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Production of Galactose Oxidase Inside the Fusarium fujikuroi Species Complex and Recombinant Expression and Characterization of the Galactose Oxidase GaoA Protein from Fusarium subglutinans

  • Carla Bertechini Faria
  • Fausto Fernandes de Castro
  • Damaris Batistão Martim
  • Camila Agnes Lumi Abe
  • Kelly Valério Prates
  • Marco Aurelio Schuler de Oliveira
  • Ione Parra Barbosa-TessmannEmail author
Original paper
  • 9 Downloads

Abstract

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of d-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s−1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M−1 s−1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides d-(+)-galactose, the purified enzyme also acted against d-(+)-raffinose, α-d-(+)-melibiose, and methyl-α-d-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

Keywords

Galactose oxidase Heterologous expression Fusarium subglutinans Fusarium fujikuroi 

Notes

Acknowledgments

The authors thank CNPq (Conselho Nacional de Pesquisa e Desenvolvimento—Ministry of Science and Technology—Brazil) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Grant 001—Ministry of Education, Brazil) for the students’ scholarships. The authors are also thankful to the Department of Biochemistry from the Federal University of Paraná, Brazil, for the use of its MS facility.

Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Carla Bertechini Faria
    • 1
  • Fausto Fernandes de Castro
    • 1
  • Damaris Batistão Martim
    • 1
  • Camila Agnes Lumi Abe
    • 1
  • Kelly Valério Prates
    • 1
  • Marco Aurelio Schuler de Oliveira
    • 1
  • Ione Parra Barbosa-Tessmann
    • 1
    Email author
  1. 1.Department of BiochemistryUniversidade Estadual de MaringáMaringáBrazil

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