Purification and characterization of a diacetyl reductase from Leuconostocpseudomesenteroides

A diacetyl reductase (acetoin:NAD (+) oxidoreductase EC 1.1.1.5) was purifi ed to homogeneity from a cell-free extract of Leuconostoc pseudomesenteroid es by sequentially using anion exchange chromatography, hydrophobic interac tion chromatography and gel-filtration. The enzyme was optimally active for the reduction of diacetyl at pH 5.5, while optimum activity for the oxidat ion of meso-2,3-butanediol by the enzyme was at pH 7.5. The temperature opt imum of the enzyme was 40 degreesC. The molecular mass of the enzyme was 26 .91, 30 and 95 kDa, as determined by matrix-assisted laser desorption/ioniz ation mass spectrometry (MALDI-MS), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and gel-filtration, respectively, indicatin g that the native enzyme exists as a trimer or tetramer. The enzyme used on ly NADH as coenzyme for the reduction of diacetyl, 2,3-pentanedione, pyruvi c acid methyl ester and methyl glyoxal; NADH and NADPH functioned equally w ell as coenzyme for the reduction of acetoin by the enzyme. The enzyme oxid ized meso-2,3-butanediol and (2S, 3S)-(+)-2,3-butanediol but not (2R, 3R)-( +)-2,3-butanediol. The apparent K-m for the reduction of diacelyl, 2,3-pent anedione and acetoin were 5.1, 5.57 and 0.34 mM, respectively. (C) 2001 Els evier Science Ltd. All rights reserved.