摘要
通过PCR方法从扣囊复膜孢酵母基因组DNA中克隆获得α-淀粉酶基因成熟肽编码区(SfA),插入乳酸克鲁维酵母表达载体pKLACl的α因子信号肽下游,构建重组表达载体pKLAClSfA。重组载体转化乳酸克鲁维酵母GG799,筛选获得表达α-淀粉酶SfA水平较高的重组菌。酶活检测和SDS-PAGE电泳检测均显示,重组菌分泌重组酶SfA到发酵液中。酶学性质研究表明:SfA最适温度为45℃,最适pH5.0,在pH4.5~5.5、50℃条件下保持稳定。Ca~(2+)等二价金属离子对SfA酶活有激活作用,EDTA强烈的抑制SfA活性。HPLC分析显示SfA水解糊精获得麦芽寡糖和少量葡萄糖,其中麦芽三糖是主要产物,占水解产物总量的52%。
The SfA gene that encoded a-amylase was amplified from the genomic DNA of Saccharomycopsis fibuligera and inserted into the downstream of the alpha-mating factor signal of the pKLAC1,an expression vector of Kluyveromyces lactis,to produce a recombinant pKLAC1-SfA recombinant plasmid.K.lactis GG799 was selected as an expression host.A transformant with a relatively high amylase activity was designated as K.lactis GG799/pKLACl-SfA YW07 and selected for further analysis.Results of enzyme activity analysis and SDS-PAGE showed that recombinant a-amylase of S.fibuligera(SfA)expressed by YW07 was secreted into medium,the molecular weight of the purified recombinant SfA was approximately 60 kDa.The optimum pH and temperature were 5.0 and 45°C,respectively.SfA was then kept stable at 50°C and pH 4.5~5.5.SfA was activated by most of the divalent cations such as Ca~(2+),and strongly inhibited by EDTA.High-performance liquid chromatography revealed that SfA hydrolyzed dextrin to form malt oligosaccharides and some glucose molecules.
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