title: Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli.
abstract: A gene encoding a galactose oxidase (GalOx) was isolated from Fusarium sambucinum cultures and overexpressed in Escherichia coli yielding 4.4mg enzyme per L of growth culture with a specific activity of 159Umg(-1). By adding a C-terminal His-tag the enzyme could be easily purified with a single affinity chromatography step with high recovery rate (90%). The enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 68.5kDa. The pH optimum for the oxidation of galactose was in the range of pH 6-7.5. Optimum temperature for the enzyme activity was 35°C, with a half-life of 11.2min, 5.3min, and 2.7min for incubation at 40°C, 50°C, and 60°C, respectively. From all tested substrates, the highest relative activity was found for 1-methyl-β-galactopyranoside (226Umg(-1)) and the highest catalytic efficiency (kcat/Km) for melibiose (2700mM(-1)s(-1)). The enzyme was highly specific for molecular oxygen as an electron acceptor, and showed no appreciable activity with a range of alternative acceptors investigated. Different chemicals were tested for their effect on GalOx activity. The activity was significantly reduced by EDTA, NaN3, and KCN.
authors.affiliation: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Regina
authors.initials: R
authors.lastname: Paukner
authors.affiliation: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Staudigl
authors.affiliation: Department of Food Technology, Ramkhamhaeng University, Bangkok, Thailand.
authors.forename: Withu
authors.initials: W
authors.lastname: Choosri
authors.affiliation: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria. Electronic address: christian.leitner@boku.ac.at.
authors.forename: Christian
authors.initials: C
authors.lastname: Leitner
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D005692:Galactose Oxidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D005692:Galactose Oxidase
keywords: Alternative electron acceptors; Fusarium sambucinum; Galactose oxidase; Gene cloning; Heterologous expression; Steady-state kinetics