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Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization
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SYSNO ASEP 0440666 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization Author(s) Paukner, R. (AT)
Staudigl, P. (AT)
Choosri, W. (TH)
Sygmund, Ch. (AT)
Halada, Petr (MBU-M) RID, ORCID
Haltrich, D. (AT)
Leitner, CH. (AT)Number of authors 7 Source Title PLoS ONE. - : Public Library of Science - ISSN 1932-6203
Roč. 9, č. 6 (2014)Number of pages 8 s. Language eng - English Country US - United States Keywords galactose oxidase ; gene ; Fusarium ; gene expression Subject RIV CE - Biochemistry Institutional support MBU-M - RVO:61388971 UT WOS 000338280800025 Annotation 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-beta-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 degrees C, respectively, and the enzyme was thermoinactivated at temperatures above 50 degrees 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. Workplace Institute of Microbiology Contact Eliška Spurná, eliska.spurna@biomed.cas.cz, Tel.: 241 062 231 Year of Publishing 2015
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