E.coli BL21(DE3)/pET28a(+)-cr羰基还原酶分离纯化及酶学性质研究

Purification and Enzymatic Characterization of E. coli BL21(DE3)/p ET28a(+)-cr Carbonyl Reductase

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.coli BL21(DE3)/p ET28a(+)-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 k Da。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℃,p H7.5;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.coli BL21(DE3)/p ET28a(+)-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 engineered E. coli BL21 （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 of t-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 rate Vmax and apparent Michaelis-Menten constants Km^CHOHB of the purified carbonyl reductase for CHOHB are 54.3 μmol.mg^-1.min^-1 and 4.4 mmol.L^-1 respectively. For COBE, the Vmax and Km^COBE are 36.5 gmol.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.