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- Title
Galactose Oxidase from <i>Fusarium oxysporum</i> - Expression in <i>E. coli</i> and <i>P. pastoris</i> and Biochemical Characterization.
- Authors
Paukner, Regina; Staudigl, Petra; Choosri, Withu; Sygmund, Christoph; Halada, Petr; Haltrich, Dietmar; Leitner, Christian
- 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.
- Subjects
GALACTOSE oxidase; FUSARIUM oxysporum; GENE expression; ESCHERICHIA coli; CLONING; PYRANOSIDE
- Publication
PLoS ONE, 2014, Vol 9, Issue 6, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0100116