地衣芽胞杆菌ATCC9945A中γ-聚谷氨酸降解酶基因的克隆、表达及降解性能鉴定

目的:验证在地衣芽胞杆菌ATCC9945A中存在着γ-聚谷氨酸降解酶基因(ywtD),为下一步解决在γ-聚谷氨酸微生物发酵合成过程中产物γ-PGA降解的技术性难题提供依据。方法:通过在pET-28b(+)大肠杆菌表达系统克隆表达地衣芽孢杆菌ATCC9945A中的ywtD基因,对诱导表达条件进行优化,采用SDS-PAGE和Western Blot方法检测目的蛋白的表达,并体外酶解实验验证其活性。结果:PCR扩增得到了一个1 245bp的基因片段,预期编码414个氨基酸,诱导表达后得到一个分子量大小约为45.6 kDa的表达产物。Western Blot分析结果表明ywtD基因得到了有效表达。体外酶解实验表明该表达产物具有降解γ-PGA的活性。结论:证明在地衣芽胞杆菌ATCC9945A中存在着γ-聚谷氨酸降解酶基因。

Cloning, Expression and Degradation of the γ- PGA Degrading Enzyme Gene from Bacillus licheniformis ATCC9945A

Objective:To confirm the presence of γ-PGA degrading enzyme gene,ywtD,in the γ-PGA producing bacterium Bacillus licheniformis ATCC9945A,and seek a better solution for the degration of γ-PGA during the late stationary phase could be solved.Method:ywtD was cloned and was expressed by prokaryotic expression system,pET-28b(+).Expressing conditions was optimized.The protein YwtD was expressed and analyzed using SDS-PAGE and Western Blot.At last,its degradation property was studied.Result:The ywtD gene coding region which includes 1 245bp and encodes 415 amino acids was obtained from bacterium Bacillus licheniformis ATCC9945A by PCR,and a 45.6 kDa polypeptide,YwtD,was successfully expressed in E.coli BL21(DE3).YwtD was purified and the study on degradation property revealed that YwtD could hydrolyzes γ-PGA.Conclusion:The above result suggested the presence of γ-PGA degrading enzyme gene in the γ-PGA producing bacterium Bacillus licheniformis ATCC9945A.