Enzymatic membrane reactor(EMR) with immobilization of polyphenol oxidase(PPO) is paid more attention due to its isolation and biochemical characteristics. In this paper, a enzymatic membrane immobilized PPO was prepared based on the chelation of PPO and Cu2+ in chitosan(CS) - Cu complex (CCC) with CS membrane as the carrier. The response surface methodology was applied to investigate the influence of three factors of copper ion sequestration,QCu2+,PPO concentration, CE, pH in immobilization process on the vitality of PPO immobilized in CCC. The relative activity of PPO in the solution and immobilized in the membrane was determined respectively. The characteristics of CS-Cu PPO membrane were discussed primarily. The results show that the optimal conditions for immobilization of PPO into the CCC were temperature 40 ℃, pH 7. 67, QCu2+ 30. 07 mg/mL, CE 2.12 mg/mL. Michaelis constant and the relative activity of PPO immobilized in CCC were larger than that of the solution. Enzyme half-decline period were 30 days, and the relative activity of PPO after repeated use of eight times reduced to 79% for CS-Cu - PPO membrane. This work offers the significantly basis for development of EMR with special function.%以壳聚糖(CS)膜为载体,通过PPO与CS-Cu膜上的Cu2+鳌合,制备固定化PPO的CS-Cu酶膜.响应面法研究了酶膜在固定化过程中的Cu2+螯合量(QCu2+)、PPO浓度(CE)、pH三因素对固定化PPO的活力的影响,分别测定了溶液PPO和酶膜上固定化PPO的酶活性,并对CS-Cu-PPO膜的特性作了初步讨论.结果表明,PPO固定化的最优条件是:温度40℃,pH 7.67,QCu2+ 30.07mg/mL,CE2.12 mg/mL;酶膜中固定化PPO的米氏常数及其相对活力都比溶液PPO的大,半衰期约30 d,酶膜重复使用8次后的PPO活力降至79％.研究结果为开发特定功能的EMR提供了重要的基础.
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