Purification and characterization of α- d-mannosidase from Aspergillus sp.

Abstract

α- d-Mannosidase from Aspergillus sp. was purified to homogeneity by preparative polyacrylamide gel electrophoresis (PAGE). The native enzyme of molecular mass 412 kDa (gel filtration) is made up of six identical subunits of molecular mass 69.2 kDa (SDS-PAGE) The enzyme is acidic (p I 4.5) and a glycoprotein with a carbohydrate content of 3.8%. The pH and temperature optima of the enzyme were in the range 6.0–6.5 and 50–55°C, respectively. At pH 6.0 the enzyme was stable for 30 min at 50°C. The K m and V max for p-nitrophenyl-α- d-mannopyranoside were 83 μM and 0.2 μmol/min per mg of the enzyme, respectively. The enzyme was strongly inhibited by 1 mM Hg 2+ and Cu 2+ and partially by 30 mM glucose and mannose. The enzyme hydrolysed Man-α-(1–3)Man at a very high rate followed by Man-α-(1–2)Man, while the rate of hydrolysis was low for Man-α-(1–6)Man. The rate of hydrolysis for high mannose oligosaccharide Man-6 was higher than for Man-9 and yeast mannan was not hydrolysed at all.