Yu_2025_Int.J.Biol.Macromol__142784

Enzymes with great stability, specificity, and efficiency are essential for sustainable biocatalysis. D-amino acid oxidase (DAAO) and Epoxide hydrolase (EH) have special catalytic capabilities in green chemistry with minimum structural and functional limitations. An improved framework has been proposed which uses enzyme characterization methods for High-Performance Rare Enzyme Characterization and Evolution System (H-PRECES) for targeted cancer therapy. Under physiological environments, this study multiplied EH stability by three and DAAO activity by seven using a mix of directed evolution and high-throughput screening. Molecular docking indicated an affinity score of -2.466 kcal/mol of DAAO with d-serine, -2.372 kcal/mol of DAAO with D-alanine, and - 4.482 kcal/mol of EH with styrene oxide. In model systems, the improved enzymes also showed a considerable decrease in reaction time and an increase of 25 % in substrate specificity. The research aimed to improve the biostability and catalytic efficiency of epoxide hydrolase (EH) and D-amino acid oxidase (DAAO) for specific anticancer uses. Specific results, such as a 2.8-fold enhancement in catalytic efficiency (kcat/Km), a 6.7 degreesC increase in thermal stability (Tm), and improved substrate selectivity verified by docking and kinetic studies. Cytotoxicity assays further confirmed selective tumor cell inhibition with over 70 % cell viability retention in normal cells. Future research should focus on developing specific delivery methods to improve therapeutic accuracy and enzyme stability under physiological circumstances.