Co-Immobilization of Enzyme Conjugates on Monolithic Hierarchical Large-Pore Mesoporous Silica for Efficient Cascade Reactions

Abstract

Monolithic large-pore mesoporous silica (LPMS) with two hierarchically structured interconnected mesopore systems of MCM-41- (2.4 – 5.5 nm) and larger mesopores (20-40 nm), high specific surface area (up to 1121 m2 g-1) and pore volume (up to 1.16 cm3 g-1) were prepared via partial pseudomorphic transformation. The relative fraction of the MCM-41 mesopores can be tuned from 0-1 by variation of the molar C18TAOH/Br ratio as structure-directing agent. The interconnectivity of the two mesopore systems was verified by PFG NMR diffusometry. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were sequentially co-immobilized on the hierarchical LPMS materials. The co-immobilized GOx (303-350 mg g-1) and HRP (95-132 mg g-1) efficiently cooperate as a multi-enzyme conjugate in an activity assay oxidizing glucose to hydrogen peroxide which oxidizes 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) to its cation. The immobilized enzymes exhibit up to 143 % of the activity of the free enzymes in solution when supported on hierarchical LPMS monoliths with particles sizes above 125 µm. Michaelis-Menten kinetics support the high enzyme activity and efficient cooperation after immobilization without diffusion limitations. The enzymes are concluded to be located within the mesopores of the monoliths.