Comparison of catalytic properties of free and immobilized cellobiase novozym 188.

The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75 degrees C at pH 3.0-7.0 for FE and from 40 to 70 degrees C at pH 2.2-7.0 for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65 degrees C: 17.8 and 2.2 micromol of glucose/(min x mg of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5-5, showing apparent diffusional limitation for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40 to 70 degrees C for FE and 40 to 75 degrees C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55 degrees C. The IE has shown higher stability, retaining its activity in the same test up to 60 degrees C. Half-life experimental results for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70 degrees C, respectively, whereas IE at the same temperatures had half-lives of 245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 kcal/mol for the free enzyme and 85.2 kcal/mol for the IE, suggesting stabilization by immobilization.

Modeling cellobiose hydrolysis with integrated kinetic models.

The enzyme cellobiase Novozym 188, which is used for improving hydrolysis of bagasse with cellulase, was characterized in its commercial available form and integrated kinetic models were applied to the hydrolysis of cellobiose. The specific activity of this enzyme was determined for pH values from 3.0-7.0, and temperatures from 40-75 degrees C, with cellobiose at 2 g/L. Thermal stability was measured at pH 4.8 and temperatures from 40-70 degrees C. Substrate inhibition was studied at the same pH, 50 degrees C, and cellobiose concentrations from 0.4-20 g/L. Product inhibition was determined at 50 degrees C, pH 4.8, cellobiose concentrations of 2 and 20 g/L, and initial glucose concentration nearly zero or 1.8 g/L. The enzyme has shown the greatest specific activity, 17.8 U/mg, at pH 4.5 and 65 degrees C. Thermal activation of the enzyme followed Arrhenius equation with the Energy of Activation being equal to 11 kcal/mol for pH values 4 and 5. Thermal deactivation was adequately modeled by the exponential decay model with Energy of Deactivation giving 81.6 kcal/mol. Kinetics parameters for substrate uncompetitive inhibition were: Km = 2.42 mM, Vmax = 16.31 U/mg, Ks = 54.2 mM. Substrate inhibition was clearly observed above 10 mM cellobiose. Product inhibition at the concentration studied has usually doubled the time necessary to reach the same conversion at the lower temperature tested.