶基因amy在大肠杆菌BL21（DE3）中的高效表达。经多步纯化，重组酶AMY及AMYD的比活分别达到312．7U／mg蛋白和354．6U／mg蛋白，纯化倍数分别为75．90和83．83，获得凝胶电泳条带单一的蛋白样品，经SDS-PAGE检测，AMY及AMYD酶分子质量均为63．5ku。重组酶AMY的最适温度80℃，最适反应pH为6．5，在温度低于90℃，反应pH5．5～7时，酶活较稳定。重组酶AMYD的最适温度80℃，最适反应pH为4．5，在温度低于90℃，反应pH4．0～6．5时，酶活较稳定。

In this research, the acid-resistant and heat-stable a-amylase mutation gene （amyd） and heatstable α-amylase gene （ amy ） were cloned into expression vector pET-30a constituting recombination vector pET-amdy and pET-- amy which were transformed into BL21 （DE3）. Positive transformant was cultivated and IPTG was added into culture to induce expression of the α-amylase. By multi-step purification, the specific activity of AMY and AMYD were 312.7U/mg and 357.6U/mg, they were purified to 75.90 and 83.83 folds respectivetly. Analysed by SDS-PAGE, the recombinant enzyme AMY and AMYD had a molecular mass of 63.5KDa. The optimum reaction temperature of AMY and AMYD were 80℃ and the 80% of their maximal activity were retained below 90℃. The AMY and AMYD were optimally active at pH 6.5 and pH 4.5 and showed stability at pH range of 5.5 to 7 and 4.0 to 6. 5.