Immobilization of Chloroperoxidase in Metal Organic Framework and Its Catalytic Performance
Received date: 2016-11-09
Revised date: 2017-02-05
Online published: 2017-03-23
Supported by
Project supported by the National Natural Science Foundation of China (No. 21176150).
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%.
Zhao Ruinan , Hu Mancheng , Li Shuni , Zhai Quanguo , Jiang Yucheng . Immobilization of Chloroperoxidase in Metal Organic Framework and Its Catalytic Performance[J]. Acta Chimica Sinica, 2017 , 75(3) : 293 -299 . DOI: 10.6023/A16110593
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