Cloning，expression and characterization of chiral alcohol dehydrogenase from Rhodococcus erythropolis ATCC 4277

Abstract:

Abstract:［Objective］We characterized alcohol dehydrogenase from Rhodococcus erytropolis to catalyze ketoesters or ketones.[Methods] We cloned alcohol dehydrogenase gene (adh) of 1047 bp from Rhodococcus erythropolis ATCC 4277，inserted the open reading frame of adh into vector pET-22b(+) and expressed in auto-inducing media for 24 h at 15℃. The enzyme activity was determined at 30℃ using acetophenone as substrate．［Results］Under the above conditions，the specific enzyme activity of crude extract was 2.6U/mg. The optimal pH was between 6.0 and 6.5 and the enzyme can survived up to 60℃． After incubation at 60℃ for 5 h，80% enzyme activity remained．The optimal substrate among β-ketoesters examined was ethyl acetoaetate． Ethyl 4-chloroacetoacetate was catalyzed by whole cell in aqueous phase． After chiral liquid chromatography，the product showed (R) -enantioselective． ［Conclusion］The study shows that the enzyme might have potential in β-ketoesters transformation on industrial scale．

Key words:Keywords: Chiral alcohol dehydrogenase，auto-induction，NADH