具有高谷胱甘肽合成活性重组大肠杆菌的构建及合成反应过程

以野生型大肠杆菌E.coliⅡ为宿主细胞，转化带有编码谷胱甘肽合成酶系的基因gshⅠ和gshⅡ的质粒pGH501，获得了一株谷胱甘肽合成活性、质粒稳定性和传代稳定性俱佳，并且能够重复使用的重组大肠杆菌E.coliⅡ\|1。该菌株经过甲苯处理后，能够在胞外积累4g/L左右的谷胱甘肽(GSH)。在合成反应体系中，提高L谷氨酸浓度可促进GSH合成，但L半胱氨酸浓度增大到20mmol/L后会抑制GSH的合成。根据GSH合成反应中能量辅因子的变化情况，提出E.coliⅡ\|1细胞控制的GSH合成反应机理：由谷胱甘肽合成酶(GSHⅡ)控制的第二步反应的能量供体是ADP而非ATP，该反应是整个GSH合成反应的限速步骤，高浓度ADP可能会抑制GSHⅡ的活性。在GSH合成反应体系中添加100mmol/L的L丝氨酸-硼酸钾混合物，可以有效地防止GSH的进一步降解，反应3 h后，GSH产量达到230mmol/L(约71g/L)。

CONSTRUCTION OF RECOMBINANT E. COLI WITH HIGH GLUTATHIONE BIOSYNTHETIC ACTIVITY AND THE BIOSYNTHETIC PROCESS

A recombinant strain E. coli II-1, which exhibited high glutathione (GSH) biosynthetic activity and high stability, was constructed by transforming plasmid pGH501 which contains gene gsh I and gsh II into a wild type strain E. coli II. 4 g/L GSH accumulated extracellularly by using toluene-treated cell. In GSH biosynthetic system, GSH production was improved by increasing the concentration of L-glutamate, while inhibited by L-cysteine if it's concentration was beyond 20 mmol/L. In GSH biosynthetic reaction, the apparent little consumption of L-glutamate and glycine was concluded experimentally to be that toluene-treated E. coli II-1 cells still contained high concentration of L-glutamate and glycine. According to the change of energy cofactor in the GSH biosynthetic process, a possible GSH biosynthetic mechanism controlled by E. coli II-1, was proposed: the energy donator of reaction catalyzed by glutathione synthetase (GSH-II) was ADP but not ATP, the reaction was rate-limited step within the whole GSH biosynthetic process, high concentration of ADP might inhibit the activity of GSH-II. Further degradation of GSH was prevented by the addition of 100 mmol/L L-serine and potassium borate mixture. In such case, 23.0 mmol/L (about 7.1 g/L) GSH accumulated at 3 h.