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

• DOI: 10.1016/j.procbio.2007.02.008
• Publication Date: 2007-02-27T20:51:32Z
• Authors: Shih-Kuang Hsu; Hsueh-Hsia Lo; Wei-De Lin; I-Chieh Chen; Chao-Hung Kao; Wen-Hwei Hsu
• Keywords: Racemization; Bioconversion

Abstract N -Acylamino acid racemase (NAAAR) gene of Deinococcus radiodurans BCRC12827 was cloned into expression vector pQE30 to generate pQE- 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 -homophenylalanine (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 °C 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.