Pyridine as novel substrate for regioselective oxygenation with aromatic peroxygenase from Agrocybe aegerita

AbstractAgrocybe aegerita peroxidase (AaP) is a versatile extracellular biocatalyst that can oxygenate aromatic compounds. Here, we report on the selective oxidation of pyridine (PY) yielding pyridine N-oxide as sole product. Using H218O2 as co-substrate, the origin of oxygen was confirmed to be the peroxide. Therefore, AaP can be regarded as a true peroxygenase transferring one oxygen atom from peroxide to the substrate. To our best knowledge, there are only two types of enzymes oxidizing PY at the nitrogen: bacterial methane monooxygenase and a few P450 monooxygenases. AaP is the first extracellular enzyme and the first peroxidase that catalyzes this reaction, and it converted also substituted PYs into the corresponding N-oxides

Conversion of Milled Pine Wood by Manganese Peroxidase from Phlebia radiata

Purified manganese peroxidase (MnP) from the white-rot basidiomycete Phlebia radiata was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tw-een 20, Mn2+, bon-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from P. radiata (MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37 degreesC with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to > 6.5. An aromatic nonphenolic lignin structure (beta -O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols

Degradation of Benzo[a]pyrene by the Litter-Decomposing Basidiomycete Stropharia coronilla : Role of Manganese Peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn2+) supplied at a concentration of 200 {micro}M stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn2+ than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn2+. Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter-1 was converted in an in vitro reaction solution containing 1 U of MnP ml-1 within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter-1) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene)

Degradation of Humic Acids by the Litter-Decomposing Basidiomycete Collybia dryophila

The basidiomycete Collybia diyophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic C-14-Iabeled humic acid (14 C-HA) prepared from [U-C-14] catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. RA decomposition was considerably enhanced in the presence of Mn 21 (200 muM), leading to 75% conversion of LHA and 50% mineralization of C-14-HA (compared to 60% and 20%, respectively, in the absence of Mn 21). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn2+-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C diyophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C diyophila which colonize forest litter and soil are involved in humus turnover by their recycling of highmolecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process

Synthesis of 1‐Naphthol by a Natural Peroxygenase engineered by Directed Evolution

This is the peer reviewed version of the following article, which has been published in final form at 10.1002/cbic.201500493. This article may be used for non-commercial purposes in accordance With Wiley-VCH Terms and Conditions for self-archivingThere is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2O2, this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity.We thank Paloma Santos Moriano (ICP, CSIC, Spain) for assistance with the HPLC and LC/MS analysis, and Jesper Vind (Novozymes, Denmark) and Angel T. Martinez (CIB, CSIC, Spain) for helpful discussions. This work was supported by the European Commission projects Indox-FP7-KBBE-2013-7-613549 and Cost-Action CM1303-Systems Biocatalysis, and the National Projects Dewry [BIO201343407-R], Cambios [RTC-2014-1777-3] and OXYdesign [CTQ2013-48287-R].Peer ReviewedPostprint (author's final draft

Cell-free production of the bifunctional glycoside hydrolase GH78 from Xylaria polymorpha

The ability to catalyze diverse reactions with relevance for chemical and pharmaceutical research and industry has led to an increasing interest in fungal enzymes. There is still an enormous potential considering the sheer amount of new enzymes from the huge diversity of fungi. Most of these fungal enzymes have not been characterized yet due to the lack of high throughput synthesis and analysis methods. This bottleneck could be overcome by means of cell-free protein synthesis. In this study, cell-free protein synthesis based on eukaryotic cell lysates was utilized to produce a functional glycoside hydrolase (GH78) from the soft-rot fungus Xylaria polymorpha (Ascomycota). The enzyme was successfully synthesized under different reaction conditions. We characterized its enzymatic activities and immobilized the protein via FLAG-Tag interaction. Alteration of several conditions including reaction temperature, template design and lysate supplementation had an influence on the activity of cell-free synthesized GH78. Consequently this led to a production of purified GH78 with a specific activity of 15.4 U mg− 1. The results of this study may be foundational for future high throughput fungal enzyme screenings, including substrate spectra analysis and mutant screenings

Genome and secretome of Chondrostereum purpureum correspond to saprotrophic and phytopathogenic life styles

We thank R. Ullrich for his mycological expertise and K. Eismann for useful technical assistance.The basidiomycete Chondrostereum purpureum (Silverleaf fungus) is a saprotroph and plant pathogen commercially used for combatting forest “weed” trees in vegetation management. However, little is known about its lignocellulose-degrading capabilities and the enzymatic machinery that is responsible for the degradative potential, and it is not yet clear to which group of wood-rot fungi it actually belongs. Here, we sequenced and analyzed the draft genome of C. purpureum (41.2 Mbp) and performed a quantitative proteomic approach during growth in submerged and solid-state cultures based on soybean meal suspension or containing beech wood supplemented with phenol-rich olive mill residues, respectively. The fungus harbors characteristic lignocellulolytic hydrolases (GH6 and GH7) and oxidoreductases (e.g. laccase, heme peroxidases). High abundance of some of these genes (e.g. 45 laccases, nine GH7) can be explained by gene expansion, e.g. identified for the laccase orthogroup ORTHOMCL11 that exhibits a total of 18 lineage-specific duplications. Other expanded genes families encode for proteins more related to a pathogenic lifestyle (e.g. protease and cytochrome P450s). The fungus responds to the presence of complex growth substrates (lignocellulose, phenolic residues) by the secretion of most of these lignocellulolytic and lignin-modifying enzymes (e.g. alcohol and aryl alcohol oxidases, laccases, GH6, GH7). Based on the genetic and enzymatic constitution, we consider the ‘marasmioid’ fungus C. purpureum as a ‘phytopathogenic’ white-rot fungus (WRF) that possesses a complex extracellular enzyme machinery to accomplish efficient lignocellulose degradation during both saprotrophic and phytopathogenic life phases.The work was financially and scientifically supported by the European Union [integrated projects INDOX – KBBE-7-2013-613549; ENZOX2 – 720297], by the DFG project PeroxiDiv HO 1961/8-1 and the AiF project PeroxyMEER IGF 19636 BG/3. The work has been partly funded by the DFG Priority Program 1374 "Infrastructure-Biodiversity-Exploratories" with the projects HO 1961/6-1, KE 1742/2-1 and JE 724/7-4 (AOBJ: 635952) and the Spanish Ministry of Economy and Competitiveness [project AGL2012-32873]. RR thanks the JAE-Program of the Spanish National Research Council [CSIC] and EA thanks MINECO and FEDER Co-Funds [RyC-2013-12481]

How experiments and molecular simulations can help understand selective C25-hydroxylation of vitamin D by fungal peroxygenases

Postprint (published version

Production of Manganese Peroxidase and Organic Acids and Mineralization of 14C-Labelled Lignin (14C-DHP) during Solid-State Fermentation of Wheat Straw with the White Rot Fungus Nematoloma frowardii

The basidiomycetous fungus Nematoloma frowardii produced manganese peroxidase (MnP) as the predominant ligninolytic enzyme during solid-state fermentation (SSF) of wheat straw. The purified enzyme had a molecular mass of 50 kDa and an isoelectric point of 3.2. In addition to MnP, low levels of laccase and lignin peroxidase were detected. Synthetic 14C-ring-labelled lignin (14C-DHP) was efficiently degraded during SSF. Approximately 75% of the initial radioactivity was released as 14CO2, while only 6% was associated with the residual straw material, including the well-developed fungal biomass. On the basis of this finding we concluded that at least partial extracellular mineralization of lignin may have occurred. This conclusion was supported by the fact that we detected high levels of organic acids in the fermented straw (the maximum concentrations in the water phases of the straw cultures were 45 mM malate, 3.5 mM fumarate, and 10 mM oxalate), which rendered MnP effective and therefore made partial direct mineralization of lignin possible. Experiments performed in a cell-free system, which simulated the conditions in the straw cultures, revealed that MnP in fact converted part of the 14C-DHP to 14CO2 (which accounted for up to 8% of the initial radioactivity added) and 14C-labelled water-soluble products (which accounted for 43% of the initial radioactivity) in the presence of natural levels of organic acids (30 mM malate, 5 mM fumarate)

Oxidoreductases on their way to industrial biotransformations

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 H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 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 D3, 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