Purification and Characterization of a Novel α-Mannosidase from Aspergillus saitoi1

Abstract

An α-mannosidase differing from 1,2-α-mannosidase was found to occur in Aspergillus saitoi . By a series of column chromatographies the enzyme was purified up to 1,000-fold, and its properties were studied in detail. The enzyme preparation, which was practically free from other exoglycosidases, showed a pH optimum of 5.0. In contrast to 1,2-α-mannosidase, the enzyme was strongly activated by Ca ²⁺ ions. p -Nitrophenyl α -mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new α-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Manα1→3Man linkage more than 10 times faster than the Manα1→6Man and the Manα1→2Man linkages. Furthermore, it cleaves the Manα1→6Man linkage of the Manα1→6(Mancα1→3)Man β 1→4GlcNAcβ1→4G1cNAcot only after its Manα1→3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R→Manα1→6(Manα1→3)Man β 1→4GlcNAcβ1→4 (± Fucα1→6)GlcNAc _OT from its isomeric counterparts, Manal→6(R→Manal→3)Manβ1→4GlcNAcβ1→4(± Fucα1→6)GlcNAc _OT , in which R represents sugars.