Stereoselective synthesis of L-homophenylalanine using the carbamoylase method with in situ racemization via N-acylamino acid racemase

摘要

N-Acylamino acid racemase (NAAAR) gene of Deinococcus radiodurans BCRC12827 was cloned into expression vector pQE30 to generate pQB-naaar and expressed in recombinant Escherichia coli JM109. The expressed enzyme purified from the crude cell extract of IPTG-induced E. coli JM109 (pQE-naaar) exhibited high racemization activity to N-carbamoyl-L-homophenylalanine (NCa-L-HPA) and N-carbamoyl-D-homo-phenylalanine (NCa-D-HPA) with specific activities of 1.91 U/mg protein and 1.31 U/mg protein, respectively. To develop a recombinant E. coli whole cell system for the conversion of racemic NCa-HPA to L-homophenylalanine (L-HPA), naaar gene from D. radiodurans and L-N-carbamoylase (LNCA) gene from Bacillus kaustophilus BCRC11223 were cloned and coexpressed in E. coli cells. Recombinant cells treated with 0.5% toluene at 30℃ for 30 min exhibited enhanced NAAAR and LNCA activities, which are about 20- and 60-fold, respectively, higher than those of untreated cells. Using toluene-permeabilized recombinant E. coli cells, a maximal productivity of 7.5 mmol L-HPA/l h with more than 99% yield could be obtained from 150 mmol racemic NCa-HPA. Permeabilized cells also showed considerable stability in the bioconversion process using 10 mmol racemic NCa-HPA as substrate, no significantly decrease in conversion yield for L-HPA was found in the eight cycles.