Biocatalytic membranes anchored with zeolitic imidazolate framework‑8 for enhanced trypsin loading and efficient proteolysis

ZIF-8 was uniformly anchored onto the membrane pores, and then trypsin was immobilized via chelation. • Asymmetric interconnected pores enhance trypsin-substrate contact and reduce fouling. • The trypsin loading per unit mass of ZIF‑8 embedded in membranes was 1.58-fold that of ZIF‑8 crystals. • EVAL/ZIF-8-TR membrane exhibits superior affinity and reaction rate. • BSA degradation efficiency was significantly improved during filtration. Proteolysis plays an important role in various fields but suffers from several limitations, including autolysis, time consumption, low efficiency, and difficulty in enzyme recovery. Biocatalytic membranes integrate enzyme immobilization and membrane separation to enhance stability and reusability, but their application in proteolysis remains challenging due to unstable enzyme loading and high mass transfer resistance. Herein, biocatalytic membranes were fabricated by embedding zeolitic imidazolate framework‑8 (ZIF‑8) into ethylene vinyl alcohol copolymer (EVAL) via the non-solvent induced phase separation (NIPS) method, followed by trypsin incorporation. The ZIF‑8 nanocrystals were anchored on membrane pores, enhancing the stability of ZIF‑8 within the porous structure, preventing aggregation, and providing abundant adsorptive sites. The asymmetric structures of the membranes were obtained by NIPS, which was beneficial for reducing fouling and provided a large surface area for enzyme loading and enhanced substrate-enzyme contact. Trypsin was loaded onto ZIF‑8 through chelation, and the high porosity and connectivity of the membrane were retained. The trypsin loading reached 0.34 mg/cm², and the loading per unit mass of ZIF‑8 embedded in membranes was 1.58-fold that of ZIF‑8 crystals. Higher affinity and reaction rate of biocatalytic membranes during dynamic filtration were obtained when compared to free trypsin from Michaelis-Menten kinetic parameters. Moreover, pressure‑driven convective transport overcame diffusion limitations, significantly improving bovine serum albumin hydrolysis efficiency. This work holds great promise for enzyme-immobilized biocatalytic membranes, and offers a green and sustainable solution for high-efficiency proteolysis. Download: Download high-res image (178KB) Download: Download full-size image