KINETIC-ANALYSIS OF THE ACTIVE-SITE OF AN INTRACELLULAR BETA-GLUCOSIDASE FROM CELLULOMONAS-BIAZOTEA

Purified beta-glucosidase from Cellulomonas biazotea had an apparent K -m and V for 2-nitrophenyl beta-D-glucopyranoside (oNPG) of 0.416 mmol /L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of beta-glucosidase was 65 kJ/mol. The inhibition b y Mn2+ vs. oNPG of parental beta-glucosidase was of mixed type with ap parent inhibition constants of 0.19 and 0.60 mu mol/L for the enzyme a nd enzyme-substrate complex, respectively. Ethanol at lower concentrat ions activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK(a)'s at different temperatures and i n the presence of 30 % dioxane indicated two carboxyl groups which con trol the V value. The thermal stability of beta-glucosidase decreased in the presence of 10 % ethanol. The half-life of beta-glucosidase in 1.75 mol/L urea at 35 degrees C was 145 min, as determined by 0-9 mol/ L transverse urea gradient-PAGE.