壳聚糖固定化海洋低温碱性蛋白酶YS-80-122的研究

Study on chitosan-immobilized marine low-temperature alkaline proteinase YS-80-122

以壳聚糖为固定化载体,采用吸附交联法,以戊二醛为交联剂,对海洋低温碱性蛋白酶YS-80-122进行固定化。通过单因素和正交试验优化得到最佳固定化条件为pH7·0,戊二醛终浓度0·2%,吸附时间50min,酶载体比例40mg/g,交联时间16h,交联温度10℃,固定化酶酶活回收可达47·52%。固定化酶和游离酶的最适作用温度均为30℃,最适作用pH分别为9·0和10·0,相比游离酶,固定化酶可在更宽的温度和pH范围内发挥高活性,且其温度和pH稳定性得到显著提高。室温下经冷冻干燥的固定化酶贮存90d后活性基本保持不变,贮藏半衰期约为190d。以酪蛋白为底物,重复使用7次后酶活仍保留有79·2%,操作半衰期约为21次使用率。

The immobilization of marine low-temperature alkaline proteinase YS-80-122 and its characteristic were studied in this paper. Immobilization of YS-80-122 was done through adsorptioncrosslinking method, by using chitosan as the carrier and glutaraldehyde as the crosslinking agent. Single factor experiments and orthogonal experiments showed that pH 7.0, final concentration of glutaraldehyde of 0. 2%, adsorption time of 50min, enzyme and carrier ratio of 40mg/g, crosslinking time of 16h at a temperature of 10℃ were the optimum condition for immobilization, and the ratio of activity recovery was 47. 52% under the optimal conditions. The optimum reaction temperature for immobilized and free enzymes was 30℃, while their optimum reaction pH were 9. 0 and 10.0, respectively. Compared with free enzyme, immobilized enzyme had higher activities in wider ranges of pH and temperature, showing significantly enhanced stability. The activity of freeze-dried immobilized enzyme was unchanged after 90 d storage at room temperatures, and its storage half-life was 190d. Using casein as substrate, the immobilized enzyme retained 79.2% of its activity after repeated reaction for 7 times, and its operation half-life was 21 times.