氨基化大孔硅胶共价固定Pancreas porcine脂肪酶的研究

Covalent Immobilization of Pancreas Porcine Lipase on Aminated Mesoporous Silica

本研究以大孔硅胶为材料,对其进行环氧基和氨基两步活化,然后利用碳化二亚胺(EDC)将氨基化硅胶与Pancreas porcine脂肪酶(PPL)进行共价固定化。通过优化固定化条件,在EDC与蛋白摩尔比为50:1;载体与粗酶质量比为10:1,固定化pH为6.0,固定化时间为8 h的条件下获得最大固定化酶酶活为86.67 U/g,远远高于未修饰硅胶物理吸附固定PPL所得固定化酶酶活(33.33U/g)。此外,本文还利用扫描电镜(SEM)和傅立叶变换光谱仪(FT-IR)对固定化前后硅胶进行了结构表征,固定化后硅胶表面出现明显的丝状蛋白,且固定化酶在1543 cm-1处出现酰胺Ⅱ带,说明PPL成功共价固定于氨基化硅胶上。通过对所得固定化酶性质进行分析,结果表明,固定化酶最适反应pH向碱偏移一个单位,最适反应温度提高了5℃,热稳定性明显提高。

In this study mesoporous silica gel was activated by epoxy-group and amino-group for covalent immobilization of pancreas porcine lipase （PPL） using water-soluble carbodiimide. At the optimum conditions （molar ratio of EDC to protein 50：1, weight ratio of support to rude enzyme 10：1, pH 6.0 and immobilization time 8h）, the maximum enzyme activity （86.67 U/g） was obtained, which was much higher than that of PPL immobilized onto silica gel without modification （33.33 U/g）. The immobilization PPLs were also characterized by scanning electron microscopy （SEM） and Fourier transform infrared spectroscopy （FT-IR）. Many filiform substances were observed on the surface of enzyme-immobilized support. Moreover, the absorption peak at 1543 cm-1 （amideⅡ） could be seen in the spectra of immobilized PPL, which confirmed that the PPL had been successfully immobilized onto the supports. In addition, the properties of immobilized PPL were studied. The optimum pH of the immobilized PPL was shifted to a more alkaline range, and the optimum temperature was determined to be 5 ℃ higher than that of free enzyme. Meanwhile, the thermal stability of modified PPL was significantly improved.