Characterization of three .BETA.-mannanases of an alkalophilic Bacillus sp.

Three extracellular β-mannanases (M-I, M-II, and M-III) of an alkalophilic Bacillus sp. (AM001) were purified to an electrophoretically homogenous state. Molecular weights and pi values of the purified enzymes (M-I, M-II, and M-III) were 58, 000, 59, 000, and 42, 000 by SDS-PAGE and 5.9, 5.7, and 5.1 by isoelectric focusing, respectively. These enzymes were most active at pH 9.0 and 60°C (M-I and M-II) and pH 8.5 and 65°C (M-III). The enzymes were activated slightly by cysteine, and-inhibited strongly by Ag+ and N-bromosuccinimide. Michaelis constants (Km) of the M-I enzyme for β-mannans from copra, locust bean, and konjak were 2.0, 3.8, and 7.7mg/ml, and maximum velocities (Vmax) for these saccharides were 730, 1470, and 1880U/mg-protein, respectively. The kinetic properties of M-II and M-III enzymes were almost the same as those of M-I. About 22, 16, 15, and 2:5% of the β-1, 4-mannosidic linkages in β-mannans from copra, konjak, locust bean, and guar bean were hydrolyzed by the M-I enzyme, and the major components in the digests were di-, tri-, and tetra-saccharides. These enzymes hydrolyzed β-1, 4-mannooligosaccharides larger than mannotriose.