Purification and characterization of a diacetyl reductase from Leuconostoc pseudomesenteroides

• Published in: International dairy journal 2000, Vol.10 (11), p.781-789
• Main Authors: Rattray, Fergal P., Walfridsson, Mats, Nilsson, Dan
• Format: Article
• Language: English
• Subjects: Acetoin reductase; Biological and medical sciences; Biotechnology; Diacetyl reductase; Enzyme engineering; Food industries; Fundamental and applied biological sciences. Psychology; Improved methods for extraction and purification of enzymes; Leuconostoc pseudomesenteroides; Methods. Procedures. Technologies; Milk and cheese industries. Ice creams
• ISSN: 0958-6946; 1879-0143
• DOI: 10.1016/S0958-6946(00)00103-5

A diacetyl reductase (acetoin:NAD + oxidoreductase EC 1.1.1.5) was purified to homogeneity from a cell-free extract of Leuconostoc pseudomesenteroides by sequentially using anion exchange chromatography, hydrophobic interaction chromatography and gel-filtration. The enzyme was optimally active for the reduction of diacetyl at pH 5.5, while optimum activity for the oxidation of meso-2,3-butanediol by the enzyme was at pH 7.5. The temperature optimum of the enzyme was 40°C. The molecular mass of the enzyme was 26.91, 30 and 95 kDa, as determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and gel-filtration, respectively, indicating that the native enzyme exists as a trimer or tetramer. The enzyme used only NADH as coenzyme for the reduction of diacetyl, 2,3-pentanedione, pyruvic acid methyl ester and methyl glyoxal; NADH and NADPH functioned equally well as coenzyme for the reduction of acetoin by the enzyme. The enzyme oxidized meso-2,3-butanediol and (2 S, 3 S)-(+)-2,3-butanediol but not (2 R, 3 R)-(+)-2,3-butanediol. The apparent K m for the reduction of diacetyl, 2,3-pentanedione and acetoin were 5.1, 5.57 and 0.34 m m, respectively.