Fabrication of Polymer Membrane Enzyme Reactors with a Dual-Responsive Feature for Cascade Enzyme Reaction Regulation

Enzymes are known to drive biological processes efficiently and uniquely. However, the integration of multienzymes and immobilization technology to improve the stability of enzymes and boost the catalytic efficiency in cascade reactions by controlling the biocatalysis process in complex conditions via an external stimulus is still facing a significant challenge. Herein, we demonstrate a concept for cascade enzyme reaction regulation by controlling the catalytic efficiency step by step based on a dual-stimuli-responsive porous polymer membrane enzyme reactor (DPMER). Poly(styrene-maleicanhydride-acrylic acid-4-[(4-methacryloyloxy)phenylazo] benzoic acid) was designed for the fabrication of the DPMER with glutaminase and alanine aminotransferase as the model modified enzymes. Under UV light irradiation and by regulating the solution pH, the cascade enzyme reaction was carried out step by step. The polymer membrane surface demonstrated a configuration change while improving the enzymolysis efficiency of the DPMER. The enzymatic kinetics of the DPMER was investigated by a chiral capillary electrophoresis technique, and the cascade enzyme reaction regulation capability was evaluated. Under pH of 4.9 and 365 nm UV irradiation, the poly(4-[(4-methacryloyloxy)phenylazo] benzoic acid) and poly(acrylic acid) moieties changed from a "stretched state" to a "curled state" to form surface nanopores, which embedded the enzymes into the surface nanopores while causing the spatial confinement effect and enhancing the enzymolysis efficiency of the DPMER by 9.9-fold in comparison with the free enzymes. This concept provided a potential platform for cascade enzyme reaction regulation and highlighted the perspective of stimuli-responsive polymers.