Purification and characterization of a beta-glucosidase from Evernia prunastri.

Intracellular beta-glucosidase from Evernia prunastri has been purified to homogeneity using anion exchange on DEAE-Sephadex A-50, and gel filtration chromatography on Sephadex G-100 and Sepharose 6B. The purified beta-glucosidase showed a single protein band on native electrophoresis and its isoelectric point was at pH 3.12. The molecular mass, calculated from its partition coefficient on the Sepharose 6B column, was 311 kDa, being composed of several subunits of 60 and 70 kDa. The highest activity of this enzyme was attained at pH 4.0 and 60 degrees C. The enzyme showed strong resistance to thermal inactivation. Its activation energy was about 15 kJ/mol. Cellobiose, salicin, and p-nitrophenyl beta-D-glucoside, but not carboxymethylcellulose, were hydrolyzed by the enzyme, following substrate inhibition kinetics. The purified beta-glucosidase was considered a true cellobiase because of its great affinity towards cellobiose. Cellobiose inhibition does not seem to be a physiological phenomenon. Glucose inhibited enzyme activity in a competitive way (Ki = 1.26 mM). Fe3+ and Co2+ inhibited activity notably. Hg2+, Cu2+ and EDTA were practically ineffective. Even 200 mM gluconolactone did not affect enzyme activity.