### 基于金属有机骨架的固定化氯过氧化物酶的制备和性能评价

1. a 陕西师范大学化学化工学院 西安 710119;
b 陕西师范大学大分子科学陕西省重点实验室 西安 710119
• 投稿日期:2016-11-09 修回日期:2017-02-05 发布日期:2017-03-23
• 通讯作者: 蒋育澄,E-mail:jyc@snnu.edu.cn E-mail:jyc@snnu.edu.cn
• 基金资助:

项目受国家自然科学基金（No.21176150）资助.

### Immobilization of Chloroperoxidase in Metal Organic Framework and Its Catalytic Performance

Zhao Ruinana, Hu Manchenga,b, Li Shunia,b, Zhai Quanguoa,b, Jiang Yuchenga,b

1. a School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119;
b Key Laboratory of Macromolecular Science of Shaanxi Province, Shaanxi Normal University, Xi'an 710119
• Received:2016-11-09 Revised:2017-02-05 Published:2017-03-23
• Supported by:

Project supported by the National Natural Science Foundation of China (No. 21176150).

30℃水相体系中"一锅法"快速制备固定化氯过氧化物酶（CPO@ZIF-8），在构筑金属有机沸石咪唑骨架结构（ZIF-8）的同时将氯过氧化物酶（CPO）固定在其三维纳米孔道中.温和的条件为固定化酶制备过程中酶活性的保持提供了前提.结构和性能表征说明酶分子的引入并不改变ZIF-8材料的孔道结构，同时酶分子在CPO@ZIF-8中呈现出在整体骨架材料中的嵌入式均匀分布.与先构筑ZIF-8骨架材料，然后通过表面吸附来固定酶分子的方法相比，通过将酶分子引入整体骨架材料中不仅提高了酶的固载量，更主要的是利用ZIF-8材料的高比表面积提高了固定化CPO的催化效率，同时基于三维孔道提供的刚性屏蔽环境有效改善了CPO在极端反应条件下的热稳定性、酸碱稳定性和对有机溶剂的耐受性.

A rapid and efficient preparation of CPO@ZIF-8 by "one pot " method at 30℃ in aqueous solution is presented in this paper. The structure of zeolitic imidazolate frameworks (ZIF-8) was constructed while chloroperoxidase (CPO) was incorporated into the channel. Mild reaction conditions ensure maintaining the enzyme activity in the preparation of immobilized CPO. The synthesis of CPO@ZIF-8 was performed by mixing zinc nitrate solution and polyvinylpyrrolidone solution (PVP, Mw:10000, 10 mg/mL, 400 μL), chloroperoxidase (CPO) (0.214 mmol/L, 500 μL) and 2-methylimidazole (1.25 mol/L, 25 mL) and stirring for 15 min at 30℃, followed by washing and centrifuging for 3 cycles at 4℃ for 8 min. The structure of CPO@ZIF-8 was characterized by scanning electron microscope (SEM), transmission electron microscopy (TEM) and X-ray diffraction (XRD), indicating that the incorporation of enzyme molecules did not affect the crystal structure of ZIF-8. To further confirm the incorporation of enzyme into ZIF-8, CPO was labeled by fluorescent probes fluorescein isothiocyanate (FITC) and subjected to the same procedure to synthesize the FITC-CPO@ZIF-8. Confocal laser scanning microscopy (CLSM) proved that CPO was distributed evenly and embedded in the whole framework of CPO@ZIF-8. Compared with the method of preparing ZIF-8 firstly, and then immobilizing enzyme molecule by physical adsorption, the immobilization efficiency of enzyme was enhanced by introducing the enzyme into the whole framework, moreover, the catalytic efficiency of the immobilized CPO was increased due to high specific surface area of ZIF-8. The catalytic performance of the CPO@ZIF-8 was evaluated by the conversion rate of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). The rigid shielding environment provided by the three-dimensional channel of ZIF-8 effectively improved the thermal stability, pH stability, and tolerance to organic solvents of the CPO@ZIF-8 under harsh reaction conditions compared with the free enzyme. When incubated at 50℃, 60℃, 70℃, 80℃ and 90℃ for 1 h, 97.1%, 87.8%, 80.2%, 68.1% and 41.5% of the activity of CPO@ZIF-8 were reserved. When incubated at 50℃, 60℃, 70℃ and 80℃ for 3 h, there were still 91.4%, 77.8%, 64.7% and 50.3% of the activity remained. The tolerance of CPO@ZIF-8 to organic solvent DMF, methanol and methyl cyanide was enhanced to 30%~40%.