Screening and Evaluation of New Hydroxymethylfurfural Oxidases for Furandicarboxylic Acid Production

Abstract

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.

Characterization of a thermotolerant aryl-alcohol oxidase from Moesziomyces antarcticus oxidizing 5-hydroxymethyl-2-furancarboxylic acid

“…The oxidation of HMF and its derivatives makes Ma AAO quite interesting for application as biocatalyst in enzymatic synthesis of FDCA. While the chemical route to FDCA requires high temperature and pressure, organic solvents, and metal catalysts (Sajid et al 2018 ), some enzymes were shown to catalyze one or more of the individual reaction steps under mild reaction conditions without cosolvents (Carro et al 2015 ; Daou et al 2019 ; Dijkman and Fraaije 2014 ; Karich et al 2018 ; Mathieu et al 2020 ; Vinambres et al 2020 ). For example, 5-hydroxymethylfurfural oxidase (HMFO) from Methylovorus sp.…”

Advances in Electrochemical Modification Strategies of 5‐Hydroxymethylfurfural

“…With the explosive integration of biocomputational research, the comprehension of enzyme fundamentals [86] can lead to precise genetic manipulation of enzymes for more sustainable, reusable, and selective HMF oxidation. Specifically, the development of robust HMFO is a passionate research topic to boost the simplicity of HMF deep oxidation [87] . Furthermore, an abiotic electrode can be coupled with these useful enzymes to facilitate the oxidation of HMF electrochemically.…”

Pecularities and applications of aryl-alcohol oxidases from fungi

“…GMC oxidoreductases were identified in ascomycetous and basidiomycetous fungi, plants, insects, and bacteria (Cavener 1992;Iida et al 2007;Martin et al 2020). Recently, hydroxymethylfurfural oxidases (HMFOs; EC 1.1.3.47) have been discovered which also belong to the GMC oxidoreductase family and oxidize aromatic primary alcohols, aldehydes, and thiols (Dijkman and Fraaije 2014;Ewing et al 2014;Vinambres et al 2020). While AAOs are secreted by fungi, HMFOs have been found in bacteria like Methylovorus sp.…”

“…Recently, two other HMFOs from P. nitroreducens and Pseudomonas sp. strain 11/12A, both heterologously expressed in E. coli, were shown to catalyze oxidation of 5-HMF to up to 99% FDCA after 96 h (Vinambres et al 2020). Carro and coworkers found that PeAAO expressed in E. coli also catalyzed the conversion of 5-HMF to FFCA, but no subsequent oxidation of FFCA to FDCA was detected.…”