Kinetic analysis of the active site of an intracellular beta-glucosidase fromCellulomonas biazotea.

Purified beta-glucosidase fromCellulomonas biazotea had an apparentK (m) andV 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 by Mn(2+) vs. oNPG of parental beta-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 micromol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK (a)'s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV 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.