Thermoresponsive Porous Polymer Membrane as a Switchable Enzyme Reactor for d-Amino Acid Oxidase Kinetics Study

Tuning the efficiency of enzymolysis in different environments is a challenge with important implications for the use of immobilized enzymes in biochemistry, pharmacology, and life science. Here, we demonstrate the use of a thermoresponsive porous polymer membrane for the construction of switchable enzyme reactors, which exhibited membrane surface property changes in diverse temperature environments. The enzyme reactor consisted of block copolymers, poly(styrene−maleic anhydride−oligo methacrylate−methoxyethoxy ethyl methacrylate) [P(St–MAn–OE–ME)], magnetic nanoparticles, and covalently bonded d-amino acid oxidase (DAAO). By varying the temperature between 25 and 39 °C, the self-assembly properties of the enzyme reactor could be controlled. Using d,l-Met as the substrate, the enzymolysis efficiency of the thermoresponsive porous polymer membrane enzyme reactor (t-PPMER) was evaluated by the chiral capillary electrophoresis technique. At higher temperatures, DAAO was embedded in the curled P(OE–ME) chains, resulting in improved enzymolysis efficiency owing to the confinement effect. At lower temperatures, inefficient enzymolysis occurred because of the stretching of the P(OE–ME) chains, which weakened the confinement effect. Remarkably, the enzymolysis rate of t-PPMER at 37 °C (3.1 mM·min–1) was 45 times higher than that at 25 °C (0.066 mM·min–1). The proposed protocol was successfully applied to the DAAO inhibitor screening. This approach capitalizes on the advantageous characteristics of the t-PPMER and provides inspirations for the construction of switchable enzyme reactors and the screening of enzyme inhibitors as potential drugs.