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 degrees C. The K-m and V-max values for diacetyl were 2.5 mmol l(-1) and 0.026 mmol l(-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 (pI) of pH5.8, and its molecular weight was 50 kDa.