Surface charge modification of PNIPAM carriers for improved thermal stability and recyclability of co-immobilized dual-enzymes in D-xylose to rare sugar cascade conversion

Design and fabrication of multifunctional charge-tunable hydrogel carriers. • Molecular docking reveals hydrogen bonding mechanisms in charge-assisted non-covalent immobilization. • PASP/PNIPAM-immobilized enzymes retain 89.7% initial activity after six cycles. • A novel biocatalytic pathway for D-xylose enables efficient rare sugar production. Immobilization of enzymatic cascades is an important tool for the efficient production of value added products, such as rare sugars from biomass monosaccharides. However, there is a lack of biocompatible and inexpensive immobilization carriers with high reusability in immobilizing multiple enzyme proteins currently. In this study, poly-N-isopropylacrylamide (PNIPAM) hydrogels were fabricated and employed to immobilize the enzymatic cascade of glucose isomerase (GI) and D-tagatose-3epimerase (DTE) for the conversion of D-xylose to rare sugar products, D-xylulose and D-ribulose. To attain stable protein attachment and high reusability, PNIPAM microspheres were further grafted by polyaspartic acid (PASP), polyethylene glycol (PEG), polylysine (PL), and anionic polyacrylamide (APAM), obtaining composite hydrogels with tailored surface charges to achieve effective non-covalent immobilization. The enzymatic cascades immobilized by modified PNIPAM hydrogels demonstrated improved thermal stability in the range of 40–70 °C with prolonged enzyme half-lives, and higher conversion of D-xylose. After 6 recycles, PASP/PNIPAM-GI-DTE retained 89.7% initial activity which was superior in enzyme immobilization by PNIPAM. Molecular simulation studies revealed that PASP/PNIPAM formed stable hydrogen bonds with GI and DTE, rationalizing its superior performance. This work proved charge aided immobilization of proteins as a feasible strategy for the production of biomass derived high value chemicals, and provided a practical protocol for industrial scale biomanufacturing. Download: Download high-res image (204KB) Download: Download full-size image