褐色喜热裂孢菌Thermobifida fusca产麦芽糖α-淀粉酶基因的克隆表达及酶学性质研究

以T.fusca基因组DNA为模板,PCR分别扩增不包括和包括其基因前段信号肽的产麦芽糖淀粉酶的基因片段tfa和sptfa,并克隆至表达载体pSE380上,获得重组质粒pSE380-tfa和pSE380-sptfa,以大肠杆菌JM109为宿主细胞大量表达融合蛋白,利用金属镍亲和层析对菌株JM109/pSE380-tfa表达的α-淀粉酶进行纯化,SDS-PAGE显示纯化蛋白的分子量约为64ku,K m值为1.305mg/mL,最适反应温度为60℃,最适pH为7.0;检测到菌株JM109/pSE380-sptfa的培养基上清有相当一部分酶活。本研究麦芽糖α-淀粉酶与可溶性淀粉反应,经HPLC检测产物均为麦芽三糖、麦芽糖和葡萄糖的混合物。因此麦芽糖α-淀粉酶在生产高麦芽糖浆上起到了一定的作用。 

Study on gene cloning,expression and enzymatic characterization of a high maltose-producing alpha-amylase from Thermobifida fusca

The alpha-amylase genes lacking signal peptide and containing signal peptide were amplified from genomic DNA of T.fusca by PCR and cloned into expression vecor pSE380, respectively.Then constructed recombined plasmids pSE380-tfa and pSE380-sptfa were expressed in host cell of E.coli JM109.The recombinant enzyme of recombinant strains E.coli JM109 containing pSE380-tfa was purified by nickel affinity chromatography.The moleculer weight of purified protein was 64kua by the SDS-PAGE analyzing.The Km of purified enzyme was 1.305mg/mL, its optimum temperature and pH were 60℃ and 7.0, respetively.Extracellular amylase activity was observed in the supernatant fluid of recombinant strains E.coli JM109 containing pSE380- sptfa, then the supernatant fluid was concentrated by ultrafiltration.The results of HPLC showed that the hydrolysis products of soluble starch using this α-amylase all contained glucose, maltose and maltotriose.So this amylase could apply to manufacture high maltose syrup.