Dataset Information

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-?-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.

SUBMITTER: Paukner R

PROVIDER: S-EPMC4370742 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli.

Paukner Regina R Staudigl Petra P Choosri Withu W Haltrich Dietmar D Leitner Christian C

Protein expression and purification 20141224

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 ...[more]

PMID: 25543085

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Dataset Information

Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli.

Publications

Expression, purification, and characterization of galactose oxidase of Fusarium sambucinum in E. coli.

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