Purification, determination of biochemical characterization of β-glucosidase from trichoderma atroviride

In this research, the Beta-glucosidase enzyme from Trichoderma atroviride NRRL 25150 was produced by the Solid State Fermentation (SSF) method utilizing ground bean pods. Subsequently, the enzyme obtained in this way was then purified using two different methods. The first step was the determination of the optimal conditions for the production of β-glucosidase from Trichoderma atroviride in SSF medium. Citric acid monohydrate buffer (C6H8O7) as humidifying liquid, pH 5, temperature 25°C and incubation time 4 days were considered as optimal conditions. The purification yield and the fold number of the purified β-glucosidase were determined to be 17.1434 % and 6.7117 fold, respectively. Sodium Dodecyl Sulfate-Polyacrylamide Gel electrophoresis was used to determine the purity and the presence/number of subunits of β-glucosidase and it was found that the enzyme has two subunits with molecular weights of approximately 70 kDa and 60 kDa. The activity of the purified enzyme was evaluated with the use of p-nitrophenyl-β-D-glucopyranoside (pNPG). The temperature and pH at which the enzyme exhibited high activity were determined to be 65°C and pH 3 respectively. The kinetic properties of β-glucosidase were also investigated. Km and Vmax were calculated as 0.0869 mM and 434.782 EU, respectively. Furthermore, δ-gluconolactone and D(+)-glucose inhibitors were found to exert mixed inhibition on β- glucosidase enzyme in the absence of pNPG substrate. Ki and IC50 of δ-gluconolactone inhibitor were calculated as Ki(1): 0.0712x10-3, Ki(2): 0.068x10-3 and 8.4214 mM; Ki and IC50 values of D(+)-glucose inhibitor were calculated as Ki(1): 4.31x10-3, Ki(2): 29.45x10-3 and 9.7395 mM, respectively.