高校化学工程学报Issue(3)：607-615,9.DOI:10.3969/j.issn.1003-9015.2015.03.016
E. coliBL21 (DE3)/pET28a(+)-cr羰基还原酶分离纯化及酶学性质研究
Purification and Enzymatic Characterization ofE. coliBL21 (DE3)/pET28a(+)-cr Carbonyl Reductase
摘要
Abstract
A NADP(H)-dependent carbonyl reductase produced by engineeredE. coliBL21 (DE3)/pET28a(+)-cr was purified through Macro-prep High S chromatography followed by Macro-prep t-butyl HIC chromatography. It was revealed by LC-MS-QTOF that the purified carbonyl reductase has a relative molecular weight of 35.4 kDa.t-Butyl 6-cyano-(3R,5R)-dihydroxylhexanoate and (S)-4-cyano-3-hydroxybutanoate were synthesized via asymmetric reduction oft-butyl 6-cyano-(5R)-hydroxy-3-oxo-hexanoate (CHOHB) and 4-chloro-3-oxobutanoate (COBE), respectively. The enzyme also exhibits strong bioreduction activity towards ethyl pyruvate and butanedione. The carbonyl reductase is metal-independent which shows highest activity at 30℃, pH 7.5. The maximum reaction ratevmax and apparent Michaelis–Menten constants Km CHOHB of the purified carbonyl reductase for CHOHB are 54.3μmol?mg?1?min?1 and 4.4 mmol?L?1respectively. For COBE, the vmax and Km COBE are 36.5 μmol?mg?1?min?1 and 1.2×10?1 mmol?L?1,respectively. The E. coli BL21 (DE3)/pET28a(+)-cr has great commercialization potential in the synthesis of atorvastatin side chains.关键词
R-羰基还原酶/分离纯化/6-氰基-(3R,5R)-二羟基己酸叔丁酯/(S)-4-氯-3-羟基丁酸乙酯Key words
carbonyl reductase/purification/t-butyl 6-cyano-(3R/5R)-dihydroxylhexanoate/(S)-4-cyano-3-hydroxybutanoate分类
生物科学引用本文复制引用
王亚军,吴配配,罗希,郑裕国..E. coliBL21 (DE3)/pET28a(+)-cr羰基还原酶分离纯化及酶学性质研究[J].高校化学工程学报,2015,(3):607-615,9.基金项目
国家自然科学基金(21476209) （21476209）
国家重点基础研究发展计划(2011CB10800) （2011CB10800）
浙江省重点科技创新团队项目(2009R50043-06). （2009R50043-06）
