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

A beta-D-mannosidase was purified to homogeneity from visceral mass extract ofAplysiafasciata 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 degrees C and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only beta-D-mannosidase activity. The K-M and V-max values for p-nitrophenyl beta-D-mannopyranoside were determined to be 2.4 mM and 50.3 mu mol min(-1) mg(-1), respectively. The catalytic efficiency of this P-mannosidase (11,519 min(-1)) was significantly higher than those reported for beta-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 P-D-mannopyranoside, self-transfer of the mannosyl group was observed, and a 10-15% yield of a beta-1-4 disaccharide was obtained. When the reaction was performed in the presence of o-nitrophenyl alpha-D-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl beta-D-mannopyranoside, two regioisomers (85:15, 12% yield) due to the beta-mannosylation of the heteroacceptor in 4 and in 6 positions were formed. (c) 2005 Elsevier B.V. All rights reserved.