红芝LYL263所产漆酶在溶胶-凝胶体系中的固定化研究

摘要

[ Objective ] The aim was to study the immobilization of laccase from Canoderma lucidum in three different sol-gel systems to find out the optimum materials for laccase immobilization. [Method] Laccase from G. Lucidum LYL 263 was immobilized in sodium alginate-gelatin, sodium alginate-chitosan and sodium alginate-gelatin-chitosan sol-gel systems, respectively; and the laccase activity was preliminarily analyzed. [Result] The activity of laccase immobilized in sodium alginate-gelatin-chitosan was 2.14 folds and 2.75 folds respectively, compared to sodium alginate-gelatin and sodium alginate-chitosan; and this optimum system contained sodium algiiiate 2.0% , gelatin 1.0% , chitosan 0.3% and calcium chloride 36%. Benzoic acid was firstly used to embed immobilized laccase in this study, and single-factor experiments revealed that the activity of laccase could be effectively improved by suitable content of benzoic acid. The optimal conditions for laccase immobilization in the system were benzoic acid 2 mmol/L, glutaraldehyde 0.32% , cross-linking reaction time 50 min and enzyme concentration 10.0% ; and then the activity of the immobilized enzyme reached 635.7 U/g with these conditions. The thermal stability of the immobilized laccase was higher than free laccase, and the activity of immobilized enzyme treated by freeze-drying was 1.59 folds compared to the non-freeze-dried, but the freeze-dried immobilized laccase had poor operational stability. [Conclusion] The compositions of the material had significant influence on laccase immobilization and this study will provide reference to select materials for immobilization of laccase from C. Lucidum LYL 263.%[目的]对红芝LYL263漆酶进行不同溶胶-凝胶体系的固定化研究,以期找到适宜该漆酶的固定化材料.[方法]将海藻酸钠-明胶、海藻酸钠-壳聚糖和海藻酸钠-明胶-壳聚糖3种溶胶-凝胶体系用于红芝LYL263所产漆酶的固定化研究,并初步分析固定化酶的酶学性质.[结果]海藻酸钠-明胶-壳聚糖体系固定后的漆酶酶活分别是海藻酸钠-明胶和海藻酸钠-壳聚糖的2.14倍和2.75倍,该体系最佳材料组成为:海藻酸钠浓度2.0％、明胶浓度1.0％、壳聚糖浓度0.3％、氯化钙浓度36.0％.该试验首次将苯甲酸用于包埋法固定化漆酶,单因素试验结果显示,适宜的苯甲酸浓度能有效提高固定化漆酶酶活；该体系固定化漆酶最佳条件为:苯甲酸浓度2mmol/L、戊二醛浓度0.32％、交联时间50 min、酶浓度10.0％,此时固定化酶活高达635.7 U/g.对固定化漆酶酶学性质的研究表明,固定化酶的热稳定性比游离酶高,冻干的固定化酶活是未冻干固定化酶的1.59倍,但冻干的固定化酶操作稳定性较差.[结论]在酶固定化材料中,各个物质的组成对酶固定化有非常大的影响,该研究对红芝LYL263漆酶的固定化材料选择有重要意义.