During synthesis of GSH by the engineered strain E.coli BL21(pTrc-gsh) coupled with Saccharomyces cerevisiae producing ATP from adenosin,the inconsistency of two systems in the concentration of phosphate buffer was solved by decreasing concentration to 250mmol/L.The conditions under 250mmol/L phosphate buffer were optimized and the yield of GSH was 1.6g/L,which was higher than that of summation by two systems under the same conditions respectively.Addition of glycine later after glutamate and cysteine weakened the inhibition of GSH to GSHI.It made the yield of GSH reach to 2.13g/L which was 30.7% higher than the control.
The genes(gsh\|I,gsh\|II)for γ\|glutamyl\|cysteine synthetase(GSH\|I) and glutathione synthetase(GSH\|II)from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively.The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc\|gsh. E.coli BL21 was transformed by pTrc\|gsh for expression of the related enzymes.Analysis of SDS\|PAGE showed that the expected products were expressed. E.coli BL21(pTrc\|gsh) were incubated at 37℃ and pH 7.2 to OD 550 =0 5.The conditions were then switched to 34℃ and pH6.7 after the addition of 0.1mmol/L IPTG.The expressed products were up to 25% of the total protein of the bacteria.Acetone\|treated cells of the engineered strain could synthesize GSH efficiently.
