Key Laboratory of Conservation Biology for Endangered Wildlife of Ministry of Education and College of Life Sciences, Zhejiang University, Hangzhou 310058, People's Republic of China.
Appl Microbiol Biotechnol. 2012 Oct;96(1):123-32. doi: 10.1007/s00253-011-3835-9. Epub 2012 Jan 12.
Superoxide dismutase (SOD) has been widely applied in medical treatments, cosmetic, food, agriculture, and chemical industries. In industry, the immobilization of enzymes can offer better stability, feasible continuous operations, easy separation and reusing, and significant decrease of the operation costs. However, little attention has focused on the immobilization of the SOD, as well as the immobilization of thermostable enzymes. In this study, the recombinant thermostable manganese superoxide dismutase (Mn-SOD) of Thermus thermophilus wl was purified and covalently immobilized onto supermagnetic 3-APTES-modified Fe(3)O(4)@SiO(2) nanoparticles using glutaraldehyde method to prepare the Mn-SOD bound magnetic nanoparticles. The Mn-SOD nanoparticles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer analysis. The results indicated that the diameter of Mn-SOD nanoparticles was 40 (± 5) nm, and its saturation magnetization value was 27.9 emu/g without remanence or coercivity. By comparison with the free Mn-SOD, it was found that the immobilized Mn-SOD on nanoparticles exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The results showed that the immobilized Mn-SOD on nanoparticles could be reused ten times without significant decrease of enzymatic activity. Therefore, our study presented a novel strategy for the immobilization of thermostable Mn-SOD and for the application of thermostable enzymes.
超氧化物歧化酶（SOD）已广泛应用于医疗、美容、食品、农业和化工等行业。在工业中，酶的固定化可以提供更好的稳定性、可行的连续操作、易于分离和再利用，并显著降低操作成本。然而，人们对 SOD 的固定化以及热稳定性酶的固定化关注甚少。在这项研究中，从嗜热菌 Thermus thermophilus wl 中纯化并通过戊二醛法共价固定到超顺磁性 3-APTES 修饰的 Fe(3)O(4)@SiO(2)纳米粒子上，制备了锰超氧化物歧化酶（Mn-SOD）结合磁性纳米粒子。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、X 射线衍射、透射电子显微镜和振动样品磁强计分析对 Mn-SOD 纳米粒子进行了表征。结果表明，Mn-SOD 纳米粒子的直径为 40（±5）nm，其饱和磁化强度值为 27.9 emu/g，无剩磁或矫顽力。与游离 Mn-SOD 相比，发现固定在纳米粒子上的 Mn-SOD 对温度、pH 值、金属离子、酶抑制剂和洗涤剂具有更好的抗性。结果表明，固定在纳米粒子上的 Mn-SOD 可以重复使用十次而酶活没有明显下降。因此，我们的研究为热稳定性 Mn-SOD 的固定化和热稳定性酶的应用提供了一种新策略。
