Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/34060
標題: Enantioselective synthesis of L-homophenylalanine by whole cells of recombinant Escherichia coli expressing L-aminoacylase and N-acylamino acid racemase genes from Deinococcus radiodurans BCRC12827
作者: Hsu, S.K.
李東昇
Lo, H.H.
Kao, C.H.
Lee, D.S.
Hsu, W.H.
關鍵字: coexpressed d-hydantoinase;amycolatopsis sp ts-1-60;l-amino-acids;aspartate-aminotransferase;kinetic resolution;substrate-specificity;structural basis;genome sequence;purification;carbamoylase
Project: Biotechnology Progress
期刊/報告no:: Biotechnology Progress, Volume 22, Issue 6, Page(s) 1578-1584.
摘要: 
L-Homophenylalanine (L-HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N-acylamino acids for the L-HPA production, N-acylamino acid racemase (NAAAR) and L-aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N-acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict L-enantioselection to hydrolyze the NAc-L-HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc-D-HPA to L-HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc-L-HPA, in the early stage of conversion and a low productivity of 0.83 mmol L-HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in L-HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of L-HPA could be reached in 1 h, with a productivity of 10 mmol L-HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of L-HPA to be reported.
URI: http://hdl.handle.net/11455/34060
ISSN: 8756-7938
DOI: 10.1021/bp0601241
Appears in Collections:化學系所

Show full item record
 

Google ScholarTM

Check

Altmetric

Altmetric


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.