Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production.

The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxy-methylfurfural (HMF) has gained interest in recent years, as the renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species, but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp hmfo gene). This oxidase acts on both furfuryl alcohols and aldehydes being, therefore, able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with the only need of oxygen as cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative hmfo genes, followed by heterologous expression in Escherichia coli After unsuccessful trials with other bacterial hmfo genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified and characterized. The Methylovorus sp enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity) revealing differences between the three HMFOs. Also the kinetic parameters for HMF, DFF and FFCA oxidation were determined, having the new HMFOs higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different operation conditions.IMPORTANCE: HMFO is the only enzyme described to date catalyzing by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification and characterization of two new HMFOs from Pseudomonas nitroreducens and an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance to the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover number in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.