Purification and characterization of carbonyl reductase from Candida krusei SW 2026 involved in enantioselective reduction of ethyl 2-oxo-4-phenylbutyrate

Optically active ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is widely used as a key chiral building block in the synthesis of a class of angiotensin-converting enzyme (ACE) inhibitors. A highly enantioselective carbonyl reductase responsible for the reduction of ethyl 2-oxo-4-phenylbutyrate (OPBE) was identified and characterized from Candida krusei SW 2026. The enzyme was purified to homogeneity through three chromatography columns. The relative molecular mass of the enzyme was estimated to be around 45,500 by gel filtration and 46,000 by SDS-polyacrylamide gel electrophoresis. The enzyme yielded (R)-enantiomer product and utilized NADPH as the cofactor. The purified enzyme exhibited maximum activity at pH 6.0 and 30 °C, and retained over 80% of its activity over an acidic pH range of 4.5–7.0. The maximum reaction rate (Vmax) and apparent Michaelis–Menten constant (Km) for OPBE and NADPH were 18.7 μmol/(min mg) protein and 0.319 mmol, 14.9 μmol/(min mg) protein and 0.306 mmol, respectively.