Purification and enzymatic characterization of E. coli BL21 (DE3)/pET28a(+)-cr carbonyl reductase

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 KmCHOHB 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 KmCOBE 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. ©, 2015, Zhejiang University. All right reserved.