Beijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, China; Hebei University of Science and Technology, No. 26 Yuxiang road, Shijiazhuang 050018, China.
Beijing National Laboratory of Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing 100190, China; University of Chinese Academy of Sciences, No. 19A Yuquanlu, Beijing 100049, China.
Talanta. 2018 May 15;182:600-605. doi: 10.1016/j.talanta.2018.02.029. Epub 2018 Feb 9.
Alanine aminotransferase (ALT) plays significant role in biological and clinical research. In this study, a unique ALT enzyme reactor based on multifunctional polymer@magnetic nanoparticles has been constructed for the first time and the enzymolysis efficiency has been evaluated by chiral ligand exchange capillary electrophoresis technique. Poly(N-acryloxysuccinimide) has been synthesized by reversible addition-fragmentation chain transfer polymerization method and immobilized on the magnetic nanoparticles via the succinimide group in the polymer. Interestingly, the enzyme also could easily react with the succinimide group, which enables of ALT covalent bonding onto the polymer. The enzyme amount immobilized and the immobilization time have been investigated. Comparing with free ALT in solution (V of free enzyme = 0.6 mM min), the resultant enzyme reactor has exhibited good reusability and stability, and displayed about five times enhanced enzymolysis efficiency with L-alanine as the substrate (V of enzyme reactor = 3.4 mM min). Furthermore, the prepared enzyme reactor has been applied in ALT inhibitors screening. The enzyme reactors based on the multifunctional polymer@magnetic nanoparticles have depicted great potential in anti-liver drugs development, liver diseases study and ALT related biological process inspect.
丙氨酸氨基转移酶（ALT）在生物和临床研究中具有重要作用。在这项研究中，首次构建了基于多功能聚合物@磁性纳米粒子的独特 ALT 酶反应器，并通过手性配体交换毛细管电泳技术评估了其酶解效率。通过可逆加成-断裂链转移聚合方法合成了聚（N-丙烯酰氧基琥珀酰亚胺），并通过聚合物中的琥珀酰亚胺基团固定在磁性纳米粒子上。有趣的是，酶也可以很容易地与琥珀酰亚胺基团反应，这使得 ALT 能够与聚合物共价结合。研究了酶的固定量和固定时间。与溶液中的游离 ALT（游离酶 V 的值为 0.6 mM·min）相比，所得的酶反应器具有良好的可重复使用性和稳定性，并且以 L-丙氨酸为底物时显示出约五倍的增强的酶解效率（酶反应器 V 的值为 3.4 mM·min）。此外，还将制备的酶反应器应用于 ALT 抑制剂筛选。基于多功能聚合物@磁性纳米粒子的酶反应器在抗肝药物开发、肝病研究和 ALT 相关生物过程检测方面具有巨大的潜力。
