采用重叠PCR方法在麦芽糖α-淀粉酶编码基因5’端添加地衣芽孢杆菌α-淀粉酶基因信号肽编码区,获得重组基因BlMa。重组基因与芽孢杆菌表达载体pHY-P43连接后直接转化枯草芽孢杆菌,获得重组质粒pHY-P43-BlMa。枯草芽孢杆菌淀粉酶基因缺陷株1A717被用作BlMa基因表达宿主菌,重组菌命名为Bacillus subtilis/pHY-P43-BlMa。酶活检测和SDS-PAGE电泳均显示,B.subtilis/pHY-P43-BlMa表达的重组麦芽糖淀粉酶(BlMa)全部分泌到培养液中。HPLC检测表明,BlMa催化可溶性淀粉水解产物主要为麦芽糖。对B.subtilis/pHY-P43-BlMa摇瓶发酵条件进行优化。获得优化发酵培养基配方:10%玉米淀粉,2.5%药媒,0.3%(NH4)2SO4,0.03%CaCl2,0.1%NaH2PO4,在优化条件下重组菌发酵酶活为5.9 U/mL。

The gene encoding maltogenic amylase was fused with signal peptide gene encoding amylase of Bacillus licheniformis by overlapping PCR.The fused gene(BlMa) was ligated with an expression vector of Bacillus,pHY-P43.B1Ma transforms Bacillus subtilis directly and results in the recombinant plasmid pHY-P43-BlMa.Bacillus subtilis 1A717 which is deficient in amylase was selected as expression host.The recombinant strain was designated as Bacillus subtilis / pHY-P43-BlMa.Enzyme activity assay and SDS-PAGE showed that recombinant maltogenic amylase of B.licheniformis(BlMa enzyme)expressed by B.subtilis / pHY-P43-BlMa was secreted into medium.High Performance Liquid Chromatography(HPLC) identified that major hydrolysate of starch catalyzed by BlMa enzyme was maltose.Culture condition of Bacillus subtilis / pHY-P43-BlMa in flask scale was optimized for production of BlMa enzyme,the optimized medium was: 10 % corn starch,2.5 % cotton seed protein,0.3 %(NH4)2SO4,0.03 % CaCl2 and 0.1 % NaH2PO4.The maximum enzyme activity under optimized condition reached 5.9 U/mL.