Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis

Abstract

(R)-ethyl-3-hydroxyglutarate with highly optical purity (> or = 99%) can be used as a novel precursor for synthesis of chiral side chain of rosuvastatin. In this study, a novel synthesis route of (R)-ethyl-3-hydroxyglutarate by whole microorganism cells from racemic ethyl 4-cyano-3-hydroxybutyate was created. A strain ZJB-0910 capable of transforming racemic beta-hydroxy aliphatic nitrile was isolated by employing a screening method based on a colorimetric reaction of Co(2+) ion with ammonia, and identified as Rhodococcus erythropolis based on its morphology, physiological tests, Biolog, and the 16S rDNA sequence. After cultivation in a sterilized medium with composition of 20 g glucose, 5 g yeast extract, 0.5 g KH(2)PO(4), 0.5 g K(2)HPO(4), 0.2 g MgSO(4).7H(2)O per liter at 30 degrees C and 150 rpm for 48 h, the whole cells of R. erythropolis ZJB-0910 were prepared as a catalyst in (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate for synthesis of (R)-ethyl-3-hydroxyglutarate, without bearing hydrolase activity for the ester bond of ethyl 4-cyano-3-hydroxybutyate. Under the optimized biotransformation conditions of pH 7.5, 30 degrees C,and 20 mM substrate concentration, (R)-ethyl-3-hydroxyglutarate with 46.2% yield (ee>99%) was afforded, and its chemical structure was determined by ESI-MS, NMR, and IR. The apparent Michaelis constant K(m) and maximum rate Vmax for this biocatalytic reaction were 0.01 M and 85.6 micromol min(-1)g(-1), respectively.