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

ZIF-8 as multi-dentate crosslinking hub converts weak CS/γ-PGA complexes into robust dual-network hydrogel. • Ultra-large pores eliminate diffusion limitation (Da ≈ 0.001) with K m increase of only 2.2-fold. • HSP regression (R 2  = 0.76, p ≈ 0.0002) validates synergistic thermodynamic exclusion and size-sieving mechanism. • Immobilized α-Glu@PC/ZIF-8 enables ligand fishing of two α-glucosidase inhibitors via capture-wash-elute workflow. 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. Download: Download high-res image (72KB) Download: Download full-size image