A 1,2-ALPHA-D-MANNOSIDASE FROM A BACILLUS SP - PURIFICATION, CHARACTERIZATION, AND MODE OF ACTION

A 1,2-alpha-D-mannosidase was purified to homogeneity from the culture supernatant of Bacillus sp. M-90, which was isolated from soil by enr ichment culture on baker's yeast mannan. The purified enzyme had M(r) 380000 Da, and was comprised of two apparently identical 190000 Da sub units. It had a neutral optimum pH (7.0) and an isoelectric point of 3 .6. The enzyme was highly specific for alpha 1,2-linked D-mannose olig osaccharides. An N-linked high-mannose type oligosaccharide, Man GlcNA c(2), was a good substrate, yielding Man(5)GlcNAc(2), and the alpha 1, 2-linked side chains of Saccharomyces cerevisiae mannan were also spec ifically hydrolyzed by the enzyme. p-Nitrophenyl alpha-D-mannopyranosi de and 1,2-alpha-D-mannobiitol were not hydrolyzed at all. Calcium ion , 1-deoxymannojirimycin, and swainsonine had no effect on the enzyme, but the activity was completely inhibited by EDTA. The mode of action on alpha 1,2-linked mannotetraose indicated that the enzyme is an exo- 1,2-alpha-D-mannanase.