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- W2372181645 abstract "The appropriate ratio of the amount of each enzyme is very important for synthesizing the desired product in multi enzyme reaction. In order to improve the GSH synthesis capability of E.coli, gsh I( γ glutamylcysteine synthetase gene) and gsh II(glutathione synthetase gene) of E.coli B were amplified and cloned into pTrc99A before being transformed into E.coli BL21(DE3), in combination with replacing the unusual initiation codon TTG of gsh I with ATG and designing the location of two genes on pTrc99A/ gsh I gsh II. After induction (in 0.08mmol/L IPTG at 28℃ for 3 hours), the yield of GSH I and GSH II in E.coli BL21(DE3)(pTrc99A/ gsh I gsh II)was 11% of the total bacterial proteins, and the GSH synthesis capability of the engineering strain was 8.5mg/g wet weight cells, which was 11 times and 15 times as much as that of E.coli B and E.coli BL21(DE3), respectively. Then the amount of GSH I in E.coli BL21(DE3)(pTrc99A/ gsh I) and that of GSH II in E.coli BL21(DE3)(pTrc99A/ gsh II) were adjusted to be approximately equal by inducing them in 0.05 mmol/L IPTG at 28℃ for 3 hours. With maintaining the enzyme amounts constant in reaction systems, the ratio of GSH I/GSH II was adjusted in order to achieve the maximum production of GSH. The optimum ratio was determined to be 3∶1～6∶1. The ratio of GSH I/GSH II in E.coli BL21(DE3)(pTrc99A/ gsh I gsh II) was 4.5∶1 and just right in this range. So a coordinated expression system E.coli BL21(DE3)(pTrc99A/ gsh I gsh II) was acquired, which improved the GSH synthesis capability of E.coli ." @default.
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- W2372181645 date "2002-01-01" @default.
- W2372181645 modified "2023-09-25" @default.
- W2372181645 title "Construction of Coordinated Expression System of gsh I and gsh II Gene for Improving the GSH Synthesis Capability of E.coli" @default.
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