Production, purification, and determination of the biochemical properties of β-glucosidase in Trichoderma koningii via solid substrate fermentation

Abstract

The beta-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The beta-glucosidase was obtained from T. koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 degrees C and 6 days. The purification efficiency of the obtained beta-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of beta-glucosidase activity, p-nitrophenyl-beta-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which beta-glucosidase showed high activity were determined to be pH 3.0 and 75 degrees C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K-m and V-max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. It was also found that d-(+)-glucose and delta-gluconolactone inhibitors exhibited competitive inhibition of beta-glucosidase in the presence of pNPG.