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 degrees C. The enzyme activity was determined at 30 degrees C using acetophenone as substrate.
Results: Under the above conditions, the specific enzyme activity of crude extract was 2.6 U/mg. The optimal pH was between 6.0 and 6.5 and the enzyme can survived up to 60 degrees C. After incubation at 60 degrees C for 5 h, 80% enzyme activity remained. The optimal substrate among beta-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 beta-ketoesters transformation on industrial scale.