Construction of a chitosan/γ-poly(glutamic acid) composite hydrogel crosslinked by ZIF-8 for enhanced α-glucosidase...

Herein, a composite hydrogel based on two natural macromolecules-chitosan (CS) and γ-poly(glutamic acid) (γ-PGA)-was developed, crosslinked by zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, which serve as reactive crosslinking hubs to covalently connect the biopolymer chains for efficient immobilization of α-glucosidase (α-Glu). A carboxyl-terminated poly(ethylene glycol)-poly(glutamic acid) copolymer was first synthesized and its activated carboxyl groups underwent amidation with amino groups of CS to rapidly form a three-dimensional PC/ZIF-8 hydrogel. The effects of ZIF-8 content and CS concentration on micro-morphology, swelling behavior, and rheological properties were systematically investigated. ZIF-8-mediated crosslinking was essential for gel formation, enabling effective modulation of network density and pore architecture. The optimized hydrogel showed no inhibitory effect on α-Glu. Immobilized α-Glu@PC/ZIF-8 exhibited enhanced pH stability (retaining 53% and 70% activity at pH 4.0 and 9.0), outstanding storage stability (>90% after 4 weeks), excellent organic solvent tolerance (>90% in most solvents), and good reusability (near 100% activity over 6 cycles). Mass transfer studies confirmed that the designed crosslinking density provided efficient substrate/product diffusion channels, minimizing product inhibition. Hansen solubility parameter analysis revealed a synergistic "thermodynamic exclusion + size-sieving" mechanism for solvent tolerance. Coupled with HPLC, the immobilized enzyme successfully identified two α-glucosidase inhibitors-batatasin I and 2,4-dimethoxy-6,7-dihydroxyphenanthrene-from Dioscorea opposita Thunb. peel. This work demonstrates that rational chemical modification of natural macromolecular networks via MOF crosslinkers provides an effective strategy for constructing high-performance enzyme immobilization platforms.