Deep eutectic solvent modified metal-organic framework for [NiFe] hydrogenase biomimetic immobilization with enhanced...

Hydrogenases can reversibly catalyze the conversion of protons and electrons into hydrogen, which are widely applied in green biohydrogen production. However, hydrogenases face challenges of poor stability and oxygen sensitivity. In this work, conductive deep eutectic solvents (DES) modified metal-organic frameworks (MOFs) were studied as carriers for Pyrococcus furiosus [NiFe]-hydrogenase (pfSHI) immobilization to improve the stability and oxygen tolerance of the enzyme. Choline chloride-ethylene glycol (ChCl-EG) modification of MOFs stabilized the enzyme conformation. After 8 reuses, pfSHI@ChCl-EG-ZIF-67 retained 63.02 % relative activity, higher than that of pfSHI@ZIF-67 (56.18 %). This enhancement is due to ChCl-EG with dynamic hydrogen-bond network. Furthermore, ChCl-EG regulated the pore of MOFs through non-covalent host-guest interactions to create the anaerobic microenvironment, which enhances oxygen tolerance of hydrogenase. After 48 h of air exposure, pfSHI@ChCl-EG-ZIF-67 retained 45.64 % activity, representing 2.67-fold increases compared to free pfSHI. Additionally, ChCl-EG in-situ modulated the structural evolution from 3D nanoflowers (pfSHI@ZIF-67) to 2D nanosheets (pfSHI@ChCl-EG-ZIF-67) during hydrogenase immobilization. The k_cat/K_m of pfSHI@ChCl-EG-ZIF-67 was 851 s^-1·mM^-1, which was 1.49-fold of that of pfSHI@ZIF-67. The immobilized hydrogenases by DES-modified MOF exhibits enhanced stability and oxygen tolerance, which is promising for applications in biohydrogen production.