Key Laboratory of Conservation Biology for Endangered Wildlife of Ministry of Education and College of Life Sciences, Zhejiang University, Hangzhou, 310058, The People's Republic of China.
Mar Biotechnol (NY). 2013 Dec;15(6):659-67. doi: 10.1007/s10126-013-9515-2. Epub 2013 Jun 8.
Lipases have found a number of commercial applications. However, thermostable lipase immobilized on nanoparticle is not extensively characterized. In this study, a recombinant thermostable lipase (designated as TtL) from Thermus thermophilus WL was expressed in Escherichia coli and immobilized onto 3-APTES-modified Fe3O4@SiO2 supermagnetic nanoparticles. Based on analyses with tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer observation, the diameter of immobilized lipase nanoparticle was 18.4 (± 2.4) nm, and its saturation magnetization value was 52.3 emu/g. The immobilized lipase could be separated from the reaction medium rapidly and easily in a magnetic field. The biochemical characterizations revealed that, comparing with the free one, the immobilized lipase exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The K m value for the immobilized TtL (2.56 mg/mL) was found to be lower than that of the free one (3.74 mg/mL), showing that the immobilization improved the affinity of lipase for its substrate. In addition, the immobilized TtL exhibited good reusability. It retained more than 79.5 % of its initial activity after reusing for 10 cycles. Therefore, our study presented that the possibility of the efficient reuse of the thermostable lipase immobilized on supermagnetic nanoparticles made it attractive from the viewpoint of practical application.
脂肪酶已经在许多商业应用中得到了应用。然而，固定在纳米颗粒上的热稳定性脂肪酶尚未得到广泛的研究。在本研究中，来自 Thermus thermophilus WL 的重组耐热脂肪酶（命名为 TtL）在大肠杆菌中表达，并固定在 3-APTES 修饰的 Fe3O4@SiO2 超顺磁性纳米颗粒上。通过三氯乙酸-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观察分析，固定化脂肪酶纳米颗粒的直径为 18.4（±2.4）nm，其饱和磁化强度值为 52.3 emu/g。固定化脂肪酶可以在磁场中快速、轻松地从反应介质中分离出来。生化特性研究表明，与游离酶相比，固定化脂肪酶具有更好的耐热性、耐酸碱性、耐金属离子性、耐酶抑制剂性和耐表面活性剂性。固定化 TtL 的 K m 值（2.56 mg/mL）低于游离酶的 K m 值（3.74 mg/mL），表明固定化提高了脂肪酶对其底物的亲和力。此外，固定化 TtL 具有良好的可重复使用性。重复使用 10 次后，其初始活性保留率仍高于 79.5%。因此，本研究表明，固定在超顺磁性纳米颗粒上的耐热脂肪酶的高效重复使用的可能性使其在实际应用方面具有吸引力。
