Luo Ruiping, Zhou Xinrui, Chen Yan, Tuo Sicheng, Jiang Fulin, Niu Xiaodi, Pan Fengguang, Wang Hongsu
College of Food Science and Engineering, Jilin University, Changchun 130062, China.
Foods. 2019 Feb 12;8(2):67. doi: 10.3390/foods8020067.
Lysozyme is in high demand due to its many favorable characteristics such as being naturally occurring, non-toxic, and easy to digest and absorb. Recently, superparamagnetic nanoparticles with strong magnetic responsiveness have attracted significant interest for enzyme purification. The aptamer of the enzyme can be chemically synthesized rapidly at a large scale using simple and low-cost preparation methods. Therefore, Fe₃O₄ nanoparticles (Fe₃O₄ NPs) were prepared by chemical co-precipitation and were then functionalized with amino groups to produce NH₂-Fe₃O₄ NPs. The specific reaction of aldehyde and amino groups was used to attach lysozyme aptamers with specific sequences to NH₂-Fe₃O₄ NPs to produce Apt-NH₂-Fe₃O₄ NPs. The synthesized materials were characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), hysteresis loop analysis, and thermogravimetric analysis (TGA). The optimal experimental conditions for adsorption of lysozyme were investigated. The effects of initial lysozyme concentration, adsorption time, pH, reaction temperature, and ionic strength were determined. The maximum adsorption capacity and relevant activity of Apt-NH₂-Fe₃O₄ NPs was 460 mg·g¹ and 16,412 ± 55 U·mg¹ in an aqueous lysozyme solution. In addition, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis analysis, lysozyme could be separated from crude fresh egg white using Apt-NH₂-Fe₃O₄ NPs with an amount up to 113 ± 4.2 mg·g¹ and an activity up to 16,370 46 U·mg¹.
由于溶菌酶具有许多良好特性，如天然存在、无毒且易于消化吸收，因此其需求量很大。最近，具有强磁响应性的超顺磁性纳米颗粒在酶纯化方面引起了极大关注。该酶的适配体可以使用简单且低成本的制备方法大规模快速化学合成。因此，通过化学共沉淀法制备了四氧化三铁纳米颗粒（Fe₃O₄ NPs），然后用氨基对其进行功能化以制备NH₂-Fe₃O₄ NPs。利用醛基和氨基的特异性反应将具有特定序列的溶菌酶适配体连接到NH₂-Fe₃O₄ NPs上，以制备Apt-NH₂-Fe₃O₄ NPs。使用透射电子显微镜（TEM）、X射线衍射（XRD）、傅里叶变换红外光谱（FTIR）、磁滞回线分析和热重分析（TGA）对合成材料进行了表征。研究了溶菌酶吸附的最佳实验条件。测定了初始溶菌酶浓度、吸附时间、pH、反应温度和离子强度的影响。在溶菌酶水溶液中，Apt-NH₂-Fe₃O₄ NPs的最大吸附容量和相关活性分别为460 mg·g⁻¹和16,412 ± 55 U·mg⁻¹。此外，如十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析所示，使用Apt-NH₂-Fe₃O₄ NPs可以从新鲜蛋清粗品中分离出溶菌酶，分离量可达113 ± 4.2 mg·g⁻¹，活性可达16,370 ± 46 U·mg⁻¹。
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