Enzymatic Characterization Of Acetyl CoA Synthetase And Application

In this study, we would screen acetyl coenzyme A synthetase of higher activity and study its enzymatic properties, and apply to the synthesis process of mevalonate which regard acetyl coenzyme A as precursor finally. According to the existing relevant studies and reports,there is a acetyl coenzyme A synthetase gene acs in E. coli, and there are acs1 and acs2 in Acetobacter pasteurianus, and all of them play better in acetic acid metabolism. Western Blot analysis shows that the three genes successfully express in E. coli BL21(DE3) firstly.Secondly, the enzyme activity is compared among the three from different sources in the thesis,and results shows that ACS2 from A. pasteurianus has highest catalytic activity. The study of enzymatic characteristics of ACS2 shows that the optimum temperature and pH of ACS2 were 37 °C and 7~8 respectively, and its Km is 1.16 mmol/L and the maximum reaction rate(Vmax) is 20.45 mmol/(L·min) under the optimum conditions. Based on these findings, the ACS2 appling to biosynthesis of mevalonate from acetic acid under fed-batch conditions, the finally engineered strain which ACS2 overexpressed accumulated 6.59 g/L mevalonate better than the one not overexpressed. By this study we can take advantage of acetic acid or acetate instead of glucose. because they are cheap and easy to get as a fermentation substrate, It can both cut costs, realizing the recycling of resources, and protect the environment, reducing the pollution of the environment.