School of Pharmacy, Lanzhou University, Lanzhou 730000, China.
The First Hospital of Lanzhou University, Lanzhou 730000, China.
Anal Methods. 2023 Nov 23;15(45):6220-6228. doi: 10.1039/d3ay01835k.
Cellulose filter paper (CFP) is expected to be an ideal carrier for enzyme immobilization due to its sustainability and biocompatibility. However, the interaction between the carrier and enzyme might change the spatial conformation of the enzyme and its microenvironment, and thus the flexibility of the enzyme molecule or the transport of the substrate to the active site would be hampered. In this work, a two-component system of catechol and tetraethylene pentamine was used to replace dopamine, and a polydopamine-like composite layer was deposited on the surface of CFP to introduce amino groups, which was similar to the self-polymerization-adhesion behavior of dopamine. Using polyethylene glycol diglycidyl ether with flexible spacer arms as the cross-linking agent, α-glucosidase was covalently bonded to amino-modified CFP through an epoxy ring-opening reaction. The immobilized α-glucosidase exhibited greater tolerance to pH and high temperature. After 10 repeated uses, the immobilized α-glucosidase maintained relatively high enzyme activity. Its kinetic behavior was investigated to illustrate the reliability for enzyme inhibitor screening. Finally, a screening method combining an immobilized enzyme and capillary electrophoresis analysis was proposed and applied to screening inhibitors from 11 kinds of traditional Chinese medicines, among which Chebulae Fructus, Phyllanthi Fructus and Terminaliae Relliricae Fructus exhibited strong enzyme inhibitory activities.
纤维素滤纸（CFP）由于其可持续性和生物相容性，有望成为酶固定化的理想载体。然而，载体与酶之间的相互作用可能会改变酶的空间构象及其微环境，从而阻碍酶分子的灵活性或底物向活性部位的传递。在这项工作中，使用儿茶酚和四乙烯五胺的两组分体系代替多巴胺，并在 CFP 表面沉积类似聚多巴胺的复合层以引入氨基，类似于多巴胺的自聚合-粘附行为。使用具有柔性间隔臂的聚乙二醇二缩水甘油醚作为交联剂，通过环氧开环反应将α-葡萄糖苷酶共价键合到氨基改性的 CFP 上。固定化的α-葡萄糖苷酶对 pH 值和高温具有更大的耐受性。经过 10 次重复使用后，固定化的α-葡萄糖苷酶仍保持相对较高的酶活性。对其动力学行为进行了研究，以说明用于酶抑制剂筛选的可靠性。最后，提出了一种结合固定化酶和毛细管电泳分析的筛选方法，并将其应用于从 11 种中药中筛选抑制剂，其中诃子、叶下珠和使君子具有较强的酶抑制活性。
