Enhancing the Stability and Activity of Enzymes through Layer-by-Layer Immobilization with Nanocomposite Hydrogel

Abstract

In this study, a layer-by-layer immobilization strategy was meticulously designed to enhance the stability and activity of enzymes. The approach utilized calcium magnesium carbonate (CaMg(CO3)2) nanoparticles as an immobilization template for abundant binding sites for enzyme attachment but also provide a microenvironment conducive to maintaining enzyme conformation with their unique physicochemical properties. Subsequently, the polymerization of poly(2-hydroxyethyl methacrylate) (pHEMA) to form the nanocomposite hydrogel poly(2-hydroxyethyl methacrylate)-calcium magnesium carbonate (pHEMA-CaMg(CO3)2) introduces a layered architecture, enabling the spatially immobilized enzymes. The pHEMA-CaMg(CO3)2 nanocomposite hydrogel immobilized carbonyl reductase retained over 80% catalytic activity after one month and 81% after 10 cycles of aryl ketone reduction. Similarly, the immobilized lipase within the pHEMA-CaMg(CO3)2 nanocomposite hydrogel exhibited over 80% catalytic activity after one month and demonstrated an impressive 85% activity retention after 10 reuse cycles in glycerol trioctanoate hydrolysis reactions. The FTIR spectra and energy-dispersive X-ray (EDX) spectroscopy mapping showed after multiple repeated uses indicating that the secondary structure of the immobilized enzyme was well preserved, further validating the superiority of our immobilization strategy in safeguarding enzymes integrity and functionality over extended periods and repeated applications.