枯草芽孢杆菌AmyX基因信号肽性能优化研究

Studies on optimizing the signal peptide of AmyX protein from B.subtilis

从枯草芽孢杆菌1A 747菌株基因组克隆Am yX基因信号肽序列,构建枯草芽孢杆菌分泌表达载体pYGAm yX,转化枯草杆菌W B 700得W B 700(pYGAm yX)。采用重叠PCR技术,将Am yX基因信号肽突变,使其N端正电荷增加,H区疏水性降低,利用突变后信号肽序列构建枯草杆菌分泌表达载体pYGMUT,转化枯草杆菌W B 700得W B 700(pYGMUT)。对W B 700(pYGAm yX)和W B 700(pYGMUT)的表达产物进行检测,研究Am yX基因信号肽突变对枯草杆菌蛋白分泌途径的影响。结果表明,W B 700(pYGAm yX)和W B 700(pYGMUT均能表达β-半乳糖苷酶,W B 700(pYGAm yX)的最高胞外酶活性达2 300 U,W B 700(pYGMUT)的最高酶活性达5 200 U。结果表明Am yX基因信号肽突变后构建的表达载体pYGMUT分泌表达β-半乳糖苷酶明显提高,说明β-半乳糖苷酶通过枯草杆菌T at途径分泌表达。

The gene sequence of the signal peptide AmyX was cloned from B. subtilis 1A747,and the pYGAmyX expression system was constructed with the signal peptide AmyX,then it was transformed into WB700 （WB700（pYGAmyX））. The other expression system pYGMUT-,which was applied to the AmyX mutation gained by overlap PCR,was transformed into WB700（WB700（pYGMUT）） in order to study the effect of Tat pathway in Bacillus subtilis by the direct-mutation of the signal peptide AmyX. The result showed the β-galactosidase was secreted into periplasmic successfully,the highest activity level of WB700（ pYGAmyX） reached 2 300 U,and that of WB700（pYGMUT） reached 5 200 U. This testified the expression system using the rebuilding signal peptide secreted β-galactosidase by Tat pathway.