Produção, isolamento, caracterização e aplicação biotecnológica da endoglucanase do fungo filamentoso Pycnoporus sanguineus

Abstract

The growth of the world population increased investment in agribusiness and, consequently, increased the generation of agroindustrial residues, mostly lignocellulosic. Clean alternatives for the use of agro-industrial residues have been researched around the world, among them is the use of residues as a source for fungal growth for the production of enzymes of biotechnological interest. The objectives of this work were to produce, isolate, characterize and biotechnologically apply the endoglucanase produced from the filamentous fungus Pycnoporus sanguineus. Five different agro-industrial residues (wheat bran, sugarcane bagasse, wood powder, coconut fiber and filter paper) were used by solid state fermentation (FES). The enzyme was isolated through ethanol fractionation and ion exchange chromatography, the isolation was confirmed by (SDS-PAGE) in reducing and denaturing conditions. The isolated endoglucanase was characterized for temperature, pH, halotolerance, enzymatic kinetics and applied biotechnologically through the saccharification of agro-industrial residues. Endoglucanase production was higher in wheat bran (72 hs). The 80-100% fraction of the ethanol fractionation concentrated greater enzymatic activity and was subjected to ion exchange chromatography (DEAE-Sepharose), the isolated enzyme was eluted in a single fraction, as confirmed by SDS-PAGE. The optimal temperature of the isolated endoglucanase was 50°C and the thermal stability was between 20° and 60°C with up to 50% of the enzymatic activity, temperatures compatible with other fungal endoglucanases. The optimum pH was pH 5.0, with stability at pH between pH 4.5 and 8.0, demonstrating stability at slightly alkaline pH compatible with biotechnological applications in the detergent industry. The isolated enzyme remained viable with activity greater than 100% in up to 5 M NaCl, an important characteristic because some industrial processes take place in the presence of large amounts of NaCl. The enzyme presented Km 3.18 ± 100 mg/mL and the Kcat equal to 4.53 S-1 and managed to produce 273 mg/mL reducing sugars in 24 hours in rice husks, proving to be viable for the production of second-generation ethanol . The present work concluded that the fungus P. sanguineus is a producer of an endoglucanase with important biochemical characteristics, fast production and low cost.