Highly Efficient Synthesis of Glutathione via a Genetic Engineering Enzymatic Method Coupled with Yeast ATP Generation

Abstract

Glutathione is a tripeptide compound with many important physiological functions. A new, two-step reaction system has been developed to efficiently synthesize glutathione. In the first step, glutamate and cysteine are condensed to glutamyl-cysteine by endogenous yeast enzymes inside the yeast cell, while consuming ATP. In the second step, the yeast cell membrane is lysed by the permeabilizing agent CTAB (cetyltrimethylammonium bromide) to release the glutamyl-cysteine, upon which added glutathione synthetase converts the glutamyl-cysteine and added glycine into glutathione. The ATP needed for this conversion is supplied by the permeabilized yeast cells of glycolytic pathway. This method provided sufficient ATP, and reduced the feedback inhibition of glutathione for the first-step enzymatic reaction, thereby improving the catalytic efficiency of the enzyme reaction. In addition, the formation of suitable oxidative stress environment in the reaction system can further promote glutathione synthesis. By HPLC analysis of the glutathione, it was found that 2.1 g/L reduced glutathione is produced and 17.5 g/L oxidized glutathione. Therefore, the new reaction system not only increases the total glutathione, but also facilitates the subsequent separation and purification due to the larger proportion of oxidized glutathione in the reaction system.