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標題: Stereoselective synthesis of L-homophenylalanine using the carbamoylase method with in situ racemization via N-acylamino acid racemase
作者: Hsu, S.K.
Lo, H.H.
Lin, W.D.
Chen, I.C.
Kao, C.H.
Hsu, W.H.
關鍵字: angiotensin-converting enzyme inhibitor
N-acylamino acid racemase
permeabilized cell
recombinant escherichia-coli
enantioselective synthesis
enzymatic conversion
期刊/報告no:: Process Biochemistry, Volume 42, Issue 5, Page(s) 856-862.
摘要: 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 (Wa-L-HPA) and N-carbamoyl-D-homophenylalanine (Wa-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 degrees 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. (C) 2007 Elsevier Ltd. All rights reserved.
ISSN: 1359-5113
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