title: Purification and characterization of 1,3-β-D-glucan phosphorylase from Ochromonas danica.
abstract: 1,3-β-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-β-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-β-glucans.
authors.affiliation: Graduate School of Bioresources, Mie University.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yamamoto
authors.forename: Daichi
authors.initials: D
authors.lastname: Kawashima
authors.forename: Ayu
authors.initials: A
authors.lastname: Hashizume
authors.forename: Makoto
authors.initials: M
authors.lastname: Hisamatsu
authors.forename: Naoto
authors.initials: N
authors.lastname: Isono
mesh_terms: D004187:Disaccharides / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D044663:Ochromonas / Q000201:enzymology*; D006005:Phosphorylases / Q000302:isolation & purification* / Q000378:metabolism*; D011509:Proteoglycans; D013379:Substrate Specificity; D013696:Temperature; D047071:beta-Glucans / Q000378:metabolism
chemical_list: D004187:Disaccharides; D005958:Glucosephosphates; D011509:Proteoglycans; D047071:beta-Glucans; C014637:laminaribiose; C010770:polysaccharide-K; C031590:glucose-1-phosphate; D006005:Phosphorylases