Department of Applied Chemistry, National Chiayi University, 300 Syuefu Road, Chiayi City 60004, Taiwan.
Department of Food Science and Technology, Hungkuang University, 1018 Taiwan Boulevard, Shalu District, Taichung City 43302, Taiwan.
Int J Mol Sci. 2019 Jul 24;20(15):3625. doi: 10.3390/ijms20153625.
In this study, silica-coated magnetic nanoparticles (SiMNPs) with isocyanatopropyltriethoxysilane as a metal-chelating ligand were prepared for the immobilization of His-tagged prolidase (His-PepQ). Under one-hour coupling, the enzyme-loading capacity for the Ni-functionalized SiMNPs (NiNTASiMNPs) was 1.5 mg/mg support, corresponding to about 58.6% recovery of the initial activity. Native and enzyme-bound NiNTASiMNPs were subsequently characterized by transmission electron microscopy (TEM), superparamagnetic analysis, X-ray diffraction, and Fourier transform infrared (FTIR) spectroscopy. As compared to free enzyme, His-EcPepQ@NiNTASiMNPs had significantly higher activity at 70 °C and pH ranges of 5.5 to 10, and exhibited a greater stability during a storage period of 60 days and could be recycled 20 times with approximately 80% retention of the initial activity. The immobilized enzyme was further applied in the hydrolysis of two different organophosphorus compounds, dimethyl -nitrophenyl phosphate (methyl paraoxon) and diethyl -nitrophenyl phosphate (ethyl paraoxon). The experimental results showed that methyl paraoxon was a preferred substrate for His-PepQ and the kinetic behavior of free and immobilized enzymes towards this substance was obviously different. Taken together, the immobilization strategy surely provides an efficient means to deposit active enzymes onto NiNTASiMNPs for His-PepQ-mediated biocatalysis.
在这项研究中，制备了以异氰酸丙基三乙氧基硅烷为金属螯合配体的硅涂层磁性纳米颗粒（SiMNPs），用于固定组氨酸标记的脯肽酶（His-PepQ）。在 1 小时的偶联反应中，Ni 功能化 SiMNPs（NiNTASiMNPs）的酶载量为 1.5 mg/mg 载体，对应于初始酶活的约 58.6%回收率。随后通过透射电子显微镜（TEM）、超顺磁分析、X 射线衍射和傅里叶变换红外（FTIR）光谱对天然和酶结合的 NiNTASiMNPs 进行了表征。与游离酶相比，His-EcPepQ@NiNTASiMNPs 在 70°C 和 pH 范围为 5.5 到 10 时具有显著更高的活性，在 60 天的储存期内表现出更高的稳定性，并且可以回收 20 次，初始活性保留约 80%。固定化酶进一步应用于两种不同的有机磷化合物，即二甲基-硝基苯基磷酸酯（甲基对氧磷）和二乙基-硝基苯基磷酸酯（乙基对氧磷）的水解。实验结果表明，甲基对氧磷是 His-PepQ 的首选底物，游离酶和固定化酶对该物质的动力学行为明显不同。总之，固定化策略为将活性酶沉积到 NiNTASiMNPs 上用于 His-PepQ 介导的生物催化提供了一种有效的方法。
