Purification and characterization of exo-α-d-mannosidase from a Cellulomonas sp.

A Gram-positive bacterium isolated from a soil sample was found to produce a high level of α-mannosidase in culture media. The organism was identified as Cellulomonas sp. from various bacteriological characteristics. The production of the enzyme was strongly induced by yeast extract. To purify the enzyme, the bacterium was first grown in a glucose medium, and then the cells were transferred to an inducing medium containing only yeast extract. The enzyme had two different molecular weight forms. The high-molecular-weight form was purified from the inducing medium by column chromatography. The enzyme was purified to homogeneity by ultracentrifugal analysis. The enzyme was strongly inactivated by Hg²⁺, Cu²⁺, Zn²⁺, EDTA and p-chloromercuribenzoic acid. The molecular weight of the enzyme was estimated to be about 450 000 by gel filtration. The purified enzyme could liberate only mannose from native yeast mannan and the optimum pH was 6.5-8.0. The enzyme rapidly cleaved α-1,2- and α-1,6-linked mannose chains, but the hydrolysis rate for α-1,3 linkage was very low. In addition, the purified enzyme showed only slight activity towards p-nitrophenyl-α-d-mannoside and did not hydrolyze O-α-d-mannopyranosyl(1 → 2)-d-mannitol.