School of Pharmacy, Lanzhou University, Lanzhou 730000, PR China.
Gansu Institute for Drug Control, Lanzhou 730000, PR China; State Drug Administration-Key Laboratory of Quality Control of Chinese Medicinal Materials and Decoction Pieces, Lanzhou 730000, PR China; Gansu Engineering Technology Laboratory for Inspection and Testing of Chinese and Tibetan Medicine, Lanzhou, PR China.
J Chromatogr A. 2022 Nov 8;1683:463550. doi: 10.1016/j.chroma.2022.463550. Epub 2022 Oct 3.
Mussel-inspired catechol-amine co-deposition is an effective modification strategy for various materials. In this work, polyvinylidene fluoride (PVDF) membrane was employed as the carrier for α-glucosidase immobilization. By virtue of the co-polymerization of tannic acid (TA) and 3-aminopropyltriethoxysilane (APTES) and the hydrolysis of APTES, a hierarchical layer-colloidal nanospheres coating was decorated on the surface of PVDF membrane. Subsequently, α-glucosidase was covalently bound to the modified PVDF membrane through Schiff base reaction and Michael addition reaction between the residual quinine groups in the coating and the amino groups in enzyme molecules. Several parameters affecting the immobilization procedure were investigated thoroughly. The morphology and functional groups of the prepared composite were characterized by scanning electron microscopy (SEM), Fourier transform infrared-Attenuated total reflectance spectroscopy (FTIR-ATR), and X-ray photoelectron spectroscopy (XPS). Combined with capillary electrophoresis (CE) analysis, the performance, enzyme reaction kinetics and inhibition kinetics of PVDF-immobilized α-glucosidase were studied. The immobilized enzyme exhibited the enhanced tolerance to temperature and pH value. In addition, it possessed good reusability maintaining 77.1% of initial relative activity after 11 recycles, and batch-to-batch reproducibility with RSD of 4.3% (n = 10). The Michaelis-Menten constant (K) of immobilized enzyme was calculated to be 4.16 mM, and IC value of acarbose was 0.10 µM. Finally, the PVDF-immobilized α-glucosidase was applied to screening potential inhibitors from 13 kinds of traditional Chinese medicines (TCMs), among which Sanguisorba Radix exhibited the strongest inhibitory activity. The positive results suggested that TA/APTES co-deposition was a simple and mild functionalization method for chemically inert polymer membrane and the proposed screening method was a reliable approach for discovering enzyme inhibitors from TCMs.
贻贝启发的儿茶酚-胺共沉积是一种有效的修饰策略，适用于各种材料。在这项工作中，聚偏二氟乙烯（PVDF）膜被用作α-葡萄糖苷酶固定化的载体。通过单宁酸（TA）和 3-氨丙基三乙氧基硅烷（APTES）的共聚合以及 APTES 的水解，在 PVDF 膜表面修饰了分层的胶体纳米球涂层。随后，通过涂层中残留的奎宁基团与酶分子中氨基之间的席夫碱反应和迈克尔加成反应，将α-葡萄糖苷酶共价结合到改性的 PVDF 膜上。详细研究了影响固定化过程的几个参数。通过扫描电子显微镜（SEM）、傅里叶变换衰减全反射光谱（FTIR-ATR）和 X 射线光电子能谱（XPS）对制备的复合材料的形貌和官能团进行了表征。结合毛细管电泳（CE）分析，研究了 PVDF 固定化α-葡萄糖苷酶的性能、酶反应动力学和抑制动力学。固定化酶表现出对温度和 pH 值的增强耐受性。此外，它具有良好的可重复使用性，在 11 次循环后保持初始相对活性的 77.1%，批间重现性的相对标准偏差为 4.3%（n=10）。固定化酶的米氏常数（K）为 4.16 mM，阿卡波糖的 IC 值为 0.10 µM。最后，将 PVDF 固定化α-葡萄糖苷酶应用于从 13 种中药中筛选潜在抑制剂，其中地榆表现出最强的抑制活性。阳性结果表明，TA/APTES 共沉积是一种简单温和的化学惰性聚合物膜功能化方法，提出的筛选方法是从中药中发现酶抑制剂的可靠方法。
