Extraction, structural characterization, and liposome encapsulation of alcohol dehydrogenase from pig liver: Potential applications in alcohol detoxification.

Alcohol dehydrogenase (ADH) is a key enzyme in ethanol metabolism and is abundant in pig liver, an underutilized animal by-product. Here, ADH was isolated from pig liver using aqueous two-phase extraction, and its enzymatic properties, structural characteristics, and biological relevance were systematically evaluated. The extracted ADH showed optimal activity at 45°C and pH 8.5, and its activity was enhanced by K+ and Mn2+. Spectroscopic analyses indicated overall structural similarity to yeast-derived ADH, while revealing a more ordered secondary structure with higher α-helix content. Kinetic analysis toward ethanol suggested a moderate catalytic efficiency relative to commercial yeast ADH, consistent with the physiological role of liver ADH in ethanol metabolism. MALDI-TOF/TOF mass spectrometry combined with bioinformatic analyses (GO enrichment and DisGeNET) supported associations with alcohol-related liver disease pathways. Molecular docking, molecular dynamics simulations, and density functional theory calculations predicted stable ethanol binding in proximity to the catalytic Zn2+ center. For biological validation, an ethanol-induced HepG2 cell injury model was established, showing that pig liver ADH mitigated ethanol-induced cytotoxicity by improving cell viability, suppressing intracellular ROS accumulation, reducing lipid peroxidation, and preserving glutathione homeostasis. Furthermore, liposomal encapsulation (ADH-LIPS) largely preserved catalytic activity while enhancing physicochemical stability, sustained release behavior, storage stability, and resistance to simulated gastrointestinal digestion. Overall, this work demonstrates the feasibility of recovering biologically relevant ADH from pig liver by-products and supports liposomal formulation as an effective strategy to improve enzyme stability under application-relevant constraints.