MTSase和MTHase在Brevibacillus brevis中的克隆表达及应用研究

Heterologous Expression of MTSase and MTHase in Brevibacillus brevis and Its Application

为实现将来源于嗜酸热硫化叶菌(Sulfolobus acidocaldarius)ATCC 33909的麦芽寡糖基海藻糖合成酶(MTSase)和麦芽寡糖基海藻糖水解酶(MTHase)在短短芽孢杆菌(Brevibacillus brevis)中的重组表达,分别以重组质粒pET-24a(+)-treY和pET-24a(+)-treZ为模板PCR扩增得到MTSase和MTHase基因片段,通过与表达载体pSVN连接得到重组质粒,电转化表达宿主Brevibacillus brevis,成功构建重组菌Brevibacillus brevis/pSVN-treY和Brevibacillus brevis/p SVN-treZ。重组菌在TM培养基中发酵48 h,胞内酶活力分别达到MTSase 630.0 U/g(wet cell)、MTHase 170.0 U/g(wet cell)。对重组MTSase和MTHase进行酶转化实验,结果表明,当以质量浓度为100 g/L马铃薯淀粉为底物,酶转化温度为60℃,pH为5.5,加酶量为MTSase 80 U/g淀粉、MTHase 20 U/g淀粉,普鲁兰酶5 U/g淀粉和环糊精葡萄糖基转移酶(CGTase)15 U/g淀粉,反应48 h,海藻糖转化率达到80.2%。

In order to achieve efficient production of the MTSase and MTHase from(Sulfolobus acidocaldarius)ATCC 33909,gene sequence encoding MTSase and MTHase were PCR-amplified using recombined plasmid pET-24a(+)-treY and pET-24a(+)-treZ which were fused to expressional vector pSVN.The recombined plasmid was transfered into expression host Brevibacillus brevis by electroporation,The enzyme activity of MTSase and MTHase expression in Brevibacillus brevis were 630.0 U/g(wet cell)and 170.0 U/g(wet cell)after cultivated in shake flasks for 48 h.Also presented are the preliminary studies on the reaction conditions of conversion by the enzymes.The optimal conditions for the conversion are as following:the initial pH was 5.5,the reaction temperature was 60℃,initial potato starch concentration was 100 g/L,enzymes concentration were MTSase 80 U per gram of substrate、MTHase 20 U per gram of substrate、Pullulanase 5 U per gram of substrate and CGTase 15 U per gram of substrate,incubated for 48 h.Under this conditions,the yield of trehalose reached approximately 80.2%.