RESUMO
The purpose of this work was to characterize an alkaline protease from the filamentous fungus Myrothecium verrucaria and to explore its capability to degrade native poultry feathers. The enzyme was purified to homogeneity using a single chromatographic step. Recovery was high, 62%, with a specific activity of 12,851.8 U/mg protein. The enzyme is a small monomeric protein with a molecular mass of 22 +/- 1.5 kDa. It presented pH optimum of 8.3 and was stable over a broad pH range (5.0-12.0). The temperature optimum was 37 degrees C, with thermal stability at temperatures up to 45 degrees C. The enzyme presented an efficiency of 80.3% in the degradation of poultry feather meal, releasing amino acids and soluble peptides. It was able to hydrolyze beta-keratin without necessity of chemical or enzymatic reduction of the disulphide bonds. Considering that, everyday, poultry-processing plants produce feathers as a waste products, this protease can be useful in biotechnological processes aiming to improve the transformation of poultry feathers through solubilization of beta-keratin into usable peptides. Furthermore, it can also be useful in processes aiming to reduce the environmental pollution caused by the accumulation of feathers.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Endopeptidases/isolamento & purificação , Hypocreales/enzimologia , Animais , Proteínas de Bactérias/metabolismo , Biodegradação Ambiental , Endopeptidases/metabolismo , Estabilidade Enzimática , Plumas/metabolismo , Cabelo/metabolismo , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Resíduos Industriais , Queratinas/metabolismo , Unhas/metabolismo , Aves Domésticas , Inibidores de Proteases/farmacologia , Ovinos , Especificidade por Substrato , Lã/metabolismo , beta-Queratinas/metabolismoRESUMO
Myrothecium verrucaria is a nondermatophytic filamentous fungus able to grow and to produce keratinase in submerged (93.0 +/- 19 U/ml) and solid state (98.8 +/- 7.9 U/ml) cultures in which poultry feather powder (PFP) is the only substrate. The purpose of the present work was to verify how different carbon and nitrogen sources can influence the production of keratinase by this fungus. Addition of carbohydrates, such as glucose and sucrose, caused only slight improvements in keratinase production, but the addition of starch caused a significant improvement (135.0 +/- 25 U/ml). The highest levels of keratinase activity, however, were obtained by supplementing the PFP cultures with cassava bagasse, 168.0 +/- 28 U/ml and 189.0 +/- 26 U/ml in submerged and solid state cultures, respectively. Contrarily, the supplementation of PFP medium with organic or inorganic nitrogen sources, such as casein, soy bean protein, gelatin, ammonium nitrate and alanine, decreased the production of keratinase in both types of cultures (around 20 U/ml), showing that the production of keratinase by M. verrucaria is repressed by nitrogen sources. The results obtained in this work suggest that the association of the two residues PFP plus cassava bagasse could be an excellent option as a cheap culture medium for the production of keratinase in submerged and solid state cultures.