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

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

以野生型大肠杆菌E .coliⅡ为宿主细胞 ,转化带有编码谷胱甘肽合成酶系的基因gshⅠ和 gshⅡ的质粒 pGH50 1 ,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 .coliⅡ 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 2 0mmol/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 .coliⅡ 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 1 0 0mmol/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 3h后 ,GSH产量达到2 3 0mmol/L(约 7 1 g/L)。

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 20mmol/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 100mmol/L L\|serine and potassium borate mixture. In such case, 23.0 mmol/L(about 7.1 g/L) GSH accumulated at 3h.