摘要
利用PCR技术扩增编码γ-谷氨酰半胱氨酸合成酶(GSH )及谷胱甘肽合成酶(GSH )的基因,并进行了部分序列分析,分别构建了表达质粒pTrc-gsh 和pTrc-gsh ,它们在E.coliBL21中的稳定性很好。在E.coliBL21(pTrc-gsh )∶E.coliBL21(pTrc-gsh )=3∶1的情况下,谷胱甘肽的合成达3.31g/L,高于工程菌E.coliBL21(pTrc-gsh)催化合成的谷胱甘肽的量(2.51g/L)。利用E.coliBL21(pTrc-gsh )与E.coliBL21(pTrc-gsh )将γ-谷氨酰半胱氨酸的合成与谷胱甘肽的合成分步进行,谷胱甘肽的合成达3.78g/L,比利用E.coliBL21(pTrc-gsh)采用两步法合成的谷胱甘肽高42.1%,解决了GSH对GHI的反馈抑制,并降低了ADP对第二步反应的抑制程度。
The genes (gshⅠ,gshⅡ) for γglutamylcysteine synthetase (GSHⅠ) and glutathione synthetase (GSHⅡ) from E.coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively.The DNA fragments harboring gshⅡ and gshI were inserted into plasmid pTrc99A separately to get hybrid plasmids pTrcgshⅠ and pTrcghsⅡ.The hybrid plasmids pTrcgshⅠ and pTrcgshⅡ were maintained steadily in E.coli BL21.E.coli BL21(pTrcgshⅠ) together with E.coli BL21(pTrcgshⅡ) in the ratio of 3∶1 could produce more GSH than E.coli BL21(pTrcgsh) could alone.the synthesized GSH could be 3.78g/L when twostep faction is taken via E.coli BL21(pTrcgshⅠ) and E.coli BL21(pTrcgshⅡ).This is 42.1% higher than that which E.coli BL21(pTrcgsh) is used by delaying adding glycine.
出处
《西北大学学报（自然科学版）》
CAS
CSCD
北大核心
2003年第5期554-557,共4页
Journal of Northwest University（Natural Science Edition）
