Produção, purificação, caracterização e aplicações de uma nova protease e uma endoglucanase do fungo Pleurotus djamor PLO13

Abstract

Brazilian agriculture, driven by technological advances and environmental concerns, is becoming an important driver of development, however it is known that agricultural activities generate large volumes of waste and their disposal in the soil without adequate treatment leads to environmental pollution and adverse effects. negative to health. To this end, research is being carried out to take advantage of these non-commercial raw materials, such as lignocellulosic biomass, originating from agro-industrial waste, to produce useful enzymes through solid state fermentation (SSF) with fungi, such as Pleurotus djamor PLO13. Two of these enzymes, proteases and endoglucanases, are widely produced worldwide and can be applied in the food, pharmaceutical, cosmetics, sugar and alcohol industries, among others. Therefore, the present research aims to produce, purify, characterize and biotechnologically apply the protease and endoglucanase enzymes from the solid state fermentation process with the fungus P. djamor PLO13. Initially, a screening of four agro-industrial residues was carried out, sugarcane bagasse (BCA), pineapple crown (CA), coconut fiber (FC) and wheat bran (FT) as support for the growth of the fungus, and FT proved to be the most efficient. Subsequently, enzymatic assays were carried out to analyze the presence of protease and endoglucanase in the extract, and both were detected. The crude enzymatic extract (EBE) reached its maximum proteolytic activity on casein after 120 hours of cultivation. The specificity of the protease was analyzed in relation to specific synthetic substrates and its inhibition in the presence of selective inhibitors, which allowed it to be classified as a serine protease similar to metal ion-dependent elastase 2, with a specific activity of 3,976 U.mg−1. This enzyme demonstrated high efficiency in the coagulation of pasteurized whole and reconstituted skimmed milk, both in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time and calcium concentration. The endoglucanase present in the crude extract presented an optimum temperature and pH of 50 °C and 5.0, respectively, remaining stable over a wide range of temperature and pH, as well as in the presence of surfactant agents, such as SDS, triton X-100, Tween 40 and Tween 80. Its enzymatic activity was activated in the presence of EDTA, Na+ , ethanol, propanone, dichloromethane, dinitromethane, urea and β-mercaptoethanol, while Mg2+, Ca2+ and Cu2+, methanol and heptane reduced its activity. To purify the enzyme, the organic fractionation process followed by anion exchange chromatography was used. A halotolerant and halophilic endoglucanase was purified, with a molecular mass of approximately 26 kDa. The purified enzyme had an optimum temperature and pH of 50 °C and 5.0, respectively, and was stable between 30 and 70 °C, and a pH of 3.0 to 10.0. Furthermore, the enzymatic activity was activated by the presence of EDTA and Mn2+ , while the presence of Mg2+ inhibited its activity. In terms of enzymatic kinetics, the enzyme exhibited a Km of 0.0997 ± 0.0063 mg/ml and a Vmax of 0.1122 ± 0.00109 mol/min/mL, indicating its effectiveness in the hydrolysis of specific substrates. These results indicate a high potential for industrial application for these enzymes, especially in the dairy, second-generation ethanol production and detergent manufacturing sectors, due to their stability under a wide range of conditions.