[Purification and properties of beta-mannanases from alkalophilic Bacillus N16-5]

Three extracellular alkaline beta-mannanases from an alkalophilic Bacillus N16-5 were purified to electrophoretic homogeneity by (NH4)2SO4 precipitation, DEAE-Sephadex A-25 chromatography, hydroxyapatite chromatography and preparative electrophoresis. Molecular weights and pI values of the beta-mannanases (M-1, M-2 and M-3) were 51000, 38000 and 34700 by SDS-PAGE and 4.3, 2.5 and 2.5 by PAGEIEF, respectively. The optimum pH for enzyme catalysis were 9.0 for M-1 and 10.0 for M-2 and M-3, respectively. All of three enzymes were the most active at 70 degrees C. The activities of three enzymes were strongly inhibited by Ag1+, Hg2+ and Mn2+. Michaelis constants (Km) of the enzyme M-1, M-2 and M-3 for mannase from konjak were 2.9, 1.7 and 12.5 mg/ml, maximum velocities (Vmax) for the saccharide were 27500, 47500 and 15700 mumol.min-1.mg-1, respectively. Konjak was hydrolyzed by the enzyme M-1, and major components in digests were mono-, di-, tri- and tetra-saccharides.