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摘要
alpha-Mannosidase (EC 3.2.1.24) was purified from 'Iseimo', a native variety of Dioscorea opposita Thunb. Before purification, we investigated the composition of a viscous polysaccharide that interferes with column chromatography procedures. The polysaccharide consisted mainly of mannose, and also contained uronic acid. We used the cationic detergent cetylpyridinium chloride (CPC) to remove the polysaccharide. CPC treatment decreased viscosity without affecting alpha-mannosidase activity. We purified alpha-mannosidase 2,650-fold. The optimal pH of the enzyme was 6.0 and the optimum temperature was 65A degrees C. The K (m) value for p-nitrophenyl-alpha-d-mannopyranoside was 0.35 +/- A 0.03 mM. Activity was inhibited by swainsonine but not kifunensine. The enzyme cleaved alpha-1,2 linkages preferentially to alpha-1,6 and alpha-1,3 linkages. The M (r) of purified alpha-mannosidase was estimated to be 250-260 kDa by gel filtration and native-PAGE. Four polypeptides (86, 83, 80, and 28 kDa) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is unclear whether the polypeptides are encoded by one gene or multiple genes. However, N-terminal sequence analysis suggested that post-translational cleavage and/or glycosylation resulted in the three large fragments, if these amino acids were encoded by the same gene. Homology searches and characterization of the enzyme's properties indicated that Iseimo alpha-mannosidase belongs to the glycoside hydrolase family 38 proteins, and to the Class II mannosidase group.
- 出版日期2010-7
