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

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 ox...

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Veröffentlicht in:Molecular biotechnology 2019-09, Vol.61 (9), p.633-649
Hauptverfasser: Faria, Carla Bertechini, de Castro, Fausto Fernandes, Martim, Damaris Batistão, Abe, Camila Agnes Lumi, Prates, Kelly Valério, de Oliveira, Marco Aurelio Schuler, Barbosa-Tessmann, Ione Parra
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Sprache:eng
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Zusammenfassung: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, K M of 132.6 ± 18.18 mM, V max of 3.2 ± 0.18 µmol of H 2 O 2 /min, k cat of 12,243 s −1 , and a catalytic efficiency ( k cat / K M ) of 9.2 × 10 4  M −1  s −1 . In the presence of 50% glycerol, the enzyme showed a T 50 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.
ISSN:1073-6085
1559-0305
DOI:10.1007/s12033-019-00190-6