Data Mining Of A New Ketoreductase And Its Application In Asymmetric Synthesis Of Ethyl(R)-2-Hydroxy-4-Phenylbutyrate

Ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is an important building block for the production of various angiotensin-converting enzyme (ACE) inhibitors, such as benazepril and cilazapril. Asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) with ketoreductases is an effective approach to producing (R)-HPBE since it can be carried out under mild reaction conditions, resulting in a high yield and enantioselectivity.A new ketoreductase (CgKR2) from Candida glabrata was discovered by data mining for ketoreductases. CgKR2 was purified to homogeneity by a Ni-NTA column from the cell free extract of E. coli containing the recombinant CgKR2 (E. coli/pCgKR2) and its basic catalytic properties were investigated. The optimum pH of purified CgKR2 was determined at pH 6.0 in sodium phosphate buffer. The optimal temperature was detected at 45℃. CgKR2 was more stable at 30℃. The enzyme was unstable at 40 and 50℃, with a half-life of 11.3 h and 2.6 min, respectively. The enzyme displayed a lower activity upon the addition of Zn2+, Fe3+and Pb2+. The Km andat values of CgKR2 for OPBE were 0.1 mM and 11 s-1. The corresponding Vmax was 18.5μmol min-1 mg-1. This enzyme could also catalyze the asymmetric reduction of other a-ketoesters with high activity and excellent enantioselectivity. But for (3-ketoesters and aromatic ketones, CgKR2 exhibited lower activity and enantioselectivity. (R)-HPBE was produced by CgKR2 powders or cells of E. coli/pCgKR2 via reduction of OPBE at 2 M (412 g/L) in aqueous phase system (10 mL) with external adding of 0.5 mM NADP+in>99%conversion and>99%ee, as coupled with glucose dehydrogenase and glucose for NADPH regeneration. It is worthwhile to note that the reduction could also be performed at a substrate loading of up to 1 M (206 g/L) within 7 h with>99%ee without any external addition of NADP+or NADPH and was easily scaled up to 100 mL, cutting down the production cost sharply. The space-time yield of (R)-HPBE production reached 700 g·L-1d-1, making CgKR2 a more competitive and promising biocatalyst.At last, the genes for CgKR2 and GDH (a glucose dehydrogenase) from Bacillus megaterium were coexpressed in E. coli. OPBE was efficiently converted to (R)-HPBE by the cells of recombinant E. coli in 9 h, with>99%conversion and>99%ee, demonstrating the potential for industrial application.