doi:10.1007/s00253-024-13314-zengCinca-Fernando, PaulaAscaso-Alegre, ChristianSevilla, EmmaMartínez-Júlvez, MartaMangas-Sánchez, JuanFerreira, PatriciaDiscovery, characterization, and synthetic potential of two novel bacterial aryl-alcohol oxidasesART-2024-140904The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min−1 mM−1. Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.9 and 48.6 °C for ShAAO and SdAAO, respectively). Crystal structures revealed an unusual wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could be associated with differences observed in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and the ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with total turnover numbers > 38000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds. Key points • Identification and characterization of two novel bacterial aryl-alcohol oxidases • Crystal structures reveal wide-open active-site pockets, impacting substrate scope • Total turnover numbers and cell-free extracts demonstrate the synthetic potential2024http://zaguan.unizar.es/record/14710310.1007/s00253-024-13314-zhttp://zaguan.unizar.es/record/147103oai:zaguan.unizar.es:147103info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00info:eu-repo/grantAgreement/ES/MICINN/RYC2021-032021-Iinfo:eu-repo/grantAgreement/EUR/MICINN/TED2021-130803B-I00APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 108, 1 (2024)byhttps://creativecommons.org/licenses/by/4.0/deed.esinfo:eu-repo/semantics/openAccess