| Autor: |
Faria CB; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil., de Castro FF; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil., Martim DB; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil., Abe CAL; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil., Prates KV; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil., de Oliveira MAS; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil., Barbosa-Tessmann IP; Department of Biochemistry, Universidade Estadual de Maringá, Av. Colombo, 5790, Maringá, PR, 87020-900, Brazil. ipbtessmann@uem.br. |
| Abstrakt: |
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. |
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