@misc{MartinezRuizDuenasGutierrezetal., author = {Mart{\´i}nez, Angel T. and Ruiz-Due{\~n}as, Francisco J. and Guti{\´e}rrez, Ana and R{\´i}o, Jos{\´e} C. del and Alcalde, Miguel and Liers, Christiane and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin and Kalum, Lisbeth and Vind, Jesper and Lund, Henrik}, title = {Search, engineering, and applications of new oxidative biocatalysts}, series = {Biofuels, Bioproducts and Biorefining}, volume = {8}, journal = {Biofuels, Bioproducts and Biorefining}, number = {6}, issn = {1932-1031}, doi = {10.1002/bbb.1498}, pages = {819 -- 835}, abstract = {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.}, language = {en} } @misc{BabotRioCanellasetal., author = {Babot, Esteban D. and R{\´i}o, Jos{\´e} C. del and Ca{\~n}ellas, Marina and Sancho, Ferran and Lucas, F{\´a}tima and Guallar, V{\´i}ctor and Kalum, Lisbeth and Lund, Henrik and Gr{\"o}be, Glenn and Scheibner, Katrin and Ullrich, Ren{\´e} and Hofrichter, Martin and Mart{\´i}nez, Angel T. and Guti{\´e}rrez, Ana}, title = {Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study}, series = {Applied and Environmental Microbiology}, volume = {81}, journal = {Applied and Environmental Microbiology}, number = {12}, issn = {0099-2240}, doi = {10.1128/AEM.00660-15}, pages = {4130 -- 4142}, abstract = {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.}, language = {en} }