title: Enhanced production of recombinant galactose oxidase from Fusarium graminearum in E. coli.
abstract: The gene gaoA encoding the copper-dependent enzyme galactose oxidase (GAO) from Fusarium graminearum PH-1 was cloned and successfully overexpressed in E. coli. Culture conditions for cultivations in shaken flasks were optimized, and optimal conditions were found to be double-strength LB medium, 0.5% lactose as inducer, and induction at the reduced temperature of 25°C. When using these cultivation conditions ~24 mg of active GAO could be produced in shaken flasks per litre medium. Addition of copper to the fermentation medium decreased the enzyme production significantly. The His-tagged recombinant enzyme could be purified conveniently with a single affinity chromatography step. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular mass of 66 kDa and had kinetic properties similar to those of the fungal wild-type enzyme.
authors.affiliation: Department of Food Sciences and Technology, Food Biotechnology Laboratory, BOKU, University of Natural Resources and Life Sciences, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Withu
authors.initials: W
authors.lastname: Choosri
authors.forename: Regina
authors.initials: R
authors.lastname: Paukner
authors.forename: Petra
authors.initials: P
authors.lastname: Wührer
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.forename: Christian
authors.initials: C
authors.lastname: Leitner