TY - GEN A1 - Martínez, Angel T. A1 - Ruiz-Dueñas, Francisco J. A1 - Gutiérrez, Ana A1 - Río, José C. del A1 - Alcalde, Miguel A1 - Liers, Christiane A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Scheibner, Katrin A1 - Kalum, Lisbeth A1 - Vind, Jesper A1 - Lund, Henrik T1 - Search, engineering, and applications of new oxidative biocatalysts T2 - Biofuels, Bioproducts and Biorefining N2 - Most industrial enzymes are hydrolases, such as glycosidases and esterases. However, oxidoreductases have an unexploited potential for substituting harsh (and scarcely selective) chemical processes. A group of basidiomycetes are the only organisms degrading the aromatic lignin polymer, enabling the subsequent use of plant polysaccharides. Therefore, these fungi and their ligninolytic peroxidases are the biocatalysts of choice for industrial delignification and oxidative biotransformations of aromatic and other organic compounds. The latter also include oxygenation reactions, which are catalyzed with high regio/stereo selectivity by fungal peroxygenases. In search for novel and more robust peroxidases/peroxygenases, basidiomycetes from unexplored habitats were screened, and hundreds of genes identified in basidiomycete genomes (in collaboration with the DOE JGI). The most interesting genes were heterologously expressed, and the corresponding enzymes structurally-functionally characterized. The information obtained enabled us to improve the enzyme operational and catalytic properties by directed mutagenesis. However, the structural-functional relationships explaining some desirable properties are not established yet and, therefore, their introduction was addressed by ‘non-rational’ directed evolution. Then, over 100 oxidative biotransformations were analyzed. Among them, it is noteworthy to mention the regio/stereo selective hydroxylation of long/short-chain alkanes (a chemically challenging reaction), epoxidation of alkenes, and production of hydroxy-fatty acids. Concerning aromatic oxygenations, the regioselective hydroxylation of flavonoids, and stereoselective hydroxylation/epoxidation of alkyl/alkenyl-benzenes were among the most remarkable reactions, together with enzymatic hydroxylation of benzene (as an alternative for harsh chemical process). Finally, peroxidases and peroxygenases also showed a potential as delignification biocatalysts and in the decolorization of contaminant dyes from textile industries. KW - peroxygenases KW - peroxidases KW - lignin degradation KW - oxidative industrial biocatalysts KW - enzyme rational design KW - directed enzyme evolution KW - selective oxygenation Y1 - 2014 UR - http://onlinelibrary.wiley.com/doi/10.1002/bbb.1498/abstract U6 - https://doi.org/10.1002/bbb.1498 SN - 1932-1031 VL - 8 IS - 6 SP - 819 EP - 835 ER - TY - GEN A1 - Babot, Esteban D. A1 - Río, José C. del A1 - Cañellas, Marina A1 - Sancho, Ferran A1 - Lucas, Fátima A1 - Guallar, Víctor A1 - Kalum, Lisbeth A1 - Lund, Henrik A1 - Gröbe, Glenn A1 - Scheibner, Katrin A1 - Ullrich, René A1 - Hofrichter, Martin A1 - Martínez, Angel T. A1 - Gutiérrez, Ana T1 - Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study T2 - Applied and Environmental Microbiology N2 - The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally-friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, and steroid hydrocarbons and ketones were followed by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating (interestingly antiviral and other biological activities of 25-hydroxycholesterol have been recently reported). However, hydroxylation in the ring moiety and terminal hydroxylation at the side-chain was also observed in some steroids, the former favored by the absence of oxygenated groups at C3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active site geometry and hydrophobicity favors the entrance of the steroid side-chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side-chain entrance ratio could be established, that explains the varying reaction yields observed. KW - Peroxyenase Y1 - 2015 UR - http://aem.asm.org/content/early/2015/04/08/AEM.00660-15 U6 - https://doi.org/10.1128/AEM.00660-15 SN - 0099-2240 SN - 1098-5336 VL - 81 IS - 12 SP - 4130 EP - 4142 ER -