RESUMO
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.
Assuntos
Fusarium/enzimologia , Galactose Oxidase/metabolismo , Galactose/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fusarium/química , Galactose/metabolismo , Galactose Oxidase/química , Galactose Oxidase/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Concentração de Íons de Hidrogênio , Melibiose/química , Melibiose/metabolismo , Metilgalactosídeos/química , Metilgalactosídeos/metabolismo , Modelos Moleculares , Oxirredução , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Rafinose/química , Rafinose/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TemperaturaRESUMO
Galactose oxidase (GO) converts galactose to an aldehyde and has several biotechnological applications, including cancer diagnosis. It is mainly produced by Fusarium austroamericanum but is also produced by Fusarium acuminatum and by isolates of the Fusarium graminearum and Gibberella fujikuroi complexes. The F. austroamericanum GO gaoA gene has been cloned, but the GO genes from other secreting species have not been characterized. Problems associated with the F. austroamericanum GO such as high pI and low catalytic efficiency and thermostability, and the difficult purification process makes the search for homologous genes attractive. In this work, the GO genes from Fusarium verticillioides and Fusarium subglutinans, two species of the G. fujikuroi complex, were cloned, sequenced, and analyzed. New GO genes were found in databases and were used to construct a phylogenetic tree, which revealed the existence of three orthologous lineages of GO genes in Fusarium spp. In addition, RT-PCR analyses revealed that the new GO cloned gene may be endogenously expressed in F. subglutinans but not in F. verticillioides, in the used culture conditions.
Assuntos
Fusarium/enzimologia , Fusarium/genética , Galactose Oxidase/genética , Sequência de Aminoácidos , Clonagem Molecular , Análise por Conglomerados , DNA Fúngico/química , DNA Fúngico/genética , Perfilação da Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
1. Mitochondrial DNAs from Dactylium dendroides, Hypomyces rosellus, Fusarium graminearum, Gibberella fujikuroi, Fusarium tricinctum strains and a galactose oxidase (GAO)-producing mold (original strain) presented distinctive restriction enzyme fragment patterns with the endonucleases Hind III and EcoRI. 2. A small number of comigrating bands was found when the GAO-producing mold was compared with the others. The molecular size of mtDNA from the GAO-producing mold, as judged by summation of fragment sizes produced by digestion with EcoRI, Hind III and Bgl II, is 61.3 +/- 2.16 kb. 3. The results suggest that the mtDNA from the GAO-producing mold strain is distinct from that of D. dendroides and all other ascomycetes analyzed.
Assuntos
DNA Fúngico/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Galactose Oxidase/biossíntese , Fungos Mitospóricos/classificação , Polimorfismo de Fragmento de Restrição , Basidiomycota , Galactose Oxidase/genética , Fungos Mitospóricos/enzimologiaRESUMO
1. Mitochondrial DNAs from Dactylium dendroides, Hypomyces rosellus, Fusarium graminearum, Gibberella fujikuroi, Fusarium tricinctum strains and a galactose oxidase (GAO)-producing mold (original strain) presented distinctive restriction enzyme fragment patterns with the endonucleases Hind III and EcoRI. 2. A small number of comigrating bands was found when the GAO-producing mold was compared with the others. The molecular size of mtDNA from the GAO-producing mold, as judged by summation of fragment sizes produced by digestion with EcoRI, Hind III and Bgl II, is 61.3 +/- 2.16 kb. 3. The results suggest that the mtDNA from the GAO-producing mold strain is distinct from that of D. dendroides and all other ascomycetes analyzed
Assuntos
DNA Fúngico/isolamento & purificação , DNA Mitocondrial/isolamento & purificação , Galactose Oxidase/biossíntese , Fungos Mitospóricos/classificação , Polimorfismo de Fragmento de Restrição , Basidiomycota , Galactose Oxidase/genética , Fungos Mitospóricos/enzimologiaRESUMO
The effect of ethanol and tunicamycin on synthesis and secretion of galactose oxidase was studied in resting cells of Dactylium dendroides. Ethanol promoted an overall decrease in both intra- and extracellular enzyme levels to the same extent that it inhibited [14C]glucosamine incorporation into total protein. The carbohydrate content of the intracellular enzyme was also depressed (44%) with a simultaneous decrease in O-Ser linked oligosaccharides. The intracellular galactose oxidase obtained after exposure of mycelia to ethanol plus tunicamycin lost 86% of its carbohydrate moieties, whereas the extracellular form lost only 35%. In both cases, residual sugar moieties were not eliminated by mild alkaline treatment. These data suggest that ethanol affects O-glycosylation of galactose oxidase. O-Underglycosylation did not affect the S0.5 values for galactose but diminished the molar catalytic activity. The absence of O-Ser/Thr-linked saccharides turned the intracellular enzyme into a form more susceptible to proteolysis than that devoid of N-linked sugars (tunicamycin-treated). O-Underglycosylation had a significant effect on the renaturation-reactivation of the enzyme after denaturation with 2.4 M Gdn-HCl.