Recent advances in hydroxynitrile lyase discovery, evolutionary history, recombinant expression and applications.

Hydroxynitrile lyases (HNLs) are stereoselective biocatalysts that catalyze the addition of hydrogen cyanide to carbonyl compounds, yielding enantiopure cyanohydrins-valuable intermediates in pharmaceutical and agrochemical synthesis. Recent advances have expanded our understanding of HNLs across multiple dimensions, including their evolutionary origins, structural diversity, and catalytic mechanisms. High-throughput screening methods have accelerated the discovery and functional assessment of novel HNLs from diverse biological sources, revealing enzymes from lipocalin, α + β barrel, and cupin superfamilies. Structural and mechanistic studies have elucidated active site architectures, enabling rational design and protein engineering for improved stability, substrate scope, and catalytic efficiency. Recombinant expression systems now support scalable production and purification, while immobilization techniques enhance enzyme reusability for industrial applications. HNLs have proven especially valuable in the asymmetric synthesis of cyanohydrins under mild, environmentally friendly conditions. This review highlights recent progress in HNL research, emphasizing innovations in enzyme discovery, engineering, and application, while outlining key challenges and future directions for integrating HNLs into sustainable biocatalytic processes. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-025-04653-3.