用PCR方法扩增扣囊复膜孢酵母CICIM Y1037菌株真菌α-淀粉酶基因成熟肽编码区(SfA),插入表达载体pPIC9K。重组质粒pPIC9K-SfA转化巴斯德毕赤酵母GS115,筛选获得淀粉酶活力相对最高的重组菌Pichia pastoris GS115/pPIC9K-SfAmy LZ08。SDS-PAGE电泳分析纯化获得的重组酶SfA,结果显示重组酶分子质量为61 kDa左右。SfA最适反应温度为45℃、最适pH为4.5,是一种酸性α-淀粉酶。Ca2+对SfA的热稳定性有促进作用,重组酶在含5 mmol/L Ca2+,pH 4.5的溶液中,55℃保温4 h酶活仍保留80%以上。SfA水解玉米淀粉获得麦芽寡糖和少量葡萄糖,其中麦芽糖和麦芽三糖为主要产物,分别占水解物的37%和39%。重组酶SfA在麦芽三糖和麦芽寡糖糖浆生产中有一定的应用潜力。

SfA gene encoding α-amylase was amplified from the genomic DNA of Saccharomycopsis fibuligera CICIM Y1037 and inserted into pPIC9K,an expression vector of Pichia pastoris,to produce a recombinant pPIC9KSfA plasmid.P.pastoris GS115 was selected as an expression host.A transformant with a relatively high amylase activity was designated as P.pastoris GS115 / pPIC9K-SfA LZ08 and selected for further analysis.The molecular weight of the purified recombinant SfA was approximately 61 kDa,which was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The optimum pH and temperature were 4.5 and 45 ℃,respectively.Therefore,SfA is possibly an acid-resistant amylase.SfA was then kept stable at 55 ℃ and pH 4.5 for 4 h in the presence of 5 mmol / L Ca2 +,and retained more than 80% of its activity.Results of high-performance liquid chromatography revealed that corn starch was hydrolyzed by SfA to form malt oligosaccharides and some glucose molecules.Maltose and maltotriose were the main products that accounted for up to 37% and 39% of the hydrolysates,respectively.Therefore,the recombinant SfA gene could be potentially applied in maltotriose and malto oligosaccharide syrup production.