Open AccessGalactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization
Author(s) -
Regina Paukner,
Petra Staudigl,
Withu Choosri,
Christoph Sygmund,
Petr Halada,
Dietmar Haltrich,
Christian Leitner
Publication year - 2014
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0100116
Subject(s) - pichia pastoris , escherichia coli , enzyme kinetics , biochemistry , fusarium oxysporum , heterologous expression , chemistry , cysteine , pichia , enzyme , stereochemistry , biology , active site , gene , recombinant dna , botany
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 ( k cat /K m ) was found with 1-methyl-β-D-galactopyranoside (2.2 mM −1 s −1 ). The Michaelis constant (K m ) 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.