Mixed-linker metal–azolate framework biocomposites: tuning hydrophobicity and synthesis pH enhances Candida rugosa lipase activity

Protection of enzymes with metal–azolate frameworks (MAF) often comes at the cost of activity for enzymes sensitive to pH and interfacial effects. We target Candida rugosa lipase (CRL) and show that a mixed-linker approach in MAFs controls both the initial synthesis pH and the framework polarity, enabling encapsulation of an interfacially active enzyme. Using an ammonia-free, one-pot aqueous route, we prepare sodalite MAFs from 2-methylimidazole (2-HmIm) and 3-methyl-1H-1,2,4-triazole (3-Hmtz) across a broad composition range. Acid-digestion 1H NMR quantifies the framework composition and reveals preferential 3-Hmtz incorporation, deviating from the nominal ratios used in the synthesis. The ligand mixture controls the initial synthesis pH; the highest-performance biocomposite, CM-98 (98% 3-Hmtz), forms at pH ~8.6, close to the free-enzyme optimum value. Despite moderate encapsulation efficiency (EE%~36%), CM-98 reaches ~140% activity relative to free CRL with specific activity ~6900 Ug-1. SDS/β-Aescin blocking demonstrates that CRL is encapsulated in the MAF crystal, and FTIR analyses are consistent with lid opening/interfacial activation retained under confinement. Moreover, encapsulation broadens the operational pH window (7.5–8.5) over which CRL retains its maximum catalytic activity, while the framework remains structurally stable. These results identify synthesis pH and mixed-linker polarity (2-HmIm/3-Hmtz) as coupled design parameters for high-performance enzyme@MAF biocomposites.