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.

摘要

研究了通过三相分配 (TPP) 技术纯化里氏木霉纤维素酶并将酶封装在海藻酸钙凝胶中。这两个基本实验用所有变量进行了优化，并最终确定了纯化和封装研究的最佳条件。纯化里氏木霉的理想 TPP 条件纤维素酶被确定为；50% (w/v) 硫酸铵饱和度，1:1 (v/v) 酶：叔丁醇比，pH 4.5 和 0.1 mg 蛋白质。在此条件下，纤维素酶以113%的活性产率和1.42倍的纯化倍数成功纯化。作为封装研究的结果，纤维素酶的最佳固定条件确定为 0.5 mg 酶（蛋白质量）和 2% (w/v) 藻酸盐、0.5% (w/v) 壳聚糖在 0.15 M CaCl _{2 中}, 3 小时用于固定，2.0% (v/v) 戊二醛和 2 小时用于交联。使用该条件将酶固定化，活性产率为 82%。浓缩在 TPP 中间相的纤维素酶也可以在这种条件下固定。游离和固定化纤维素酶的最适温度确定为 65 °C，最适 pH 值分别为 pH 4.0 和 pH 5.0。固定化纤维素酶也使用3次，活性产率高。生化特性和稳定性研究证实，这种固定化酶在工业中显示出前景，可以安全地使用，尤其是在饲料和食品工业以及食品加工中。