Encapsulation and characterization of cellulase purified with three-phase partitioning technique

Abstract

Purification of Trichoderma reesei cellulase by three-phase partitioning (TPP) technique and encapsulation of the enzyme in calcium alginate gel were studied. These two basic experiments were optimized with all variables and finally optimum conditions for both purification and encapsulation studies were determined. Ideal TPP condition for the purification of T. reesei cellulase was determined as; 50% (w/v) ammonium sulphate saturation, 1:1 (v/v) enzyme: t-butanol ratio, pH 4.5 and 0.1 mg of the protein. By using this condition, the cellulase enzyme was successfully purified with 113% activity yield and 1.42 purification fold. As a result of the encapsulation studies, optimum immobilization condition for cellulase was determined as 0.5 mg enzyme (protein amount) and 2% (w/v) alginate, 0.5% (w/v) chitosan in 0.15 M CaCl2, 3 h for immobilization, 2.0% (v/v) glutaraldehyde and 2 h for cross-linking. The enzyme was immobilized with 82% activity yield by using this conditions. The cellulase, which was concentrated in the middle phase of TPP, could also immobilized with this condition. The optimal temperature of the free and immobilized cellulase were determined as 65 °C and the optimum pHs were found as pH 4.0 and pH 5.0, respectively. The immobilized cellulase was also used three times with high activity yields. The biochemical characterization and stability studies confirm that this immobilized enzyme show promise for industry that could be safely used especially both in the feed and food industry and food processing.