Asymmetric reduction of α-keto esters with thermophilic actinomycete: purification and characterization of α-keto ester reductase from Streptomyces thermocyaneoviolaceus IFO 14271

An α-keto ester reductase was purified and characterized from Streptomyces thermocyaneoviolaceus IFO 14271, one of the thermophilic actinomycetes. The molecular mass of the native enzyme was estimated to be 64 kDa by gel filtration chromatography. The enzyme was a homodimer, with a 30-kDa subunit molecular mass estimated by SDS-polyacrylamide gel electrophoresis. The enzyme showed reducing activity toward aliphatic and aromatic α-keto esters exclusively and produced the corresponding (S)-alcohols with >99% enantiomeric excess (ee). The kinetic constants (Km values) for α-keto esters, RCOCO2Et (R=methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and iso-propyl) were 0.079, 0.12, 1.6, 0.85, 0.70, 0.46, and 9.0 mM, respectively. The enzyme had high stability and was stable toward a variety of additives such as organic solvents and surfactants.