Purification and characterization of a novel alpha-mannosidase from Aspergillus saitoi.
An alpha-mannosidase differing from 1,2-alpha-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-alpha-mannosidase, the enzyme was strongly activated by Ca2+ ions. p-Nitrophenyl alpha-mannopyranoside was not hydrolyzed by the enzyme. Accordingly, the substrate specificity of the new alpha-mannosidase was studied by using a variety of tritium-labeled oligosaccharides. Studies with linear oligosaccharides revealed that the enzyme cleaves the Man alpha 1----3Man linkage more than 10 times faster than the Man alpha 1----6Man and the Man alpha 1----2Man linkages. Furthermore, it cleaves the Man alpha 1----6Man linkage of the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT only after its Man alpha 1----3 residue is removed. Because of this specificity, the enzyme can be used as an effective reagent to discriminate R----Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT from its isomeric counterparts, Man alpha 1----6(R----Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAcOT, in which R represents sugars.
D001230:Aspergillus / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002845:Chromatography / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D009844:Oligosaccharides; D008361:Mannosidases; D043323:alpha-Mannosidase; D002118:Calcium