Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with HO as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating HO that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other HO generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.2.1) from the ascomycete Chaetomium globosum (CglUPO) and the basidiomycete Marasmius rotula (MroUPO), as well as with recombinant UPO of the ascomycete Humicola insolens (rHinUPO), as an alternative to chemical epoxidation that is non-selective and requires strongly acidic conditions. The three enzymes were able of converting the free fatty acids and the methyl esters from the oils into epoxide derivatives, although significant differences in the oxygenation selectivities were observed between them. While CglUPO selectively produced “pure” epoxides (monoepoxides and/or diepoxides), MroUPO formed also hydroxylated derivatives of these epoxides, especially in the case of the oil hydrolyzates. Hydroxylated derivatives of non-epoxidized unsaturated fatty acids were practically absent in all cases, due to the preference of the three UPOs selected for this study to form the epoxides. Moreover, rHinUPO, in addition to forming monoepoxides and diepoxides of oleic and linoleic acid (and their methyl esters), respectively, like the other two UPOs, was capable of yielding the triepoxides of α-linolenic acid and its methyl ester. These enzymes appear as promising biocatalysts for the environmentally friendly production of reactive fatty-acid epoxides given their self-sufficient monooxygenase activity with selectivity toward epoxidation, and the ability to epoxidize, not only isolated pure fatty acids, but also complex mixtures from oil hydrolysis or transesterification containing different combinations of unsaturated (and saturated) fatty acids.

Tyrian purple (also known as royal or imperial purple) is the oldest known commercial pigment and still one of the most expensive dyes, often associated with the wardrobes of clergy and royalty. It is a brominated derivative of indigo, a natural dye that has been used since 4000 BC. Moreover, just recently, the therapeutic value of indigoids for the treatment of several disorders was discovered. The manufacturing of indigo derivatives by the existing chemical routes has become increasingly uninteresting due to the use of aggressive reagents, expensive starting materials and high-energy costs. Thus, both dyestuff manufacturers and the pharmaceutical industry are interested in the development of gentle preparation methods of indigoids from simple precursors. Here, we describe a simple enzymatic method for the one-step synthesis of Tyrian purple and other indigo derivatives with fungal peroxygenases (UPO, EC 1.11.2.1). The reaction does not require complex co-substrates and works well in phosphate buffers with H2O2 (<0.1 wt%) and less than 5% (v/v) acetonitrile as co-solvent. We demonstrate the scaling up of the reaction to 10 Liters and established thereupon an environmentally friendly combined synthesis and in-situ dyeing process, further simplifying the manufacturing of vat-dyed fabrics. Eventually, we screened a number of halogen-substituted indoles in the search for novel indigo derivatives, which may be of interest for pharmaceutical and/or dyeing purposes.

Recent developments in enzymatic bleaching processes have led to replacement strategies of harsh chemicals by haloperoxidases. For this purpose, it is important to control the haloperoxidase-mediated formation of adsorbable organic halides (AOX). In this study, we studied the chlorination of monomeric and dimeric lignin model substrates. Guaiacol, acetovanillone, veratryl alcohol, pinoresinol and adlerol were treated with Curvularia verruculosa haloperoxidase and compared to a sodium hypochlorite treatment. High-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) analysis was employed for the characterization of the reaction products. Our results show that while treatment with haloperoxidases in the presence of sodium chloride and hydrogen peroxide leads to no improvement in AOX formation compared to chemical treatment with NaOCl, addition of ammonium chloride substantially lessens chlorination and promotes β-O-4 ether bond cleavage. The use of ammonium chloride in conjunction with enzymatic haloperoxidase-based bleaching could be a route to minimize lignin chlorination.

A novel approach for lignin and hexenuronic acid removal from cellulosic pulp based on the combination of a vanadium haloperoxidase and a tertiary amine co-catalyst.