School of Pharmacy, Lanzhou University, Lanzhou 730000, China.
College of Pharmacy, Gansu University of Chinese Medicine, Lanzhou 730000, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Mar 15;1167:122582. doi: 10.1016/j.jchromb.2021.122582. Epub 2021 Feb 14.
In this work, cellulose filter paper (CFP), which is inexpensive and commercially available, was used as the carrier, and the immobilized α-glucosidase was obtained by two steps: firstly, the surface of CFP was modified by polydopamine/polyethyleneimine (PDA/PEI) co-deposition method to obtain CFP-PDA/PEI with a uniform coating of rich positive charge; subsequently, α-glucosidase was immobilized on the CFP-PDA/PEI by electrostatic adsorption. The free enzyme and immobilized enzyme have the same optimal temperature (70℃) and pH (8.0), and their K is similar, which is 2.2 and 2.8, respectively. These results show that the immobilization process does not change the properties of the enzyme greatly. The immobilized enzyme still maintains 75.6% of its initial activity after 10 repeated uses, showing good reusability. The excellent repeatability (RSD = 2.2%, n = 5) and the verification of competitive inhibitor (acarbose) illustrates the reliability of the immobilized enzymes for enzyme inhibitor screening. Finally, combined with CE, a screening method based immobilized α-glucosidase was proposed and applied to screen the α-glucosidase inhibitory from 10 kinds of Traditional Chinese medicines (TCMs) in vitro. The results indicated that the method was a very effective tool for screening potential α-glucosidase inhibitors from TCMs.
在这项工作中，我们使用了廉价且市售的纤维素滤纸 (CFP) 作为载体，通过两步法获得固定化α-葡萄糖苷酶：首先，通过聚多巴胺/聚乙烯亚胺 (PDA/PEI) 共沉积法修饰 CFP 表面，得到带正电荷的均匀涂层的 CFP-PDA/PEI；随后，通过静电吸附将α-葡萄糖苷酶固定在 CFP-PDA/PEI 上。游离酶和固定化酶具有相同的最佳温度（70℃）和 pH（8.0），其 K 值也相似，分别为 2.2 和 2.8。这些结果表明，固定化过程并未显著改变酶的性质。固定化酶在重复使用 10 次后仍保持初始活性的 75.6%，具有良好的可重复使用性。优异的重复性（RSD=2.2%，n=5）和竞争性抑制剂（阿卡波糖）的验证表明，固定化酶在酶抑制剂筛选中具有可靠性。最后，结合 CE，提出了一种基于固定化α-葡萄糖苷酶的筛选方法，并应用于体外筛选 10 种中药中的α-葡萄糖苷酶抑制剂。结果表明，该方法是从中药中筛选潜在α-葡萄糖苷酶抑制剂的一种非常有效的工具。
