反应详情

Whole cell reductions of 2-benzyl-3-ketoglutarate were carried out using E. coli cells expressing the gene encoding KRED 1008. Cells were grown overnight at 30 C in 400 mL of a Luria Broth/glucose (2 g/L)/ampicillin (100 mg/L) media. The cells were isolated by centrifugation and re-suspended in 400 mL of a minimal salt (M9) solution containing isopropylthiogalactoside (IPTG, 0.5 mM), glucose (5 g/L), ampicillin (100 mg/L), and 2-benzyl-3-ketoglutarate diethyl ester. In independent experiments, the concentration of 2-benzyl-3-ketoglutarate diethyl ester was varied over the range of 1 gram/liter to 7.5 grams/liter. Cells continued to grow at 30 C after addition of the ketodiester substrate. Samples were removed at regular time intervals and analyzed for product formation using gas chromatography until complete reaction was observed. Although the 2-benzyl-3-ketoglutarate diethyl ester substrate did not completely dissolve in the aqueous buffer, and although no DMSO co-solvent was used, the reduction reactions were complete after 20-40 hours. In reactions that were allowed to proceed for more that 24 hours, another portion of glucose (1-3 g) and IPTG (0.4 mL of a 0.5 M solution) were added. The product 2-benzyl-3-hydroxyglutarate diethyl ester was isolated by removal of the cells by centrifugation and extraction of the reaction mixture with ethyl acetate. The yield of 2-benzyl-3-hydroxyglutarate diethyl ester in all cases was greater than 80%.

1. customThe cells were isolated by centrifugation
2. customSamples were removed at regular time intervals
3. customuntil complete reaction
4. custommore that 24 hours