利用强酸型阳离子交换层析和疏水作用层析技术,从实验室构建的羰基还原酶工程菌E. coliBL21 (DE3)/pET28a(+)-cr细胞中分离得到电泳纯NADP(H)依赖型的羰基还原酶,LC-MS-QTOF分析揭示其相对分子质量为35.4 kDa.该酶能不对称还原 6-氰基-(5R)-羟基-3-羰基己酸叔丁酯 (CHOHB)、4-氯乙酰乙酸乙酯(COBE)合成阿托伐他汀关键手性合成子6-氰基-(3R,5R)-二羟基己酸叔丁酯、(S)-4-氯-3-羟基丁酸乙酯,对丙酮酸乙酯、丁二酮也表现出较高的还原能力.该羰基还原酶的最适作用条件为:30℃,pH7.5;且活性不依赖于金属离子.它催化CHOHB还原反应的最大反应速度vmax 54.3μmol?mg?1?min?1,KmCHOHB值4.4 mmol?L?1;作用于COBE时,最大反应速度vmax 36.5μmol?mg?1?min?1,KmCOBE值1.2×10?1 mmol?L?1.E. coliBL21 (DE3)/pET28a(+)-cr羰基还原酶在他汀手性侧链制造上具有广阔的应用前景.%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.
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