Cloning and Expression of Ketoacid Reductase for Asymmetric Synthesis of Phenyllactic Acid

Abstract:

Phenyllactic acid was one of the most important compounds with wide application in pharmaceuticals and biological preservatives. Five ketoacid reductases capable of asymmetric reduction of phenylpyruvic acid（PPA） into phenyllactic acid（PLA） were obtained from genome databases，and were heterogeneously over-expressed in E. coli BL21（DE3）. Among 5 enzymes，the recombinant ketoacid reductase from Lactobacillus casei（LcKAR） displayed the best biocatalytic performance，with the highest specific activity of 0.32 U/mg of purified enzyme and enantioselectivity of >99%. This LcKAR obeys Prelog rule in the asymmetric reduction of PPA into（R）-PLA. The fermentation medium composition and culture conditions of recombinant E.coli BL21（DE3）/pET28a-LcKAR were optimized to be seed age of 6 h，3% inoculation，induced with 0.6 mmol/L IPTG at 30 ℃ for 5 h. Under above optimal conditions，the LcKAR production could reach 2.17 kU/L in a 3 L bioreactor.