title: Galactose oxidase from Fusarium oxysporum--expression in E. coli and P. pastoris and biochemical characterization.
abstract: A gene coding for galactose 6-oxidase from Fusarium oxysporum G12 was cloned together with its native preprosequence and a C-terminal His-tag, and successfully expressed both in Escherichia coli and Pichia pastoris. The enzyme was subsequently purified and characterized. Among all tested substrates, the highest catalytic efficiency (kcat/Km) was found with 1-methyl-β-D-galactopyranoside (2.2 mM(-1) s(-1)). The Michaelis constant (Km) for D-galactose was determined to be 47 mM. Optimal pH and temperature for the enzyme activity were 7.0 and 40°C, respectively, and the enzyme was thermoinactivated at temperatures above 50°C. GalOx contains a unique metalloradical complex consisting of a copper atom and a tyrosine residue covalently attached to the sulphur of a cysteine. The correct formation of this thioether bond during the heterologous expression in E. coli and P. pastoris could be unequivocally confirmed by MALDI mass spectrometry, which offers a convenient alternative to prove this Tyr-Cys crosslink, which is essential for the catalytic activity of GalOx.
authors: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Regina
authors: R
authors: Paukner
authors: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Petra
authors: P
authors: Staudigl
authors: Department of Food Technology, Ramkhamhaeng University, Bangkok, Thailand.
authors: Withu
authors: W
authors: Choosri
authors: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Christoph
authors: C
authors: Sygmund
authors: Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors: Petr
authors: P
authors: Halada
authors: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Dietmar
authors: D
authors: Haltrich
authors: Department of Food Science and Technology, BOKU-University of Natural Resources and Life Sciences, Vienna, Austria.
authors: Christian
authors: C
authors: Leitner
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D004987:Ethers / Q000737:chemistry; D005670:Fusarium / Q000201:enzymology* / Q000235:genetics; D005692:Galactose Oxidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011487:Protein Conformation
chemical_list: D004987:Ethers; D005692:Galactose Oxidase