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

A-d-mannosidasewas 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 oC and 4.5, respectively. Substrate specificity tests revealed that the enzyme exerts only -d-mannosidase activity.
The KM and Vmax values for p-nitrophenyl -d-mannopyranoside were determined to be 2.4mM and 50.3 mol min-1 mg-1,
respectively. The catalytic efficiency of this -mannosidase (11,519 min-1) was significantly higher than those reported for
-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 -d-mannopyranoside, self-transfer of the mannosyl group
was observed, and a 10-15% yield of a -1-4 disaccharide was obtained. When the reaction was performed in the presence of
o-nitrophenyl -d-2-deoxy-N-acetyl glucopyranoside in 3:1 molar ratio with respect to the p-nitrophenyl -d-mannopyranoside,
two regioisomers (85:15, 12% yield) due to the -mannosylation of the heteroacceptor in 4 and in 6 positions were formed.