Key Laboratory of Analytical Chemistry for Living Biosystems, Beijing National Laboratory for Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, No. 2 Zhongguancun Beiyijie, Beijing, 100190, China.
College of Chemistry & Environmental Science, Hebei University, Key Laboratory of Analytical Science and Technology of Hebei Province, Baoding, 071002, China.
Anal Bioanal Chem. 2018 Aug;410(21):5211-5218. doi: 10.1007/s00216-018-1169-5. Epub 2018 Jun 16.
Fabrication of polymer membranes with nanopores and a confinement effect toward enzyme immobilization has been an enabling endeavor. In the work reported here, an enzyme reactor based on a thermoresponsive magnetic porous block copolymer membrane was designed and constructed. Reversible addition-fragmentation chain transfer polymerization was used to synthesize the block copolymer, poly(maleic anhydride-styrene-N-isopropylacrylamide), with poly(N-isopropylacrylamide) as the thermoresponsive moiety. The self-assembly property of the block copolymer was used for preparation of magnetic porous thin film matrices with iron oxide nanoparticles. By covalent bonding of glutaminase onto the surface of the membrane matrices and changing the temperature to tune the nanopore size, we observed enhanced enzymolysis efficiency due to the confinement effect. The apparent Michaelis-Menten constant and the maximum rate of the enzyme reactor were determined (K = 32.3 mM, V = 33.3 mM min) by a chiral ligand exchange capillary electrochromatography protocol with L-glutamine as the substrate. Compared with free glutaminase in solution, the proposed enzyme reactor exhibits higher enzymolysis efficiency, greater stability, and greater reusability. Furthermore, the enzyme reactor was applied for a glutaminase kinetics study. The tailored pore sizes and the thermoresponsive property of the block copolymer result in the designed porous membrane based enzyme reactor having great potential for high enzymolysis performance. Graphical abstract ᅟ.
制备具有纳米孔和限制酶固定化效应的聚合物膜一直是一项具有挑战性的工作。在本工作中，设计并构建了一种基于温敏磁性多孔嵌段共聚物膜的酶反应器。可逆加成-断裂链转移聚合被用来合成嵌段共聚物，聚（马来酸酐-苯乙烯-N-异丙基丙烯酰胺），其中聚（N-异丙基丙烯酰胺）为温敏部分。嵌段共聚物的自组装特性被用于制备具有氧化铁纳米颗粒的磁性多孔薄膜基质。通过将谷氨酰胺酶共价键合到膜基质的表面，并通过改变温度来调节纳米孔的大小，我们观察到由于限制效应而提高了酶解效率。通过手性配体交换胶束电动色谱法，以 L-谷氨酰胺为底物，确定了酶反应器的表观米氏常数和最大反应速率（K = 32.3 mM，V = 33.3 mM min）。与溶液中的游离谷氨酰胺酶相比，所提出的酶反应器表现出更高的酶解效率、更好的稳定性和更好的可重复使用性。此外，该酶反应器还用于谷氨酰胺酶动力学研究。嵌段共聚物的定制孔尺寸和温敏性质导致设计的多孔膜基酶反应器具有高酶解性能的巨大潜力。
