Qu Zhuangzhuang, Li Mingjie, Ablat Ayzohra, Hu Yikao, Wang Yuan, Shu Peng, Liao Xun
HBN Research Institute and Biological Laboratory, Shenzhen Hujia Technology Co., Ltd., Shenzhen 518000, China; Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China; University of Chinese Academy of Sciences, Beijing 100049, China.
HBN Research Institute and Biological Laboratory, Shenzhen Hujia Technology Co., Ltd., Shenzhen 518000, China.
Int J Biol Macromol. 2025 Sep;321(Pt 1):146179. doi: 10.1016/j.ijbiomac.2025.146179. Epub 2025 Jul 19.
Ligand fishing with immobilized enzymes offers a promising approach for screening natural active compounds in complex extracts. Key challenges in enzyme immobilization include maintaining structural integrity and enhancing loading capacity. This study employed hydrogen-bonded organic frameworks (HOFs), eco-friendly porous materials synthesized via hydrogen bonding, to immobilize elastase (ELA) through self-assembly for affinity screening of ELA inhibitors from Coreopsis tinctoria Nutt. The successful fabrication of ELA@HOF was validated using techniques such as SEM, TEM, FT-IR, XRD, XPS, TGA, and BET. HOFs significantly improved enzyme loading capacity (524.4 mg/g), immobilization rate (85.7%), specific activity (25.7 U/mg) and stability (notable acid resistance) compared to conventional methods. ELA@HOF demonstrated remarkable repeatability for ligand fishing which was reusable for 12 cycles. Seven inhibitors of ELA were extracted from extracts of C. tinctoria, which were identified by UPLC-MS combined with comparison with authentic samples as isookanin, taxifolin, marein, 7,3',5'-trihydroxyflavanone, okanin, eriodictyol, and sulfuretin. The ELA inhibitory activity and enzymatic kinetic study were further investigated, revealing a significant level of inhibition. Molecular docking technique was used to simulate the interaction between the ligand and ELA. These findings suggest the great potential of hydrogen-bonded organic frameworks for the rapid screening of active natural compounds.
用固定化酶进行配体垂钓为筛选复杂提取物中的天然活性化合物提供了一种有前景的方法。酶固定化的关键挑战包括维持结构完整性和提高负载能力。本研究采用氢键有机框架（HOFs），即通过氢键合成的环保型多孔材料，通过自组装固定弹性蛋白酶（ELA），用于从金鸡菊中亲和筛选ELA抑制剂。通过扫描电子显微镜（SEM）、透射电子显微镜（TEM）、傅里叶变换红外光谱（FT-IR）、X射线衍射（XRD）、X射线光电子能谱（XPS）、热重分析（TGA）和比表面积分析（BET）等技术验证了ELA@HOF的成功制备。与传统方法相比，HOFs显著提高了酶的负载能力（524.4 mg/g）、固定化率（85.7%）、比活性（25.7 U/mg）和稳定性（显著的耐酸性）。ELA@HOF在配体垂钓方面表现出显著的可重复性，可重复使用12个循环。从金鸡菊提取物中提取了七种ELA抑制剂，通过超高效液相色谱-质谱联用（UPLC-MS）并与标准品比较鉴定为异鼠李素、紫杉叶素、圣草酚、7,3',5'-三羟基黄烷酮、奥卡宁、圣草素和硫磺菊素。进一步研究了ELA抑制活性和酶动力学，结果显示出显著的抑制水平。使用分子对接技术模拟配体与ELA之间的相互作用。这些发现表明氢键有机框架在快速筛选活性天然化合物方面具有巨大潜力。
