Enzyme immobilization on a pH-responsive porous polymer membrane for enzymatic kinetics study

Abstract

Immobilization of enzymes onto carriers is a rapidly growing research area aimed at increasing the stability, reusability and enzymolysis efficiency of free enzymes. In this work, the role of phase-separation and a pH-responsive “hairy” brush, which greatly affected the topography of porous polymer membrane enzyme reactors (PMER), was explored. The porous polymer membrane was fabricated by phase-separation of poly(styrene-co-maleic anhydride-acrylic acid) and poly(styrene-ethylene glycol). Notably, the topography and pores size of the PMER could be controlled by phase-separation and a pH-responsive “hairy” brush. For evaluating the enzymolysis efficiency of d-amino acid oxidase (DAAO) immobilized carrier (DAAO@PMER), a chiral ligand exchange capillary electrophoresis method was developed with d-methionine as the substrate. The DAAO@PMER showed good reusability and stability after five continuous runs. Notably, comparing with free DAAO in solution, the DAAO@PMER exhibited a 17.7-folds increase in catalytic velocity, which was attributed to its tailorable topography and pH-responsive property. The poly(acrylic acid) moiety of poly(styrene-co-maleic anhydride-acrylic acid) as the pH-responsive “hairy” brush generated topography changing domains upon adjusting the buffer pH, which enable the enzymolysis efficiency of DAAO@PMER to be tuned based upon the well-defined architectures of the PMER. This approach demonstrated that the topographical changes formed by phase-separation and the pH-responsive “hairy” brush indeed made the proposed porous polymer membrane as suitable supports for enzyme immobilization and fitting for enzymolysis applications, achieving high catalytic performance.