Purification and characterization of diacetyl reductase from Kluyveromyces marxianus

Diacetyl reductase from Kluyveromyces marxianus NRRL Y-1196 was purified 27.5-fold with a yield of 13% by ammonium sulphate fractionation, DEAE-anion exchange chromatography, hydroxyapatite chromatography and chromatofocusing. The purified enzyme was most active at pH 7.0 and exhibited optimal activity at 40°C. The K _m and V _max values for diacetyl were 2.5 mmol 1^-1 and 0.026 mmol 1^-1 min^-1, respectively. The enzyme did not react with monoaldehydes or monoketones, but reduced acetoin, diacetyl and methylglyoxal with NADH as a cofactor. The enzyme had an isoelectric point (pl) of pH 5.8, and its molecular weight was 50 kDa.