Purification and characterization of a beta-D-mannosidase from the marine anaspidean Aplysia fasciata.
A beta-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 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 micromol min(-1)mg(-1), respectively. The catalytic efficiency of this beta-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 beta-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.
Andreotti
Giuseppina
G
Istituto di Chimica Biomolecolare, Consiglio Nazionale delle Ricerche, Via Campi Flegrei 34, 80072 Pozzuoli, Naples, Italy. gandreotti@icmib.na.cnr.it
Giordano
Assunta
A
Tramice
Annabella
A
Mollo
Ernesto
E
Trincone
Antonio
A
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001048:Aplysia / Q000201:enzymology*; D002236:Carbohydrate Conformation; D002384:Catalysis; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008362:Mannosides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D044902:beta-Mannosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D008362:Mannosides; C014124:4-nitrophenyl-N-acetyl-2-deoxyglucopyranoside; C036119:4-nitrophenyl-beta-D-mannopyranoside; D044902:beta-Mannosidase; D000117:Acetylglucosamine