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.
Subject(s)
Fusarium/enzymology , Galactose Oxidase/metabolism , Galactose/chemistry , Recombinant Proteins/metabolism , Amino Acid Sequence , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Fusarium/chemistry , Galactose/metabolism , Galactose Oxidase/chemistry , Galactose Oxidase/genetics , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Hydrogen-Ion Concentration , Melibiose/chemistry , Melibiose/metabolism , Methylgalactosides/chemistry , Methylgalactosides/metabolism , Models, Molecular , Oxidation-Reduction , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Raffinose/chemistry , Raffinose/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , TemperatureABSTRACT
Filamentous fungi produce a great variety of enzymes, and research on their biotechnological potential has recently intensified. The objective of this work was to identify, at the species level, using DNA barcoding, 46 fungal isolates obtained from maize grains with rot symptoms. We also analyzed the production of extracellular amylases, cellulases, proteases and lipases of 33 of those fungal isolates. The enzymatic activities were evaluated by the formation of a clear halo or a white precipitate around the colonies in defined substrate media. The found fungi belong to the genera Talaromyces, Stenocarpella, Penicillium, Phlebiopsis, Cladosporium, Hyphopichia, Epicoccum, Trichoderma, Aspergillus, Irpex, Fusarium, Microdochium, Mucor and Sarocladium. In the genus Fusarium, the species Fusarium verticillioides was predominant and this genus presented the highest diversity, followed by the genera Aspergillus. The best genera for lipase production were Cladosporium and Penicillium; while Cladosporium, Aspergillus and Penicillium were best for cellulase activity; Hyphopichia, Aspergillus and Irpex for amylase activity; and Cladosporium and Sarocladium for proteases activity. In conclusion, a collection of fungi from maize seeds presenting rotten symptoms were obtained, among which exist important producers of hydrolases.
