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.

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The immobilization of biocatalysts on magnetic nanomaterial surface is a very attractive alternative to achieve enzyme nanoderivatives with highly improved properties. The combination between the careful tailoring of nanocarrier surfaces and the site-specific chemical modification of biomacromolecules is a crucial parameter to finely modulate the catalytic behavior of the biocatalyst. In this work, a useful strategy to immobilize chemically aminated lipase B from Candida antarctica on magnetic iron oxide nanoparticles (IONPs) by covalent multipoint attachment or hydrophobic physical adsorption upon previous tailored engineering of nanocarriers with poly-carboxylic groups (citric acid or succinic anhydride, CALB EDA @CA-NPs and CALB EDA @SA-NPs respectively) or hydrophobic layer (oleic acid, CALB EDA @OA-NPs) is described. After full characterization, the nanocatalysts have been assessed in the enantioselective kinetic resolution of racemic methyl mandelate. Depending on the immobilization strategy, each enzymatic nanoderivative permitted to selectively improve a specific property of the biocatalyst. In general, all the immobilization protocols permitted loading from good to high lipase amount (149 < immobilized lipase < 234 mg/g Fe ). The hydrophobic CALB EDA @OA-NPs was the most active nanocatalyst, whereas the covalent CALB EDA @CA-NPs and CALB EDA @SA-NPs were revealed to be the most thermostable and also the most enantioselective ones in the kinetic resolution reaction (almost 90% ee R-enantiomer). A strategy to maintain all these properties in long-time storage (up to 1 month) by freeze-drying was also optimized. Therefore, the nanocarrier surface engineering is demonstrated to be a key-parameter in the design and preparation of lipase libraries with enhanced catalytic properties.