Purification and characterization of a β-d-mannosidase from the marine anaspidean Aplysia fasciata

A β-d-mannosidase was purified to homogeneity from visceral mass extract of Aplysia fasciata a mollusc belonging to the order Anaspidea. The purified enzyme is a homodimer with a subunit mass of 130 kDa. Temperature and pH optima of this enzyme were 45 °C and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only β-d-mannosidase activity. The KM and Vmax values for p-nitrophenyl β-d-mannopyranoside were determined to be 2.4 mM and 50.3 μmol min−1 mg−1, respectively. The catalytic efficiency of this β-mannosidase (11,519 min−1) was significantly higher than those reported for β-mannosidases from other sources. It was verified that this is an exo-acting glycosyl hydrolase with transglycosidase activity. When the enzyme was incubated in the presence of p-nitrophenyl β-d-mannopyranoside, self-transfer of the mannosyl group was observed, and a 10-15% yield of a β-1-4 disaccharide was obtained. When the reaction was performed in the presence of o-nitrophenyl α-d-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl β-d-mannopyranoside, two regioisomers (85:15, 12% yield) due to the β-mannosylation of the heteroacceptor in 4 and in 6 positions were formed.