地衣芽孢杆菌生麦芽糖α-淀粉酶的基因克隆与鉴定

Gene Cloning and Characterization of Bacillus licheniformis Maltogenic α-amylase

以地衣芽孢杆菌ATCC14580基因组DNA为模板，PCR扩增出1．7kb大小的麦芽糖淀粉酶基因amyM，克隆入表达载体pET-28a（＋），转化Escherichia．coliBL21（DE3），经IPTG诱导，测定麦芽糖淀粉酶酶活性．结果表明，麦芽糖淀粉酶基因amyM获得了活性表达，酶活力为3．797U／mL，SDS—PAGE电泳结果显示出相对分子质量约为67×10^3的特异性蛋白质条带．酶学性质分析表明，重组麦芽糖淀粉酶的最适反应温度为45℃，最适反应pH为6．5，在45℃以下的中低温环境保持稳定，且能在比较宽的pH范围内（pH4．5～8．5）稳定．

The gene amyM was amplified by the method of PCR with genomic DNA of Bacillus licheniformis ATCC14580 as template, and expressed in Escherichia coli BL21 （DE3） by yielding hybrid plasmid pET-28a-amyM. Induced with IPTG, the E. coli （DE3） harboring pET-28a-amyM successfully expressed the active maltogenic a-amylase, and the enzyme activity was 3.797 U/mL. The recombinant maltogenic a-amylase showed a molecular mass of 67×10^3 by SDS-PAGE. The recombinant enzyme showed an activity optimum at 45℃ and pH at 6.5. It was stable below 45℃ after incubated at pH 6.5 for 1 h and stable at pHs ranging from 4.5 to 8.5. Fig 3, Tab 1, Ref 16