Purification and biochemical characterization of two soluble -mannosidases from Candida albicans

Abstract

Two soluble α-mannosidases, E-I and E-II, were purified from C.albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-α-d-mannopyranoside and p-nitrophenyl-α-d-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42°C. Apparent Km values for hydrolysis of 4-methylumbelliferyl-α-d-mannopyranoside and p-nitrophenyl-α-d-mannopyranoside by E-I were 0.83 µM and 2.4 mM, respectively. Corresponding values for E-II were 0.25 µM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-α-d-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-α-d-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+ and Ca2+ in the range of 0.5–2 mM. At the same concentrations, Mg2+ was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved α-1,6 and α-1,3 linkages, respectively.