Purificação, caracterização bioquímica e imobilização de uma β-glucosidase de Rasamsonia composticola

Abstract

β-glycosidases are enzymes that are part of the cellulolytic complex with the ability to act on lignocellulosic materials, promoting their hydrolysis. These enzymes are produced by microorganisms, mainly filamentous fungi. They have varied applications such as in the pulp and paper industry, food, animal feed and in the production of second generation ethanol. The present work involves the purification, biochemical characteristics and immobilization of a β-glucosidase resistant to the thermophilic fungus Rasamsonia composticola. The enzyme was purified using two chromatographic steps, including DEAE-Fractogel and hydrophobic Phenyl-Sepharose ion exchange. It was purified 19 times with a final yield of 14%. A purified β-glucosidase having an optimum temperature of 70 °C, remaining stable at a temperature of 65 to 70 °C for up to 8 hours, pH 5.0, and a molecular mass of approximately 45 kDa. The enzyme was not stimulated by most of the chemical compounds tested, however it was slightly stimulated by the detergents saponin and triton X-100, in addition to the chelators EDTA and EGTA and the reducing agent Temed. The enzyme has activity on the synthetic substrates pNP-Glc and pNP-Xyl, and is shown to be glucose tolerant and stimulated by xylose (1-100 mM). The estimated kinetic parameters for a β-glucosidase were Km and Vmax of 2.3 mM and 0.0221 µmol / min / mg for pNP-Glc. The catalytic efficiency (Kcat / Km) of the enzyme was 7.7x10-11. For pNP-Glc + glucose and pNP-Glc + xylose, the enzyme presented Km and Vmax of 2.33 mM and 0.0271 µmol / min / mg and 2.02 mM and 0.0255 µmol / min / mg, respectively . The enzyme was immobilized in glyoxyl agarose about 40% and 56% in the presence of glucose for 60 minutes. It demonstrated optimal activity at pH 5.0 and 75 °C. The immobilized enzyme is stable at temperatures from 75 to 80 °C during 60 minutes of reaction. The results demonstrate that the fungus Rasamsonia composticola can be a candidate to contribute to the knowledge of the properties of a β-glucosidase with great potential for biotechnological application.