RESUMO
In this work, the extracellular protease Eap1 from Sporisorium reilianum was characterized in solid and liquid cultures using different culture media. The results showed that Eap1 was produced in all media and under all culture conditions, with the most activity in solid culture at an acidic pH of 3-5. Following purification, the 41 kDa protease demonstrated aspartyl protease activity. The enzyme was stable at a wide range of temperatures and pH values, but 45°C and pH 3 were optimal. The K(m) and V(max( values obtained were 0.69 mg/mL and 0.66 µmol/min, respectively, with albumin as the substrate. Eap1 degraded hemoglobin as well as proteins obtained from corn germ, roots, stems and slides at pH 3 and also had milk-clotting activity. Sequencing analysis showed that this protein has 100% similarity to the peptide sequence theoretically obtained from the sr11394 gene, which encodes an aspartyl protease secreted by S. reilianum.
Assuntos
Ácido Aspártico Proteases/química , Ácido Aspártico Proteases/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ustilaginales/enzimologia , Albuminas/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Meios de Cultura/química , Meios de Cultura/metabolismo , Espaço Extracelular , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Dados de Sequência Molecular , Alinhamento de Sequência , Temperatura , Ustilaginales/químicaRESUMO
The objective of the present work was to evaluate the water hyacinth (WH) as a substrate for the production of hydrolytic enzymes (cellulases and hemicellulases) of 100 strains of filamentous fungi under conditions of solid growth. Five fungal strains, identified as Trichoderma harzianum, Trichoderma atroviride, Penicillium griseofulvum, Penicillium commune and Aspergillus versicolor, were selected and studied for their ability to grow on water hyacinth as a substrate and carbon source only, evaluating hydrolytic enzymatic activities (α-l-arabinofuranosidase, cellulase, xylanase and ß-d-xylopyranosidase) and extracellular protein per g of water hyacinth dry matter (gdm). The five strains selected were able to produce the four enzymes studied; however, T. harzianum strain PBCA produces the highest xylanase (149.3 ± 14.3 IU/gdm at 108 h), cellulase (16.4 ± 0.6 IU/gdm at 84 h) and ß-d-xylopyranosidase (127.7 ± 14.8 IU/gdm at 48 h). In contrast, the fungus with the highest α-l-arabinofuranosidase activity was A. versicolor, with 129.8 ± 13.3 IU/gdm after 108 h. In conclusion, T. harzianum showed the best production of the hydrolytic enzymes studied, using as a matrix and carbon source, water hyacinth. In addition, catalytic activities of arabinofuranosidase and xylopyranosidase were reported for the first time in T. versicolor and T. harzianum.