A periplasmic D-malate:cytochrome c oxidoreductase (DMCO) was identified in Ectopseudomonas oleovorans CECT5344, utilizing 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) as an artificial electron acceptor. The assay was adapted for a spectrophotometric or native polyacrylamide gel electrophoresis (PAGE) analysis. The DMCO-encoding gene (BN5_4044) was cloned and expressed in Escherichia coli, enabling a partial purification and biochemical characterization. In addition to D-malate, the enzyme oxidizes D-2-hydroxyglutarate and, to a lesser extent, D-lactate, with cytochrome c also serving as an electron acceptor. DMCO requires Zn²⁺ for activity and exists as a dimer, as determined by gel filtration. The in vitro reconstitution of the electron transfer from D-malate to oxygen was achieved using spheroplasts, enriched periplasmic fractions, and cytochrome c. This extracytoplasmic respiration, unique among homologs of this protein, may eliminate the need for a dedicated inner membrane transporter, thereby avoiding potential upstream respiratory bottlenecks. In the context of bioremediation, and particularly regarding the cyanide metabolism, this D-malate oxidation to oxaloacetate facilitates detoxification by forming the corresponding cyanohydrin, which can be subsequently assimilated for growth.

The development of sustainable alternatives to traditional chemical oxidation processes represents a primary objective in modern green chemistry. In this work, we report the biocatalytic potential of the strain Ectopseudomonas oleovorans CECT 5344 R1D for the selective, chemo- and regioselective oxidation of a diverse range of aldehydes into their corresponding high-value carboxylic acids. Our results demonstrate that this biocatalyst exhibits remarkable versatility, successfully transforming a broad library of aromatic and heterocyclic aldehydes under mild, aqueous conditions, thus bypassing the need for hazardous metal catalysts or stoichiometric oxidants. We performed a systematic evaluation of the substrate scope, assessing the system’s robustness by testing substrate concentrations ranging from 2 to 100 mM. While complete conversion (100%) was achieved for most substrates at lower loads, higher concentrations revealed specific limitations in the biocatalytic performance, which were characterised and discussed. Notably, the strain achieved complete assimilation of furfural, further expanding its unique biotechnological profile as a potential detoxification agent in biofuel production. Structural identification and kinetic monitoring of the reaction products were conducted via ¹H NMR analysis, providing a precise dual-analytical approach. These findings highlight E. oleovorans CECT 5344 R1D as a robust, stable, and efficient whole-cell “cell factory” for the production of value-added chemicals. Ultimately, this research provides a scalable and sustainable framework for the valorisation of bio-based aldehydes, offering a superior synthetic strategy compared to conventional, energy-intensive oxidation methodologies.