RESUMO
Fusarium graminearum strains are well known for their role as plant pathogens and for their production of mycotoxins, and less known for their secretion of galactose oxidase, a well-studied and useful enzyme. Three galactose oxidase-producing isolates of F. graminearum were grown on rice to identify the production of zearalenone and trichothecenes through the use of thin layer chromatography and gas chromatography coupled to mass fragmentation. Detection and identification of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone were accomplished.
Assuntos
Fusarium/patogenicidade , Galactose Oxidase/biossíntese , Micotoxinas/análise , Tricotecenos/análise , Zearalenona/análise , Fusarium/enzimologiaRESUMO
Fifty-two isolates of Fusarium species and one of Gibberella fujikuroi were tested for galactose oxidase (GO) production. Five Fusarium isolates contained GO activity in the culture filtrate: three F. graminearum and one each F. moniliforme f. sp. subglutinans and F. acuminatum. This is the first time F. acuminatum is reported to be a producer of GO enzyme. GO enzyme activity produced by isolates was assayed through a time course. Moreover, GO protein was partially purified from the most productive four isolates to show that the activity measured in the culture filtrates was due to the presence of GO protein.
Assuntos
Fusarium/classificação , Fusarium/enzimologia , Galactose Oxidase/biossíntese , Meios de Cultura , Galactose Oxidase/isolamento & purificação , Fatores de TempoRESUMO
The galactose oxidase-producing fungus Dactylium dendroides was re-identified as a Fusarium species. Fungi of this genus are well known for the production of mycotoxins. Verification of growth of this fungus on rice, corn and liquid medium described for the production of galactose oxidase is provided to determine whether the fungus could produce Fusarium toxins, namely, moniliformin, fusaric acid, fumonisin, zearalenone and the trichothecenes, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenone, nivalenol, diacetoxyscirpenol, neosolaniol, and toxin T-2. Under the culture conditions used, deoxynivalenol, 3-acetyldeoxynivalenol and zearalenone were detected in the fungal culture medium. The finding is consistent with the hypothesis that the fungus is in fact a Fusarium species.
Assuntos
Fusarium/metabolismo , Galactose Oxidase/biossíntese , Micotoxinas/biossíntese , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Fusarium/enzimologia , Fusarium/isolamento & purificação , Cromatografia Gasosa-Espectrometria de Massas , Masculino , Micotoxinas/análise , Micotoxinas/toxicidade , Ratos , Ratos Wistar , Pele/efeitos dos fármacos , Testes de Irritação da Pele , Tricotecenos/análise , Tricotecenos/biossíntese , Tricotecenos/toxicidade , Zearalenona/análise , Zearalenona/biossíntese , Zearalenona/toxicidadeRESUMO
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.