Comparison of catalytic properties of free and immobilized cellobiase Novozym 188

The enzyme cellobiase from Novo was immobilized in controlled pore silica p articles by covalent binding with the silane-glutaraldehyde method with pro tein and activity yields of 67 and 13.7%, respectively. Tl;e activity of th e free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75 degreesC at pH 3.0-7.0 for FE and from 40 to 70 degreesC at pH 2.2-7.0 for IE. At FH 4.8 the maximum specific activity for the FE and IE occurred at 65 degreesC: 17.8 and 2.2 micromol of glucose/(mi n mg of protein), respectively. For all temperatures the optimum pH observe d for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activa tion was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5-5, showing appa rent 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 4 0 to 70 degreesC for FE and 40 to 75 degreesC for IE. Free cellobiase maint ained its activity practically constant for 240 min at temperatures up to 5 5 degreesC. The IE has shown higher stability, retaining its activity in th e same test up to 60 degreesC. Half-life experimental results for FE were 1 1.1, 2.1, and 0.17 h at 60, 65, and 70 degreesC, 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 kca l /mol for the IE, suggesting stabilization by immobilization.