Search

17 páginas.- 4 figuras.- 4 tablas.- 46 referencias.- Supplementary Materials: The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antiox11050915/s1, Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO (by substituting one, two, or up to six residues of its access channel by alanines) improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α–linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate. © 2022 by the authors. Licensee MDPI, Basel, Switzerland., This research was funded by BioBased Industries Joint Undertaking under the European Unions Horizon 2020 Research and Innovation Programme, grant number 792063 (SusBind project; https://susbind.eu; to A.G and A.T.M.); the BIO2017-86559-R project of the Spanish Ministry of Science and Innovation (co-financed by FEDER funds; to A.T.M.); the PID2020-118968RB-I00 project by the Spanish MCIN/AEI/10.13039/501100011033 (to A.G.); the CSIC projects PIE-202040E185 (to A.G.) and PIE-202120E019 (to A.T.M.); the CSIC SusPlast platform (to A.T.M.); and the CSIC program for the Spanish Recovery, Transformation, and Resilience Plan funded by the Recovery and Resilience Facility of the European Union, established by the Regulation (EU) 2020/2094.

12 páginas.- 2 figuras.- 2 tablas.- 55 referencias.-Supplementary Materials: The following supporting information can be downloaded at: https:www.mdpi.com/article/10.3390/antiox11030522/s1, Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, fla-voring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosub-strate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosub-strate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, nota-ble differences were observed between them, with rCciUPO being responsible for the highest sub-strate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies. © 2022 by the authors. Licensee MDPI, Basel, Switzerland, This research was funded by BioBased Industries Joint Undertaking under the European Union’s Horizon 2020 Research and Innovation Programme, grant number 792063 (SusBind project; https://susbind.eu, accessed on 1 February 2022; to A.G., A.T.M. and M.H.); the PID2020-118968RB-100 project of the Spanish MCIN/AEI/10.13039/501100011033 to A.G.; the CSIC projects PIE-202040E185 (to A.G.) and PIE-202120E019 (to A.T.M.); the CSIC SusPlast platform (to A.T.M.); and the CSIC program for the Spanish Recovery, Transformation and Resilience Plan funded by the Recovery and Resilience Facility of the European Union, established by the Regulation (EU) 2020/2094

9 p.-8 fig.-2 tab., 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., This work has been funded by the BBI-JU (www.bbi-europe.eu) project SusBind (on “Sustainable biobinders”; H2020-BBI-JTI-2017-792063; https://susbind.eu), together with the CTQ2016-79138-R and BIO2017-86559-R (GENOBIOREF) projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

19 páginas.- 8 figuras.- 5 tablas.- 174 referencias, Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as “dream biocatalysts” of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases. © 2021 The Authors, This work has received funding from the Bio Based Industries Joint Undertaking under the European Union's Horizon 2020 research and innovation programme under Grant Agreement No 792063 (Development and pilot production of sustainable binder systems for wood based panels, https://susbind.eu), and from the Spanish Ministry of Industry, Economy and Competitiveness project BIO2017-86559-R, cofinanced by FEDER funds, and the Consejo Superior de Investigaciones Científicas projects PIE-201620E081 and PIE-202040E185

12 páginas.- 6 figuras.- 1 tabla.- 84 referencias.- The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscatal.0c03165, Epoxidation of unsaturated fatty acids by unspecific peroxygenases (UPOs) of the best-known long-UPO subfamily, including the Agrocybe aegerita (AaeUPO) and Coprinopsis cinerea enzymes, is reported here. To understand the different oxygenation patterns by members of the long-UPO and short-UPO subfamilies, the latter represented by the Marasmius rotula enzyme (MroUPO), fatty-acid diffusion into their heme pockets was simulated with the adaptive PELE software. Computational results shed light on the inability of AaeUPO to epoxidize oleic acid (C18:1), opposed to MroUPO, due to steric hindrances to harbor (with a good interaction energy) the substrate with the Δ9 C10 atom at a catalytically relevant distance (99%) formation of cis,cis-15,16-epoxyoctadeca-9,12-dienoic acid. The nearly complete conversion of α-linolenic acid by the two enzymes was transferred to a small preparative scale, the yield of purified product was estimated, its chemical structure analyzed by NMR, and more interestingly, stereoselective production of the 15(R),16(S) enantiomer (80-83% ee) assessed by chiral HPLC. This enzymatic synthesis overcomes the unspecificity of chemical epoxidation where the reaction cannot be restricted to the formation of monoepoxides as found during m-perchlorobenzoic acid oxidation of α-linolenic acid. Moreover, the variant was able to produce the unsaturated dicarboxylic fatty acid, together with subterminal oxygenation products, during partial conversion of oleic acid. These two noteworthy reactions had not been reported for any UPO described to date. © 2020 American Chemical Society. All rights reserved., This work has received funding from the Bio Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No 792063 (“Development and pilot production of sustainable binder systems for wood based panels”, https://susbind.eu ), the CTQ2016-79138-R and BIO2017-86559-R projects of Spanish MINECO, the Secretaria d’Universitats i Recerca of Generalitat de Catalunya, and the European Social Fund (ESF-2019-FI-B-00154). The authors thank Novozymes A/S for supplying r CciUPO. MM acknowledges a Catalan Government doctoral grant.

9 páginas.- 6 figuras.- 5 tablas.- 37 referencias.- The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.0c01019, Apocarotenoids are among the most highly valued fragrance constituents, being also appreciated as synthetic building blocks. This work shows the ability of unspecific peroxygenases (UPOs, EC1.11.2.1) from several fungi, some of them being described recently, to catalyze the oxyfunctionalization of α- and β-ionones and α- and β-damascones. Enzymatic reactions yielded oxygenated products such as hydroxy, oxo, carboxy, and epoxy derivatives that are interesting compounds for the flavor and fragrance and pharmaceutical industries. Although variable regioselectivity was observed depending on the substrate and enzyme, oxygenation was preferentially produced at the allylic position in the ring, being especially evident in the reaction with α-ionone, forming 3-hydroxy-α-ionone and/or 3-oxo-α-ionone. Noteworthy were the reactions with damascones, in the course of which some UPOs oxygenated the terminal position of the side chain, forming oxygenated derivatives (i.e., the corresponding alcohol, aldehyde, and carboxylic acid) at C-10, which were predominant in the Agrocybe aegerita UPO reactions, and first reported here., This work was supported by the EnzOx2 (H2020-BBI-PPP-2015-2-1-720297) EU-project and the CSIC (201740E071) project.

26 páginas.- 28 figuras.- 71 referencias, Deracemisation methods have demonstrated their importance in the preparation of chiral compounds in the last few years. In order to resolve a racemic mixture in a dynamic sense, one enantiomer of the starting material can be converted to the other through a deracemisation procedure, that can be achieved by different mechanisms based on stereoinversion or enantioconvergence, often involving two‐opposite half reactions, with at least one of the reactions being enantioselective enough to finally obtain an enantioenriched chiral compound. The focus of this comprehensive review is on the application of deracemisation procedures in the present century in order to obtain optically active valuable compounds when employing non‐metallic catalysts. Thus, the review will mainly focus on the use of different enzymatic preparations (purified enzymes, cell‐free extracts or whole cell systems) and organocatalysts for the deracemisation of racemic mixtures., We thank Martin G. López-Vidal for his collaboration.G.d.G. thanks MINECO for Ramón y Cajal Program and CTQ2016-76908-C2-1,2-P projects. G.O-M. and F.R.B. ac-knowledge INFIQC-CONICET and Universidad Nacionalde Cordoba (UNC) and also thank FONCyT as well asSECyT-UNC for research funding.

25 páginas.- 2 figura.- 4 tablas.- 21 diagramas- 102 referencias, The application of biocatalysts to perform reductive/oxidative chemical processes has attracted great interest in recent years, due to their environmentally friendly conditions combined with high selectivities. In some circumstances, the aqueous buffer medium normally employed in biocatalytic procedures is not the best option to develop these processes, due to solubility and/or inhibition issues, requiring biocatalyzed redox procedures to circumvent these drawbacks, by developing novel green non-conventional media, including the use of biobased solvents, reactions conducted in neat conditions and the application of neoteric solvents such as deep eutectic solvents

202 páginas.- Tutor Dr. D. José María Fernández-Bolaños Catedrático de la Universidad de Sevilla, In this Thesis, the selective synthesis of different products of industrial interest such as antioxidants derived from trans-stilbene and other stilbenoids, flavour-and-fragrance additives derived from α-isophorone, and epoxides from unsaturated fatty acids (FA) and their methyl esters (FAME) catalyzed by fungal unspecific peroxygenases (UPOs, EC.1.11.2.1) was studied. With this purpose, several UPOs were tested, including those from the basidiomycetes Agrocybe aegerita (AaeUPO), Marasmius rotula (MroUPO) as well as the recombinant enzyme from Coprinopsis cinerea (rCciUPO) and those from the ascomycetes Chaetomium globosum (CglUPO) and the recombinant enzyme from Humicola insolens (rHinUPO). The first reaction studied was the oxygenation of trans-stilbene and other stilbenoids such as pinosylvin (Pin) and resveratrol (RSV). In trans-stilbene oxygenation, different reactivities were found depending on the UPO used. AaeUPO, MroUPO and rCciUPO selectively hydroxylated the aromatic rings to form the compound of interest 4,4′-dihydroxy-trans-stilbene (DHS). DHS is a RSV analogue whose preventive effects on cancer invasion and metastasis has recently been shown. The kinetic studies revealed AaeUPO as the most efficient enzyme with one and two orders of magnitude higher catalytic efficiencies (kcat/Km) for the first and second hydroxylation steps, respectively, compared to MroUPO and rCciUPO. Likewise, AaeUPO achieved the highest total turnover numbers (TTN) of up to 200 000. In addition, AaeUPO gave also the best results in Pin and RSV hydroxylation reactions with higher regioselectivity and substrate conversions. However, CglUPO failed to hydroxylate the aromatic ring and instead epoxidized the double bond of the alkenyl moiety forming trans-stilbene epoxide as the only product. True peroxygenative activity was demonstrated by incorporation of 18O from H218O2 in the oxidation products. The second reaction studied comprises the α-isophorone hydroxylation by UPOs to produce 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are flavor-and-fragrances molecules commonly used as additives and as intermediates in the synthesis of vitamins and carotenoids. rHinUPO and CglUPO selectively transformed the substrate (10 mM) in the compounds of interest attaining TTN of up to 5500. Interestingly, although AaeUPO was found to be less selective since this enzyme oxygenated positions 4 and 7 of α-isophorone, it was the only stereoselective enzyme producing the S-enantiomer of 4HIP with an enantiomeric excess of 88%. On the other hand, using the racemic 4HIP as substrate, a faster conversion of the S-enantiomer by rHinUPO and CglUPO was observed, leading to a kinetic resolution of the racemate with 60-75% recovery of the R-enantiomer. Surprisingly, MroUPO and rCciUPO failed to transform α-isophorone. These differences in regioselectivities could be explained by computational analysis carried out with adaptive PELE software, where differences in the distances between the substrates atoms and the oxo-heme of reactive Compound I and different binding energies were observed. The last reaction studied included the oxygenation of FAs and FAMEs to form valuable reactive epoxides. A series of mono- and poly-unsaturated FAs and FAMEs were transformed by MroUPO and CglUPO with high conversion yields, generally attaining better selectivity towards the desired epoxide with the latter enzyme. The differences were observed during oleic acid oxygenation reactions, where CglUPO showed one order of magnitude higher catalytic efficiency and higher TTN (8000) compared to MroUPO (4000). The regioselectivity observed is different from the previously described subterminal hydroxylation by the well-known rCciUPO and AaeUPO, which do not epoxidize unsaturated FAs. In conclusion, biotransformations using these novel and appealing biocatalysts would represent a potential more environmentally friendly alternative to chemical synthesis in the oxyfunctionalization reactions described during this Thesis., Esta Tesis se ha llevado a cabo en el Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS) del Consejo Superior de Investigaciones Científicas (CSIC) con un contrato predoctoral del Ministerio de Economía y Competitividad asociado al Proyecto Nacional “Modificación enzimática de lignina y lípidos en las biorrefinerías de la lignocelulosa” (BIORENZYMERY, AGL2014-53730-R, cofinanciado con fondos FEDER). Esta Tesis ha sido financiada también por los Proyectos Europeos “Industrial oxidoreductases” (INDOX, GA-KBBE-2013-7-613549) y “New enzymatic oxidation /oxyfunctionalization technologies for added value bio-based products” (EnzOx2, H2020-BBI-PPP-2015-2-1-720297).

8 páginas.-- 4 figuras.-- 4 tablas.-- 34 referencias.-- Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cy00272j, This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200000) than MroUPO (26000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV., This study was funded by the EnzOx2 (H2020-BBI-PPP-2015-2-1-720297) EU-project, and the BIORENZYMERY (AGL2014- 53730-R) project of the Spanish MINECO (co-financed by FEDER). H. Lund (Novozymes) is acknowledged for rCciUPO