Characterization of a strictly specific acid beta-galactosidase from Achatina achatina.
An acid beta-galactosidase was isolated from the digestive juice of Achatina achatina and purified to homogeneity by anion exchange, gel-filtration and hydroxyapatite chromatographies. This enzyme is soluble, as are the cytosolic beta-galactosidases, functions at acid pH like the lysosomal enzymes but differs from the other soluble animal beta-galactosidases in that it is highly specific for the beta-D-galactosyl residue. In addition, it cleaves the beta1-4 linkage much faster than the beta1-3 and beta1-6 linkages. The enzyme is a monomeric glycoprotein with a molecular mass of 120-125 kDa and the carbohydrate moiety makes up approximately 6% (w/w) of the protein. The amino acid composition displays an important amount of acidic/amide and hydroxy amino acid residues and a low content of basic residues. The enzyme activity is markedly affected by the ionic strength of the medium and the rate-pH curve was shifted towards higher pH values in the presence of added salt. Acid beta-galactosidase is capable of catalysing transgalactosylation reactions. The yields of galactosylation of hydroxy amino acid derivatives, catalysed by the enzyme in the presence of lactose as the glycosyl donor, were higher than those reported previously with conventional sources of beta-galactosidases. In addition, the pH optimum is different for the hydrolysis (pH 3.2) and transgalactosylation (pH 5.0) reactions. On the basis of this work, the enzyme could be used as a tool in the structural analysis of D-galactose-containing oligosaccharide chains, as well as for the synthesis of glycoconjugates.
Leparoux
S
S
Laboratoire de Biochimie, UPRES 2161, Centre de Recherche de Biologie et Physico-chimie Cellulaires, Faculté des Sciences et des Techniques, Nantes, France.
Padrines
M
M
Placier
G
G
Colas
B
B
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004064:Digestive System / Q000201:enzymology; D017886:Durapatite; D005690:Galactose; D006031:Glycosylation; D007700:Kinetics; D008970:Molecular Weight; D012908:Snails / Q000201:enzymology*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D017886:Durapatite; D001616:beta-Galactosidase; D005690:Galactose