Carro et al. Biotechnol Biofuels (2018) 11:86 https://doi.org/10.1186/s13068-018-1091-2 Biotechnology for Biofuels RESEARCH Open Access Self‑sustained enzymatic cascade for the production of 2,5‑furandicarboxylic acid from 5‑methoxymethylfurfural Juan Carro1, Elena Fernández‑Fueyo1, Carmen Fernández‑Alonso1, Javier Cañada1, René Ullrich2, Martin Hofrichter2, Miguel Alcalde3, Patricia Ferreira4 and Angel T. Martínez1* Abstract Background: 2,5-Furandicarboxylic acid is a renewable building block for the production of polyfurandicarboxylates, which are biodegradable polyesters expected to substitute their classical counterparts derived from fossil resources. It may be produced from bio-based 5-hydroxymethylfurfural or 5-methoxymethylfurfural, both obtained by the acidic dehydration of biomass-derived fructose. 5-Methoxymethylfurfural, which is produced in the presence of methanol, generates less by-products and exhibits better storage stability than 5-hydroxymethylfurfural being, therefore, the industrial substrate of choice. Results: In this work, an enzymatic cascade involving three fungal oxidoreductases has been developed for the pro‑ duction of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural. Aryl-alcohol oxidase and unspecifc peroxyge‑ nase act on 5-methoxymethylfurfural and its partially oxidized derivatives yielding 2,5-furandicarboxylic acid, as well as methanol as a by-product. Methanol oxidase takes advantage of the methanol released for in situ producing H­ 2O2 that, along with that produced by aryl-alcohol oxidase, fuels the peroxygenase reactions. In this way, the enzymatic cascade proceeds independently, with the only input of atmospheric ­O2, to attain a 70% conversion of initial 5-meth‑ oxymethylfurfural. The addition of some exogenous methanol to the reaction further improves the yield to attain an almost complete conversion of 5-methoxymethylfurfural into 2,5-furandicarboxylic acid. Conclusions: The synergistic action of aryl-alcohol oxidase and unspecifc peroxygenase in the presence of 5-meth‑ oxymethylfurfural and ­O2 is sufcient for the production of 2,5-furandicarboxylic acid. The addition of methanol oxidase to the enzymatic cascade increases the 2,5-furandicarboxylic acid yields by oxidizing a reaction by-product to fuel the peroxygenase reactions. Keywords: 2,5-Furandicarboxylic acid, 5-Methoxymethyl furfural, Enzyme cascade, Biocatalysis, Oxidase, Peroxygenase, Renewable polyesters Background biodegradable bioplastics. Polyester formed by the con- Fossil resources are fnite and the need for substi- densation of this building block with ethylene glycol, tuting petroleum-based materials with renewable known as poly(ethylene-2,5-furandicarboxylate) (PEF), materials is increasing in recent years [1]. 2,5-Furandi- is expected to substitute for other polyesters produced carboxylic acid (FDCA) is nowadays regarded as a prom- from fossil fuels, thanks to their renewable origin and ising precursor for the production of renewable and their mechanical and gas barrier properties, which are even better than those of conventional poly(ethylene terephthalate) (PET) [2, 3]. Terefore, it is expected that *Correspondence: [email protected] PEF will be able to compete with PET not only in eco- 1 Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E‑28040 Madrid, Spain nomic but also in environmental terms since its produc- Full list of author information is available at the end of the article tion lowers the balance of green-house gases emissions © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Carro et al. Biotechnol Biofuels (2018) 11:86 Page 2 of 10 [4]. Te frst report on PEF enzymatic hydrolysis, which of complex molecules like in the whole conversion of permits the recycling of its monomers, has been brought HMF to FDCA which comprises three sequential oxida- to light recently [5]. tion steps [13]. FDCA can be obtained from precursors that are MMF conversion into FDCA can involve three or four formed upon the acidic dehydration of fructose, directly oxidation steps depending on whether the ether break- obtained from plants (as monosaccharide, in sucrose down leaves an alcohol or a carbonyl function in the fur- disaccharide and in inulin-type polymers) or by isomeri- fural molecule (Fig. 1 scheme, pathways 1/3–5 or 1/2/5, zation of glucose from hydrolysis of disaccharides (e.g., respectively). In the present study, a new self-sustained sucrose) or polysaccharides (e.g., in lignocellulosic mate- enzymatic cascade was developed for the production rials). Tese precursors are mainly 5-hydroxymethylfur- of FDCA from MMF—combining aryl-alcohol oxidase fural (HMF) and more recently 5-methoxymethylfurfural (AAO) [16], unspecifc peroxygenase (UPO) [17], and (MMF). Te latter is obtained when fructose is dehy- methanol oxidase (MOX) [18]—identifed the intermedi- drated in the presence of methanol or by HMF etherifca- ate products by gas chromatography-mass spectrometry tion [6–8]. MMF is more stable upon storage than HMF, (GC–MS), estimated the conversion yields and estab- and fructose dehydration in methanol yields less side- lished the oxidation pathway. products than when it takes place in water for HMF pro- duction. Successful attempts have been made to obtain Results polyesters from MMF and its derivatives [9], and a joint Hydration of the carbonyl group in MMF venture between BASF and Avantium, Synvina (www. Comparison of 1H-NMR spectra in deuterated water and synvi na.com), has been created for sustainable industrial deuterated dimethylsulfoxide (DMSO-d6) allows detec- production of PEF from stable MMF. tion of the aldehyde and the geminal diol signals to meas- In the above context, several patents [10–12] present ure the degree of hydration at equilibrium. Te MMF methods for the production of FDCA from MMF, but all spectrum in DMSO-d6 showed six signals assigned of them use oxidation catalysts such as bromide, cobalt, to the aldehyde (9.6 ppm), the furanic ring (7.5 and or manganese, along with other metals. Moreover, they 6.7 ppm), methylene ether (4.5 ppm), methyl (3.3 ppm), describe processes that take place at high temperatures and residual DMSO (2.5 ppm) protons. On the contrary, (in the range of 100–220 °C) and pressures (3–15 bar). the spectrum in sodium phosphate (pD 7.0) gave 8 sig- Te advantage of enzymes, which work under mild nals assigned to the aldehyde proton (9.6 ppm) and its conditions (in aqueous solution, at room temperature shifted counterpart hemiacetalic hydrated form (small and under atmospheric pressure), for the production of signal at 6.8 ppm), the two ring protons (7.7 and 6.9 ppm) FDCA has gained momentum and several reports on and their shifted counterparts (small signal at 6.5 ppm), the enzymatic oxidation of HMF to FDCA are available the methylene ether (4.7 ppm) and methyl (3.5 ppm) pro- [13–15]. Particularly, the use of the natural portfolio of tons, as well as the water protons (4.9 ppm). Integration oxidases and peroxygenases in synthetic chemistry is of the aldehyde signal and its small shifted counterpart very timely. While the former can perform selective oxi- points towards a degree of MMF hydration ≤ 10%. dations producing H2O2 from atmospheric O2, the latter can use the released H2O2 to complete the full oxidation MMF MMFA FFCA FDCA HMFCA Fig. 1 Scheme of the possible pathways for the oxidation of MMF into FDCA. MMF, 5‑methoxymethylfurfural; MMFA, 5‑methoxymethylfurancarboxylic acid; HMFCA, 5‑hydroxymethyl‑2‑furancarboxylic acid; FFCA, 5‑formylfurancarboxylic acid; and FDCA, 2,5‑furandicarboxylic acid Carro et al. Biotechnol Biofuels (2018) 11:86 Page 3 of 10 MMF oxidation by AAO release, by coupling the reaction of horseradish peroxi- AAO may oxidize aldehydes to acids if their carbonyl dase (HRP) and a reagent that gives a colored product groups are partially hydrated to gem-diols [19]. To test when peroxide is available (Table 1). Comparison with the ability of AAO from Pleurotus eryngii to oxidize the kinetic constants estimated for related furfurals the gem-diol form of MMF, the compound was incu- shows that the methoxyl moiety in MMF decreases the bated with the enzyme and the reaction was analyzed enzyme afnity (Km is increased) and lowers reactivity by GC–MS. Te reaction was completed after 15 h, (kcat is slightly reduced). Both efects together result in using a substrate/enzyme ratio of 300 under the con- much lower AAO catalytic efciency for MMF than for ditions described above (Fig. 2a). 5-(Methoxymethyl)- the other two furfurals assayed. 2-furancarboxylic acid (MMFA) accumulated over time Residual activity estimations showed that AAO was and additional products were not detected, confrming active during the whole time of the reaction (dashed line that AAO does not show any activity on the resulting in Fig. 2a) displaying a half-life of around 6 h that guar- molecule. Te above results show that the small hydra- anteed the full conversion of MMF into MMFA. Cata- tion degree shown by NMR was enough for AAO oxi- lytic performance parameters of AAO, including residual dation of the MMF molecule to MMFA (step 1 in Fig. 1 activity together with turnover number (TON), turno- scheme). ver frequency (TOF), and total turnover number (TTN) Te kinetic constants for the AAO oxidation of MMF values under the described conditions, are provided in and related furfurals were indirectly measured as H2O2 Table 2. Fig. 2 Time course of the reaction of MMF (1.5 mM) with: a AAO; b AAO, UPO and H2O2; c AAO, UPO and MOX; and d AAO, UPO, MOX and methanol (at 28 °C in 100 mM phosphate, pH 7).