Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins.
An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae) has been purified 166-fold. Apparent molecular weights of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were found to be 51,000 and 42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of (1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal activity toward these substrates at pH 4.6. Based on the mode of the liberation of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase capable of catalyzing the sequential hydrolytic release of single D-galactosyl residues from the nonreducing termini. The extent of the hydrolysis of the carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased with their decreasing branching. Isolation and characterization of the major products formed from the proteoglycans indicated the action pattern of the enzyme to include the capability of bypassing the branching points. Consequently, the side chains carrying an additional D-galactosyl group at the reducing termini are released as neutral (1----6)-linked beta-D-galactooligosaccharides and their acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the nonreducing end-group. The specificity and the mode of action showed the enzyme to be a useful tool for analyzing the fine structure of type II arabinogalactans and arabinogalactan-protein conjugates.
Tsumuraya
Y
Y
Department of Biochemistry, Faculty of Science, Saitama University, Urawa, Japan.
Mochizuki
N
N
Hashimoto
Y
Y
Kovác
P
P
D000596:Amino Acids / Q000032:analysis; D001487:Basidiomycota / Q000201:enzymology*; D002241:Carbohydrates / Q000032:analysis; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005685:Galactans / Q000378:metabolism*; D005696:Galactosidases / Q000302:isolation & purification*; D006023:Glycoproteins / Q000378:metabolism*; D006026:Glycoside Hydrolases; D006882:Hydroxyapatites; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D011124:Polyporaceae / Q000201:enzymology*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D005685:Galactans; D006023:Glycoproteins; D006882:Hydroxyapatites; D017886:Durapatite; D005696:Galactosidases; D006026:Glycoside Hydrolases; C063901:exo-beta-(1--3)-D-galactanase; D001616:beta-Galactosidase; C005653:arabinogalactan