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. waitthe reduction reactions were complete after 20–40 hours
5. custommore that 24 hours