菌株Arthrobacter sp.DNS10降解酶固定化条件的响应面优化

Optimization for the immobilization of the atrazine degrading enzyme extracted from strain Arthrobacter sp.DNS10 using response surface methodology

以前期分离得到阿特拉津降解菌株Arthrobacter sp.DNS10为供试菌株,选取海藻酸钠作为固定化材料,采用响应面法优化该菌株所包含的降解酶的固定化条件。借助Plackett-Burman设计筛选确定海藻酸钠浓度、固定化体系的p H值、加酶量和Ca Cl2溶液质量分数4个因素作为影响固定化酶比酶活的典型因素。借助Box-Behnken设计及响应面回归分析拟合,确定适宜上述降解酶的最佳包埋固定化条件及方法为:在每10 m L海藻酸钠浓度为1.93%,p H为8.5的固定化基质中加入983μL的降解酶液(蛋白浓度为88μg·L-1),然后利用注射器将上述混合溶液滴加到质量分数为2.7%的Ca Cl2溶液中即可制备出比酶活最高的固定化酶,实际测定固定化酶的最优比酶活为0.190 2 U·mg-1(预测值为0.187 4U·mg-1)。上述固定化酶平均粒径约为(0.44±0.01)cm,其在连续6次的使用过程中比酶活仍能保持在初始值的77.5%以上。上述固定化处理可有效的改善降解酶的环境贮存特性,固定化酶在常温下保存35 d后比酶活仍能保持在其初始状态的12.34%,而游离酶则无活性检出。

The atrazine degrading strain （Arthrobacter sp. DNS10） has been isolated from farm soil in the northeastern part of China, which has a long history of atrazine application. This strain was selected as the exper- imental strain to study the immobilization of the atrazine degrading enzyme, when the algin is the immobilized matrix, using the response surface methodology. The significant factors selected by the Plackett-Burman （P-B） design included the algin concentration, pH of the matrix, the amount of enzyme, and the CaC12 concentration. Furthermore, the optimal immobilized condition and the method for the degrading enzyme of strain DNS10 were ： 10 mL mixed immobilized matrix （pH was 8.5） , which contained 1.93% algin, and 983 μL degrading enzyme （protein concentration was 88 μg· L-1 ） dropped into a 2.7% CaC12-water solution to form the optimal immobi- lized enzyme （the predicted specific enzyme activity was 0. 187 4 U · mg -1 ）. The actual specific enzyme activity of the immobilized enzyme was 0. 190 2 U · mg-1 These results suggest that the optimal immobilized condition for the degrading enzyme was accurate and feasible. The average diameter of the immobilized atrazine degrading enzyme, which was produced according to the optimized technology mentioned above, was （0.44 ＋ 0.01 ）cm. The specific enzyme activity of the immobilized degrading enzyme, which was used six times, retained more than 77.5% of the initial specific enzyme activity. In addition, the immobilized atrazine degrading enzyme exhibited superior substrate catalyzing ability when it was stored at room temperature （20℃ ） and 4 ℃. Furthermore, the immobilized atrazine degrading enzyme retained 12.34% of the initial specific enzyme activity after storage at room temperature for 35 days.