Synthesis of hydrogel microspheres with tunable pore size and their application in alkaline protease immobilization

Hydrogels, as the carrier of immobilized enzymes, are beneficial for the aqueous phase catalysis of enzymes, and have attracted extensive attention in maintaining enzymatic activity and improving stability. However, their network structure is relatively large and low molecular weight enzymes are prone to leakage, which affects the catalytic performance of enzymes. In this study, poly(hydroxyethyl methacrylate-acrylamide) hydrogel microspheres (P(H-A)HMs) with adjustable pore sizes (14.52 nm, 5.41 nm and 2.00 nm) are designed and synthesized as a carrier material for alkaline protease immobilization. It has been found that there are significant advantages in using a carrier with a pore size closer to the enzyme during enzyme immobilization. This carrier could effectively prevent enzyme molecules from being limited to small pores or losing to large pores, and maximize the loading capacity of immobilized enzymes. The resultant alkaline protease-poly(hydroxyethyl methacrylate-acrylamide) hydrogel microspheres (Alk-P(H-A)HMs) not only effectively maintain the original activity of the enzyme compared with free alkaline protease but also exhibit improved stability against harsh conditions. Alk-P(H-A)HM2 shows good reusability, which remains at 41.74% of its initial activity after 14 cycles. This strategy breaks the pore size limitation of different enzyme molecules and prepares controlled mesopores, showing great prospects and significance for a series of applications such as enzyme immobilization and biomolecule delivery. Alkaline protease has been successfully immobilized in poly(hydroxyethyl methacrylate-acrylamide) hydrogel microspheres (P(H-A)HMs) with three different pore sizes.