Characterization of β-Mannosidase of an Alkalophilic Bacillus sp.

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The cell-associated β-mannosidase of an alkalophilic Bacillus sp. (AM-001) was purified to the electrophoretically homogenous state. The relative molecular mass of the purified enzyme was 94, 000 daltons by SDS-PAGE and the pi was 5.5 by isoelectric focusing. The enzyme was most active at pH 6.0 and 50°C. This β-mannosidase was inhibited by some metal ions and chemical reagents as follows: Ag⁺, Cd²⁺, Cu²⁺, Hg²⁺, Zn²⁺, PCMB, SDS, DBS, and NBS, but was not inhibited by D-mannose, D-mannitol, or D-mannonic acid-γ-lactone. Using synthetic substrates such as p-nitrophenyl glycopyranosides, this enzyme showed strong activity only with p-nitrophenyl β-D-mannopyranoside, and no activity with p-nitrophenyl α-D-mannopyranoside. The Michaelis constant (Km) of the enzyme for p-nitrophenyl β-D-mannopyranoside was 1.3mM. The enzyme hydrolyzed β-1, 4-mannooligosaccharides larger than mannobiose.