Low-Temperature Water-Based De novo Synthesis of Composites Based on Nanosized ZIF-8 Polymorph and Embedded Candida rugosa Lipase

In this study, Candida rugosa lipase (CRL) was embedded within nano-zeolitic imidazolate framework-8, nZIF-8 (1:35), synthesized with a zinc(II) ion to 2-methylimidazole (Zn 2+ : HmeIM) ratio of 1:35 using a water-based, de novo synthesis method at ambient temperature without any modulating agents. The optimization of the embedding process focused on three parameters: the molar ratio of zinc(II) ion to 2-methylimidazole (Zn 2+ : HmeIM), the concentration of partially purified lipase, and the duration of embedding process. Various physicochemical characterization techniques were employed to confirm and analyze the embedded lipase, including powder X-ray diffraction (PXRD), transmission electron microscopy (TEM), and energy dispersive X-ray (EDX) analysis. Additional characterization methods such as thermogravimetric analysis (TGA), N 2 adsorption-desorption measurements, and Fourier transform infrared spectroscopy (FTIR) were also conducted for comprehensive analysis. The amount of lipase successfully embedded was quantified using the standard Bradford assay, with CRL 3 @nZIF-8(1:35) ( Candida rugosa lipase (3.0 mg) immobilized on nano ZIF-8 synthesized with a Zn²⁺:HmeIM ratio of 1:35) achieving the highest immobilization of 89% with loading capacity of 17.7 mg/g. To evaluate the functional performance of the immobilized enzyme, a p-nitrophenyl palmitate ( p -NPPal) hydrolysis assay was performed. The results showed a six-fold increase in enzymatic activity compared to the soluble lipase. These findings highlight the potential of nZIF-8(1:35) as a promising support material for lipase immobilization, functioning as a robust protective nanocomposite and as an enhancer of enzymatic performance, with the added capability of reusability in the CRL 3 @nZIF-8(1:35) system.