Oxidative conversion of 5-hydroxymethylfurfural (HMF) is of biotechnological interest for the production of renewable (lignocellulose-based) platform chemicals, such as 2,5-furandicarboxylic acid (FDCA). To the best of our knowledge, the ability of fungal aryl-alcohol oxidase (AAO) to oxidize HMF is reported here for the first time, resulting in almost complete conversion into 2,5-formylfurancarboxylic acid (FFCA) in a few hours. The reaction starts with alcohol oxidation, yielding 2,5-diformylfuran (DFF), which is rapidly converted into FFCA by carbonyl oxidation, most probably without leaving the enzyme active site. This agrees with the similar catalytic efficiencies of the enzyme with respect to oxidization of HMF and DFF, and its very low activity on 2,5-hydroxymethylfurancarboxylic acid (which was not detected by GC-MS). However, AAO was found to be unable to directly oxidize the carbonyl group in FFCA, and only modest amounts of FDCA are formed from HMF (most probably by chemical oxidation of FFCA by the H 2 O 2 previously generated by AAO). As aldehyde oxidation by AAO proceeds via the corresponding geminal diols (aldehyde hydrates), the various carbonyl oxidation rates may be related to the low degree of hydration of FFCA compared with DFF. The conversion of HMF was completed by introducing a fungal unspecific heme peroxygenase that uses the H 2 O 2 generated by AAO to transform FFCA into FDCA, albeit more slowly than the previous AAO reactions. By adding this peroxygenase when FFCA production by AAO has been completed, transformation of HMF into FDCA may be achieved in a reaction cascade in which O 2 is the only co-substrate required, and water is the only byproduct formed.

Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Both enzyme types share the ability to perform selective oxygenation reactions. Moreover, the Marasmius rotula UPO (MroUPO) catalyzes reactions of interest compared with the previously described UPOs, including formation of reactive epoxy fatty acids. To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme channel were predicted using binding and molecular dynamics simulations. Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both the native (wild-type recombinant) MroUPO and the mutated variants were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography–mass spectrometry in combination with kinetic analyses. Interestingly, a small modification of the channel shape in the I153T variant increased the ratio between epoxidized oleic acid and its additionally hydroxylated derivatives. A fully opposite effect was attained with the double I153F/S156F variant that completely abolished the ability of the MroUPO to epoxidize oleic acid (while no activity was detected for the I153V variant). The rationale for these results was revealed by the substrate positioning in the above computational simulations, which predict a shorter distance between the oleic acid double bond and the oxygen atom of the peroxide-activated heme (compound I) in the I153T variant than in the native enzyme, promoting epoxidation. In contrast, the I153F/S156F double mutation fully prevents the approach of oleic acid in the bent conformation required for double-bond epoxidation, although its (sub)­terminal hydroxylation was predicted and experimentally confirmed. The I153T mutation also increased the UPO selectivity on polyunsaturated fatty acid epoxidation, strongly reducing the ratio between simple epoxides and their hydroxylated derivatives, with respect to the native UPO.

Background 5-Hydroxymethylfurfural (HMF) is a highly valuable platform chemical that can be obtained from plant biomass carbohydrates. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a renewable substitute for the petroleum-based terephthalic acid in polymer production. Results Aryl-alcohol oxidase (AAO) from the white-rot fungus Pleurotus eryngii is able to oxidize HMF and its derivative 2,5-diformylfuran (DFF) producing formylfurancarboxylic acid (FFCA) thanks to its activity on benzylic alcohols and hydrated aldehydes. Here, we report the ability of AAO to produce FDCA from FFCA, opening up the possibility of full oxidation of HMF by this model enzyme. During HMF reactions, an inhibitory effect of the H2O2 produced in the first two oxidation steps was found to be the cause of the lack of AAO activity on FFCA. In situ monitoring of the whole reaction by 1H-NMR confirmed the absence of any unstable dead-end products, undetected in the HPLC analyses, that could be responsible for the incomplete conversion. The deleterious effect of H2O2 was confirmed by successful HMF conversion into FDCA when the AAO reaction was carried out in the presence of catalase. On the other hand, no H2O2 formation was detected during the slow FFCA conversion by AAO in the absence of catalase, in contrast to typical oxidase reaction with HMF and DFF, suggesting an alternative mechanism as reported in some reactions of related flavo-oxidases. Moreover, several active-site AAO variants that yield nearly complete conversion in shorter reaction times than the wild-type enzyme have been identified. Conclusions The use of catalase to remove H2O2 from the reaction mixture leads to 99% conversion of HMF into FDCA by AAO and several improved variants, although the mechanism of peroxide inhibition of the AAO action on the aldehyde group of FFCA is not fully understood.

Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds. IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.