Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701, USA.
Int J Biol Macromol. 2017 Sep;102:384-395. doi: 10.1016/j.ijbiomac.2017.03.153. Epub 2017 Mar 29.
Euphorbia tirucalli peroxidase (ETP) was immobilized on chitosan beads having magnetic properties for the ease of separation and increasing the reusability of ETP for cost effective assay conditions. The present work reports immobilization of ETP on polymeric support chitosan-cobalt oxide beads subsequently activated with 0.05% cynuric chloride. The magnetic immobilized enzyme was characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) analysis and scanning electron microscopy (SEM). The immobilized ETP can be reused up to 10 cycles with retention of more than 60% activity. The optimum pH was shifted from 6.0 to 5.5 for soluble ETP to immobilized ETP and optimum temperature from 50°C and 55°C for the immobilized ETP. Based on response surface methodology, the optimal immobilization conditions obtained were: enzyme concentration, 2mg/286mg beads; optimal pH, 4.93; temperature, 28.88; cynuric chloride concentration, 0.17%; reaction time, 14.4h, which resulted 74.51% maximum immobilization. The enzyme magnetic nanoparticles could be separated magnetically for easy reuse. Immobilization of ETP onto the magnetic nanoparticles could be useful for biotechnological applications and bioassay due to its reusability and improved stability.
辣根过氧化物酶（ETP）被固定在具有磁性的壳聚糖珠上，以方便分离，并提高 ETP 的重复使用性，从而降低成本，实现有效的分析条件。本工作报道了 ETP 在聚合物载体壳聚糖-氧化钴珠上的固定，随后用 0.05%三聚氰胺氯进行了活化。通过傅里叶变换红外光谱（FTIR）、X 射线衍射（XRD）分析和扫描电子显微镜（SEM）对固定化酶进行了表征。固定化酶可重复使用 10 次以上，保留超过 60%的活性。与可溶性 ETP 相比，固定化 ETP 的最适 pH 从 6.0 转移到 5.5，最适温度从 50°C 和 55°C 转移到固定化 ETP。基于响应面法，获得的最佳固定化条件为：酶浓度为 2mg/286mg 珠；最适 pH 值为 4.93；温度为 28.88；三聚氰胺氯浓度为 0.17%；反应时间为 14.4h，最大固定化率为 74.51%。酶磁性纳米颗粒可通过磁场分离，便于重复使用。由于其可重复使用性和提高的稳定性，将 ETP 固定在磁性纳米颗粒上可用于生物技术应用和生物测定。
