Immobilization of Chloroperoxidase in Metal Organic Framework and Its Catalytic Performance

Abstract

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, M w : 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%.

A Systematic Study of the Stability of Enzyme/Zeolitic Imidazolate Framework‐8 Composites in Various Biologically Relevant Solutions

“…The conditions used in the synthesis (i. e. in aqueous solution, at mild pH and ambient temperature and for short periods of time) are also compatible with enzymes. A wide range of enzymes have been encapsulated in ZIF-8, including cytochrome c, [5][6][7] horseradish peroxidase, [5,6,[8][9][10] lipase, [5,6,[11][12][13] glucose oxidase (GOx), [9,14] laccase, [12,15,16] catalase, [17] urease, [18] glucoamylase, [19] chloroperoxidase [20] and phenylethanol dehydrogenase. [21] It is also possible to encapsulate multiple enzymes in the same ZIF-8 for cascade reactions.…”

“…[22] Encapsulation of enzymes in ZIF-8 provides several advantages. Compared with their free counterparts, the encapsulated enzymes generally display higher stability under harsh conditions, including at elevated temperatures, [6,8,15,16,[18][19][20] in the presence of organic solvents, [6,9,15,20] or at extreme pH. [17] The encapsulation also shields the enzymes from protease digestions.…”

Study on Construction and Performance of Immobilized Enzyme Reactors by Carboxyl-functionalized Fe3O4

“…共固定化酶的荧光标记, 参照文献 [32] , 分别用异硫 氰酸荧光素(FITC)对 CPO、罗丹明 B(RhB)对 GOx 进行 标记处理, 用 FITC-CPO 和 RhB-GOx 代替合成步骤中的 CPO 和 GOx 制备共固定化酶反应器, 进行激光共聚焦 测试. [ ]…”

Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe3O4-SiO2-NH2-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin

“…The effects of various metal cations on the catalytic efficiency of immobilized enzymes have not been studied thoroughly [32][33][34]. Based on known interactions between free enzymes and metal ions, the effects of metal cations on the activities of FSNcZ8-TpeRha and free TpeRha were determined at pH 5.0 and at 90°C.…”