Cellobiose hydrolysis usingPichia etchellsii cells immobilized in calcium alginate

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Abstract
The rate of celluose degradation, limited due to the inhibition by cellobiose, can be increased by the hydrolysis of cellobiose to glucose using immobilized β‐glucosidase. Production of β‐glucosidase in four yeasts was studied and a maximum activity of 1.22 IU/mg cells was obtained in cells of Pichia etchellsii when grown on 3% cellobiose as the sole carbon source. A study of the immobilization of β‐glucosidase containing cells of Pichia etchellsii on various solid supports was conducted and immobilization by entrapment in calcium alginate gel beads was found to be the most simple and efficient method. A retention of 96.5% of initial activity after ten sequential batch uses of the immobilized preparation was observed. The pH and temperature optima for free and immobilized cells were the same, i.e., 6.5 (0.05M Maleate buffer) and 50°C, respectively. Even though the temperature optimum was found to be 50°C, the enzyme exhibits a better thermal stability at 45°C. Beads stored at 4°C for six months retain 80% of their activity. Kinetic studies performed on free and immobilized cells shown that glucose is a noncompetitive product inhibitor. The immobilized preparation was found to be limited by pore diffusion but exhibited no film‐diffusion resistance during packed bed column indicated by a low dispersion number of 0.1348. A model for reaction with pore diffusion for a noncompetitive type of inhibited system was developed and applied to the cellobiose hydrolysis system. The rate of reaction with diffusional limitations was determined by using the model and effectiveness factors were calculated for different particle sizes. An effectiveness factor of 0.49 was obtained for a particle diameter of 2.5 mm. The modified rate expression using the effectiveness factor represented batch and packed bed reactor operation satisfactorily. The productivity in the packed bed column was found to fall rapidly with increase in conversion rate indicating that the operating conditions of the column would have to be a compromise between high conversion rates and reasonable productivity. A half‐life of over seven days was obtained at the operating temperature of 45°C in continuous operation of the packed bed reactor. However, the half‐life in the column was found to be greatly affected by temperature, increasing to over seventeen days at a temperature of 40°C and decreasing to less than two days at 50°C.

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