Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [145]-167).

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references. Also available online.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009. / Committee Chair: Ragauskas, Arthur; Committee Member: Bunz, Uwe; Committee Member: Cairney, John; Committee Member: Collard, David; Committee Member: Singh, Preet. Part of the SMARTech Electronic Thesis and Dissertation Collection.

La biomasse végétale est une alternative durable et écologique pour les ressources fossiles. L'exploitation et la valorisation de cette biomasse sont rendues possibles grâce à la capacité naturelle des enzymes fongiques à dégrader et modifier cette biomasse. Parmi ces enzymes, les glyoxal oxydases génératrices de H2O2 (GLOX) restent un groupe peu étudié avec un seul exemple de protéine caractérisée dans la littérature à partir d’un champignon dégradant le bois.Dans cette thèse, trois GLOX, précédemment identifiées dans le génome du champignon dégradant le bois Pycnoporus cinnabarinus (PciGLOX), ont été sélectionnées, produites par voie hétérologue et caractérisées. La caractérisation a révélé des différences entre les trois PciGLOX dans la stabilité des protéines, la spécificité du substrat et l’efficacité catalytique. Les protéines PciGLOX sont produites sous leur forme inactive et leur mécanisme d'activation a été étudié. La capacité des GLOX à catalyser la réaction d'oxydation du 5-hydroxyméthylfurfural (HMF), d’intérêt industriel, a été étudiée pour la première fois dans ce travail. Le HMF a été oxydé par PciGLOX en acide 5-hydroxyméthyl-2-furancarboxylique (HMFCA) comme produit principal. Le HMFCA est difficile à produire par catalyse chimique et est utilisé dans la production de polyesters et de produits pharmaceutiques. PciGLOX ont également été capables de produire l’acide furandicarboxylique (FDCA), qui est un précurseur dans les procédés de production du bioplastique. Ce travail ouvre de nouvelles perspectives pour étudier plus en détail le rôle de GLOX dans la dégradation de la lignocellulose, et dans les applications biotechnologiques. / Plant biomass is a sustainable and eco-friendly alternative for fossil fuels. The exploitation and valorisation of plant biomass is possible through biotechnological processes that rely on the natural ability of fungal enzymes to degrade and modify this biomass. Among these enzymes are H2O2-generating glyoxal oxidases (GLOX), which haven’t been extensively studied with only one example in the literature on GLOX from wood-degrading fungi. In this thesis three GLOX, previously identified in the genome of the wood-degrading fungus Pycnoporus cinnabarinus (PciGLOX), were heterologously produced and characterisation. The three PciGLOX showed differences in their stability, substrate preferences and catalytic properties. The ability of GLOX to catalyse the biotechnologically important oxidation reaction of 5-hydroxymethylfurfural (HMF) was investigated for the first time in this work. PciGLOX oxidized HMF to 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), which is difficult to produce via chemical catalysis and is used in polyesters and pharmaceutical products production. PciGLOX were also able to oxidize HMF derivatives leading to the formation of the final product furandicarboxylic acid (FDCA), which is a bioplastic precursor. PciGlOX proteins are produced in their inactive form and their activation mechanism was investigated in this thesis. This work opens new prospects to investigate more the role of GLOX in plant biomass degradation and biotechnology, and the possible optimization techniques of the catalytic properties of this enzyme.

O capítulo 1 descreve o estudo da biorredução do grupo carbonílico de cetoazidas e β-ceto-1,2,3-triazois para a produção de β-hidroxi-1,2,3-triazois enantiomericamente puros ou enriquecidos. Cinco linhagens de fungos de origem marinha foram avaliadas para a redução da 2-azido-1-feniletanona 1 e duas linhagens, A. sydowii CBMAI 935 e M. racemosus CBMAI 847, foram selecionadas também para a biorredução das 2-azido-1-feniletanonas 2-4 para a produção dos (R)- e (S)-2-azido-1-feniletanois 2a-4a. Os azidoálcoois enantiomericamente enriquecidos obtidos 1a-4a das reações biocatalíticas foram empregados como material de partida para a síntese dos β-hidroxi-1,2,3-triazois 7a-10a enantiomericamente enriquecidos através da click reaction entre a azida terminal e o alcino, fenilacetileno. Uma segunda abordagem para a obtenção de β-hidroxi-1,2,3-triazois enantiomericamente enriquecidos foi o estudo da biorredução de β-ceto-1,2,3-triazois, que são cetonas contendo dois substituintes volumosos. Uma triagem inicial para a biorredução do β-ceto-1,2,3-triazol 7 foi realizada com seis linhagens de fungos de origem marinha, na qual a linhagem do fungo P. citrinum CBMAI 1186 foi selecionada para estudos de otimização da reação biocatalítica. Estudos com variação do meio reacional, utilização de co-solvente e efeito do pH mostraram que as condições ótimas de reação foram utilizando-se tampão fosfato (Na2HPO4/KH2PO4, 0,07 M) em pH 5 e metanol 5% (v/v) como co-solvente. O fungo P. citrinum CBMAI 1186 foi empregado na biorredução dos β-ceto-1,2,3-triazois 8-12 com excelentes resultados de rendimento e seletividade para a produção dos (S)-β-hidroxi-1,2,3-triazois 8a-12a. O Capítulo 2 apresenta a síntese multienzimática da diidropinidina, um alcaloide de origem natural. A nonano-2,6-diona utilizada como material de partida foi obtida através da descarboxilação do dicetoéster, 2-butiril-5-oxo-hexanoato de etila. Estudos para a otimização tanto da síntese do dicetoéster quanto da etapa de descarboxilação foram realizados. Condições ótimas de produção do 2-butiril-5-oxo-hexanoato de etila foram obtidas através da reação da but-3-em-2-ona com o 3-oxo-hexanoato de metila catalisada por CeCl3/NaI. A descarboxilação do dicetoéster foi avaliada através do método de Krapcho empregando-se sais de cloro e água em altas temperaturas, entretanto, a elevada formação de subprodutos estimulou a busca por uma diferente metodologia para a obtenção da nonano-2,6-diona. Foram avaliadas diferentes lipases e esterases para a hidrólise enzimática do dicetoéster seguida por descarboxilação por HCl, na qual a esterase de fígado de porco foi selecionada e promoveu a hidrólise de até 1,6 M de dicetoéster para a produção da nonano-2,6-diona. Diferentes transaminases (TAs) foram estudadas para a aminação redutiva assimétrica da nonano-2,6-diona e duas linhagens foram selecionadas para a produção da (R)- e (S)-2-metil-6-propil-2,3,4,5-tetra-hidropiridina, as TAs de Arthrobacter sp. e Arthrobacter citreus, respectivamente empregando-se isopropilamina como amino doador. As (R)- e (S)-2-metil-6-propil-2,3,4,5-tetra-hidropiridina foram avaliadas pela redução assimétrica para a síntese da diidropinidina por imina redutases (IREDs) e duas linhagens foram selecionadas, a IRED de Mesorhizobium sp. e Norcardiopsis alba, respectivamente. TAs e IREDs foram acopladas em um sistema one-pot multienzimático utilizando como material de partida a nonano2,6-diona (100 mM) para a obtenção dos isômeros cis da diidropinidina com excelentes excessos diastereoisoméricos. / The Chapter 1 describes the bioreduction of the carbonyl group of ketoazides and β-keto-1,2,3-triazoles to produce enantiomerically pure or enriched β-hydroxy-1,2,3-triazoles. Five marine-derived fungi strains were screened to perform the reduction of 2-azido-1-phenylethanone 1. The strains from A.sydowii CBMAI 935 and M. racemosus CBMAI 847 were selected for the bioreduction of the 2-azido-1-phenylethanones 2-4 to yield the (R)- and (S)-2-azido-1-phenylethanols 2a-4a. The enantiomerically enriched azidoalcohols 1a-4a obtained from biocatalytical reactions were used as starting materials for the synthesis of enantiomerically enriched β-hydroxy-1,2,3-triazoles 7a-10a through the click reaction between the terminal azide and phenylacetylene. A second approach for obtaining enantiomerically enriched β-hydroxy-1,2,3-triazoles was the bioreduction of β-keto-1,2,3-triazoles, which are ketones with two bulky substituents. The screening for the bioreduction of the β-keto-1,2,3-triazol 7 was performed with six marine-derived fungi strains and P. citrinum CBMAI 1186 was selected for the optimization studies for the biocatalytic reduction of β-keto-1,2,3-triazoles 8-12.Studies about the composition of reaction medium, use of cosolvent and pH effect showed that the optimal conditions was in phosphate buffer (Na2HPO4/KH2PO4, 0.07 M) at pH 5 and methanol 5% (v/v) as cosolvent. P. citrinum CBMAI 1186 was applied to the bioreduction of β-keto-1,2,3-triazoles 8-12 and good yields and selectivities were obtained for the (S)-β-hydroxy-1,2,3-triazoles 8a-12a. The Chapter 2 describes the multienzymatic synthesis of dihydropinidine, a natural alkaloid. The nonane-2,6-dione used as starting material was obtained through the reduction of the diketoester, methyl butyryl-5-oxohexanoate, and the optimization studies for both diketoester synthesis and decarboxylation reaction were performed. Optimal conditions for the synthesis of methyl butyryl-5-oxohexanoate were obtained by the reaction between but-3-en-2-one and 3-oxohexanoate catalyzed by CeCl3/NaI. The diketoester decarboxylation step was evaluated by the Krapcho method using chlorine and water at high temperatures. However, because of the production of side products by this method, a different procedure for the synthesis of nonane-2,6-dione was studied. Different enzymes (lipases and esterases) were evaluated for the diketoester hydrolysis followed by decarboxylation by HCl. The porcine liver esterase was selected to promote the diketoester hydrolysis up to 1.6 M, yielding nonane-2,6-dione. Different transaminases (TAs) were applied to the asymmetric reductive amination of the nonane-2,6-dione and TAs from Arthrobacter sp. e Arthrobacter citreus were selected for the production of (R)- and (S)-2-methyl-6-propyl-2,3,4,5-tetrahydropyridine, respectively, using isopropylamine as the amine donor. The asymmetric reduction of (R)- and (S)-2-methyl-6-propyl-2,3,4,5-tetrahydropyridine by imine reductases (IREDs) was evaluated and the IREDs from Mesorhizobium sp. and Norcardiopsis alba were selected. TAs and IREDs were coupled in multienzymatic one-pot system using nonane-2,6-dione (100 mM) as starting material for the syntheses of cis isomers of dihydropinidine in excellent diastereoisomeric excess.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T18:00:04Z (GMT). No. of bitstreams: 1 Costa_BrunaZucolotoda_D.pdf: 6212345 bytes, checksum: ec8b84a214cbc377e0f9f13d33ddc561 (MD5) Previous issue date: 2011 / Resumo: Os processos biocatalíticos foram abordados nesta tese de doutorado deste a triagem de micro-organismos para a seleção de biocatalisadores adequados até o uso de enzimas isoladas em reações de interesse biotecnológico. O primeiro capítulo apresenta o isolamento, identificação e a triagem enzimática de bactérias heterotróficas isoladas de rejeitos de mineração de cobre. A partir destas amostras foi possível isolar 189 bactérias, as quais apresentaram uma diversificada atuação catalítica. As bactérias isoladas foram identificadas por MALDI-TOF e por sequenciamento do gene RNAr 16S sendo encontrados diversos gêneros como Bacillus, Acinetobacter, Pseudomonas, Delftia, Stenotrophomonas, Hydrogenophaga, Rhodococcus, entre outros. O segundo capítulo apresenta o estudo do potencial catalítico de uma nova epóxido hidrolase de Aspergillus brasiliensis CCT1435, recombinante e expressa em E. coli. Esta EH é ativa em uma ampla faixa de pH e temperatura, apresentando um desempenho ótimo em pH 6,0 e 30 °C. Este trabalho ainda permitiu uma avaliação detalhada da aplicação biocatalítica desta EH na hidrólise do óxido de estireno em meio aquoso e bifásico. Por fim, o terceiro capítulo apresenta resultados preliminares envolvendo um processo quimio-enzimático para a produção de alcalóides pirrolidínicos a partir das respectivas 1,4-dicetonas. Esta metodologia é promissora uma vez que diversas 1,4-dicetonas foram convertidas nas suas respectivas iminas cíclicas, sendo que a subsequente etapa de redução com NaBH3CN promoveu a formação das pirrolidinas de interesse. Portanto, esta tese de doutorado apresenta um estudo amplo e diversificado envolvendo processos biocatalíticos aplicados em uma série de reações de interesse biotecnológico / Abstract: Biocatalytic processes have been addressed in this thesis from the microorganism screening for the selection of suitable biocatalysts to the use of isolated enzymes in biotechnological reactions. The first chapter presents the isolation, identification and enzymatic screening of heterotrophic bacteria isolated from copper mine drainages. From these samples, 189 bacteria were isolated, which showed diverse catalytic activities. The isolated bacteria were identified by MALDI-TOF and 16S rRNA gene sequencing and several genera were found: Bacillus, Acinetobacter, Pseudomonas, Delftia, Stenotrophomonas, Hydrogenophaga, Rhodococcus, among others. The second chapter presents the catalytic potential of a new epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH), recombinant and expressed in E. coli. This EH is active in a wide pH and temperature range, with a great performance at pH 6.0 and 30 °C. This work also presents the AbEH biocatalytic application for the styrene oxide hydrolysis in aqueous and biphasic media. Finally, the third chapter presents preliminary results involving a chemo-enzymatic method for the production of pyrrolidine alkaloids from the corresponding 1,4-diketones. This approach is promising since several 1,4-diketones were converted to their respective cyclic imines, and the subsequent reduction with NaBH3CN promoted the pyrrolidines formation. Therefore, this thesis presents a broad and diverse study of biocatalytic processes applied in a number of interesting biotechnological reactions / Doutorado / Quimica Organica / Doutora em Ciências

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.