Design and characterisation of an enzyme system for inulin hydrolysis

The optimal conditions for inulin hydrolysis using a commercial inulinase preparation, either free or immobilised in activated Amberlite were established by factorial design and surface response methodology. The immobilised biocatalyst displayed highest activity at pH 5.5 and 50°C, whereas the optimum pH for the free form was slightly more acidic (4.5), and the optimum temperature was a little higher (55°C). The model system estimated optimal pH and temperature values of 5.4 and 52°C for the immobilised system and 4.9 and 56°C for the free system. Michaelis-Menten type kinetics adequately described both free and immobilised bioconversion systems, which were evaluated under the respective optimal pH and temperature conditions. The use of a non-linear regression method for the determination of the kinetic parameters provided a best fit to the experimental data, as compared to a conventional Lineweaver-Burk linearisation. The K_m for inulin of the free biocatalyst was 153 g l^-1 at 55°C and pH 4.5, whereas the apparent K_m for inulin of the immobilised biocatalyst was 108 g l^-1 at pH 5.5 and 50°C. The reutilisation of the immobilised biocatalyst throughout consecutive batches was evaluated. A significant decrease of enzyme activity was observed in the first two batches, after which the system exhibited significant stability. The low cost of the support, the stability of the immobilised biocatalyst towards pH and temperature and its high affinity for the substrate suggests its potential for inulin hydrolysis.