以地芽孢杆菌Geobacillus sp.GXS1基因组DNA为模板，PCR扩增获得α-淀粉酶基因，构建重组质粒pSE-amy，转化大肠杆菌诱导表达。SDS-PAGE电泳结果显示，有相对分子质量为58 ku的特异性蛋白得到表达。用金属镍亲和层析将重组蛋白进行分离纯化，并进行酶学性质研究。结果表明：重组酶的最适温度为65℃，最适pH 7.0，Km值为2.93 mg/mL，比活力为353.95 U/mg，Tm为75℃。金属离子Cu2+、Fe3+、Fe2+、Zn2+、Co2+、Hg2+、Ag+及金属鏊合剂EDTA对酶活有显著抑制作用， Mn2+、Ba2+对酶活有微弱的抑制作用， K+、Ca2+、Mg2+、巯基乙醇对酶有微弱的激活作用，而其它一些离子如Na+、Li+对酶活影响不大。经HPLC分析表明，重组α-淀粉酶催化淀粉的水解产物为葡萄糖、麦芽糖和麦芽三糖的混合物。%The gene encoding alpha-amylase from Geobacillus sp.GXS1 was amplified by PCR and expressed in JM109 (DE3). The recombinant protein was purified by nickel affinity chromatography.The purified enzyme presented as one protein band on SDS-PAGE with molecular weight of 58 u.The results showed that the optimum tempreture and pH of purified alpha-amylase were 65℃/7.0 respectively. The Vmax,Km,activity and Tm for soluble-starch were shown to be 2.93 mg/mL,353.95 U/mg,75 ℃. The activity of the enzyme was strongly inhibited by Cu2+, Fe3+,Fe2+,Zn2+,Co2+,Hg2+,Ag+and EDTA, while Na+,Li+had no effect on it. Mn2+,Ba2+had a little inhibition and K+,Ca2+,Mg2+had a little activation on its activity. The results of HPLC of products from starch by the amylase demonstrated that the enzyme can be used to produce maltotriose,maltose and glucose from starch.
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