About: Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization     Goto   Sponge   NotDistinct   Permalink

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  • 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.
  • 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. (en)
Title
  • Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization
  • Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization (en)
skos:prefLabel
  • Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization
  • Galactose Oxidase from Fusarium oxysporum - Expression in E. coli and P. pastoris and Biochemical Characterization (en)
skos:notation
  • RIV/61388971:_____/14:00440666!RIV15-AV0-61388971
http://linked.open...avai/riv/aktivita
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  • I
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  • 6
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  • 17890
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  • RIV/61388971:_____/14:00440666
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  • galactose oxidase; gene; Fusarium; gene expression (en)
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  • US - Spojené státy americké
http://linked.open...ontrolniKodProRIV
  • [FD295F8F031A]
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  • PLoS ONE
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  • 9
http://linked.open...iv/tvurceVysledku
  • Halada, Petr
  • Haltrich, D.
  • Choosri, W.
  • Leitner, CH.
  • Paukner, R.
  • Staudigl, P.
  • Sygmund, Ch.
http://linked.open...ain/vavai/riv/wos
  • 000338280800025
issn
  • 1932-6203
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