Bacillus stearothermophilus麦芽糖淀粉酶在Bacillus subtilis中的重组表达及发酵优化

Recombinant Expression and Fermentation Optimization of B.stearothermophilu Maltogenic Amylases in Bacillus subtilis

为了实现Bacillus stearothermophilus嗜热脂肪芽孢杆菌来源的麦芽糖淀粉酶基因amyM(EC 3.2.1.133)在枯草芽孢杆菌中的重组表达,以opt-amyM/T质粒为模板进行PCR扩增得到目的基因,与表达载体pHY300PLK进行重组连接后转入宿主菌Bacillus subtilis CCTCC M 2016536中进行表达。在TB培养基中发酵培养48 h,麦芽糖淀粉酶酶活达到250.7 U/mL。继续对重组菌氮源种类及复配、氮源质量浓度、碳源质量浓度等摇瓶发酵条件进行优化,确定其最佳产酶条件为:复合氮源为酵母浸膏25 g/L和大豆蛋白胨5 g/L、葡萄糖5 g/L、培养基初始pH 6.5、最适培养温度41℃;在此条件下麦芽糖淀粉酶酶活可达396.7 U/mL,是优化前的1.6倍。在此基础上进一步对麦芽糖淀粉酶进行酶学性质进行测定:麦芽糖淀粉酶最适温度为60℃,最适pH为5.5,半衰期为325 h,Km为0.95 g/L,比活为2646 U/mg。

To obtain the recombinant expression of gene amyM for maltogenic amylases from Bacillus stearothermophilus in Bacillus subtilis,the target gene was PCR-amplified using plasmid opt-amyM/T as template,and ligated with the expression vector pHY300PLK,then transformed into the expression host Bacillus subtilis CCTCC M 2016536.The activity of maltogenic amylases reached 250.7 U/mL after cultivated in TB culture for 48 h.By optimizing the types of nitrogen source and the complex of it,the concentration of nitrogen source and carbon source etc.The optimal fermentation condition was determined as:25.0 g/L yeast extract and 5.0 g/L soy peptone of complex nitrogen source,5.0 g/L of glycerin,initial pH of medium 6.5 and the optimal temperature was 41℃.Under optimal conditions,the production of maltogenic amylases reached 396.7 U/mL,which was 1.6 times of that before optimization.Further experiments were determined the enzymatic properties of maltogenic amylase:the optimum temperature of maltogenic amylase was 60℃,the optimum pH was 5.5,the half-life was 325 h,respectively.The Km was 0.95 g/L and the specific activity is 2646 U/mg.