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

摘要

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 phaseseparation 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 pHresponsive"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 a17.7-folds increase in catalytic velocity,which was attributed to its tailorable topography and pHresponsive 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 phaseseparation 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.