仿生硅化-磁性Fe3O4固定化木瓜蛋白酶的性质
Property of papain immobilized on magnetic ferroferric oxide by biosilicification
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摘要: 将木瓜蛋白酶（papain）交联固定在经氨基修饰过的磁性粒子上,通过papain诱导仿生硅化使磁性粒子（MNPS）表面包覆一层氧化硅,并将papain同时包埋于其中,制备出磁性氧化铁（Fe3O4）-氧化硅载体。红外光谱仪的测定表明,仿生硅化作用所形成的氧化硅能很好地包覆在磁性粒子交联酶（EMNPS）表面。当酶浓度为7.5mg·mL-1时,酶的载入量为0.24mg papain·mg-1 MNPS,此时EMNPS和EMNPS/SiO2的活性分别3.72和3.66U·mg-1 MNPS。EMNPS/SiO2的pH稳定性、温度稳定性、重复操作稳定性均优于EMNPS。与游离papain相比,EMNPS与底物的亲和力略有升高,而EMNPS/SiO2与底物的亲和力降低。EMNPS和EMNPS/SiO2的米氏常数Km分别为1.39、2.31mg·mL-1,最大反应速度Vm分别为0.04、0.038mg·mL-1·min-1。Abstract: An approach combining biomimetic mineralization with Fe3O4 magnetic particles (MNPS) was proposed to prepare immobilized papain that was easily separated from reaction mixture. The results from FT-IR spectra showed that aminosilane-modified MNPS was synthesized, and EMNPS was coated with biosilica by biomimetic silicification. When the concentrate of papain was 7.5mg·mL-1, the loading of papain was 0.24mg·mg-1MNPS and the activity of EMNPS and EMNPS/SiO2were 3.72 and 3.66U·mg-1MNPS, respectively. Furthermore, the pH stability, thermal stability and reusability of EMNPS/SiO2were better than EMNPS. Compared with free papain, EMNPS has slightly higher affinity, and EMNPS/SiO2has lower affinity with substrate. The Michaelis constant (Km) for hydrolysis of casein by EMNPS and EMNPS/SiO2were 1.39 and 2.31mg·mL-1. The maximum velocity (Vm) of EMNPS and EMNPS/SiO2were 0.04 and 0.038mg·mL-1·min-1, respectively.
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