No description available.

Yes / The crotonases comprise a widely distributed enzyme superfamily that has multiple roles in both primary and secondary metabolism. Many crotonases employ oxyanion hole-mediated stabilization of intermediates to catalyze the reaction of coenzyme A (CoA) thioester substrates (e.g., malonyl-CoA, α,β-unsaturated CoA esters) both with nucleophiles and, in the case of enolate intermediates, with varied electrophiles. Reactions of crotonases that proceed via a stabilized oxyanion intermediate include the hydrolysis of substrates including proteins, as well as hydration, isomerization, nucleophilic aromatic substitution, Claisen-type, and cofactor-independent oxidation reactions. The crotonases have a conserved fold formed from a central β-sheet core surrounded by α-helices, which typically oligomerizes to form a trimer or dimer of trimers. The presence of a common structural platform and mechanisms involving intermediates with diverse reactivity implies that crotonases have considerable potential for biocatalysis and synthetic biology, as supported by pioneering protein engineering studies on them. In this Perspective, we give an overview of crotonase diversity and structural biology and then illustrate the scope of crotonase catalysis and potential for biocatalysis. / Biotechnology and Biological Sciences Research Council, the Medical Research Council, and the Wellcome Trust

Yes / Studies on the substrate selectivity of recombinant ferrous-iron- and 2-oxoglutarate-dependent proline hydroxylases (PHs) reveal that they can catalyse the production of dihydroxylated 5-, 6-, and 7-membered ring products, and can accept bicyclic substrates. Ring-substituted substrate analogues (such hydroxylated and fluorinated prolines) are accepted in some cases. The results highlight the considerable, as yet largely untapped, potential for amino acid hydroxylases and other 2OG oxygenases in biocatalysis.

The work in this Thesis addresses the challenges associated with using redox enzymes for chemical synthesis. The use of enzymes as catalysts in the synthesis of fine chemicals is becoming more wide spread, in part due their ability to catalyse reactions with incredible selectivity under relatively mild conditions. In particular, enzymes are useful for selective reduction of ketones to enantiomerically pure alcohols or amines, and partial oxidations of alkanes to alcohols. However, a key limitation to exploiting redox enzymes in these reaction pathways is the requirement for a specialised electron source, usually the expensive nicotinamide cofactors NADH or NADPH. Existing cofactor regeneration methods use a second enzyme with a sacrificial substrate which is oxidised to generate a stoichiometric waste product; this complicates isolation of the desired product and prevents the environmental benefits of biocatalysis from being fully realised. In order to provide clean and efficient biocatalytic routes, improved recycling methods for these cofactors are crucial. This Thesis develops two novel methods for in situ cofactor recycling. The first is an electro-enzymatic system; an NAD⁺-reductase enzyme is shown to use electrons directly from an electrode for supply of NADH to a co-immobilised cofactor-dependent enzyme. The second uses a hydrogenase, NAD⁺ reductase and cofactor-dependent enzyme immobilised on conducting particles for H₂-driven NADH regeneration. This relies on the thermodynamically favourable reduction of NAD⁺ by H₂ when the hydrogenase and NAD⁺-reductase are in electronic contact, provided by the conducting particle. The electro-enzymatic approach to NAD⁺ reduction is then adapted for electrochemical devices; an enzyme catalysed fuel cell and a self-powered biosensor were considered.

Très récemment, les solvants de type « mélanges eutectiques profonds (MEP)» ont été décrits comme une alternative sérieuse et économiquement plus réaliste aux liquides ioniques. En effet, ces solvants qui consistent en un mélange d'un sel organique (ammonium ou phosphonium) et d'un donneur de liaison hydrogène peuvent également être liquides à température ambiante, non volatils et présentant une excellente stabilité thermique. De plus, contrairement aux liquides ioniques, ces nouveaux solvants sont très facilement préparés et leur innocuité ainsi que leur bonne biodégradabilité sont sensiblement améliorées. Dans le domaine des procédés enzymatiques, si la biocatalyse en milieu liquide ionique est très documentée, il n'existe que très peu de publications décrivant des réactions de biotransformation en MEP. Concernant les lipases en particulier, outre leurs applications dans le biofaçonnement des corps gras, ces enzymes sont également utilisées dans des réactions dites de lipophilisation pour la synthèse de nouvelles molécules à haute valeur ajoutée (tensioactifs, antioxydant lipophilisés). Au travers cette étude nous nous sommes investis à tester le potentiel des MEP en tant que nouveaux milieux réactionnels « verts » pour la synthèse lipasique. Ce travail n'a pas eu comme objectif de faire l'éloge de ces solvants pour leur utilisation dans le domaine de la biocatalyse, mais surtout d'évaluer leur capacité à favoriser ou non des synthèses lipasiques. Par ailleurs, nous nous sommes engagés à essayer de comprendre, d'un point de vue fondamental, l'organisation supramoléculaire de ce type de milieux pour déterminer les paramètres qui influencent le plus la réactivité et la stabilité enzymatique dans ce type d'environnement. Les variations des conditions réactionnelles (solvants et biocatalyseurs) ont permis de mettre en évidence la très nette supériorité de deux MEP (Chlorure de cholinium:Urée et Chlorure de cholinium:glycérol) pour la réalisation de réactions d'alcoolyses biocatalysées par la lipase B de Candida antarctica. Toutefois, les résultats ont montré que les réactions de biotransformations de composés phénoliques dans ces MEP sont extrêmement difficiles à réaliser sans l'addition d'eau. De profondes études (pH, activité thermodynamique de l'eau, activité et stabilité de la lipase, composition du solvant, etc.) réalisées sur des mélanges du type MEP-eau ont permis de finement adapter les conditions de réaction pour optimiser la catalyse enzymatique dans ce type de solvant. Compte tenu des difficultés rencontrées pour la lipophilisation de composés phénoliques, nous sommes toutefois parvenus à synthétiser toute une gamme de dérivés lipophiles d'acides férulique et coumarique de C4 à C16 (chaîne aliphatique) avec des rendements élevés. / With the emergence of the green chemistry concept in the 90s, many studies have been dedicated to the discovery of new reactions media both suitable and efficient for chemical/enzyme catalysis. Up to now, the main efforts have focused on the development of ionic liquids. However, recently a novel class of solvent called "deep eutectic mixtures (DES)", have been described as a serious alternative and economically stronger than ionic liquids. Such solvents are formed by mixing an organic salt (ammonium or phosphonium) with a hydrogen-bond donor. Just like ionic liquid, DES may also be liquid at room temperature, non-volatile and have excellent thermal stability. However, unlike most ionic liquids, these new solvents are biodegradable, inexpensive, and very easy to prepare. In the field of biocatalysis, whereas the studies in ionic liquid are deeply documented, the published papers describing biotransformation reactions in DES are very low, especially in lipase-catalyzed processing, where these enzymes may be used in so-called "lipophilisation reactions", for the synthesis of new molecules with high added value (surfactants or lipophilized antioxidants).The main objective of this work was to assess and test the potential of DES as new "green" reaction media for lipase-catalyzed synthesis. On a fundamental point of view, this study provides valuable information to understand how the different components involved in these mixtures could contribute to their functional properties in order to enhance their use in various applications. Changes in reaction conditions (solvents and biocatalysts) allowed us to highlight the clear superiority of two DES (chloride cholinium:Urea and chloride cholinium:glycerol) to carry out lipase-catalyzed reactions using the lipase B from Candida antarctica as biocatalyst. However, our results showed that the biotransformations of dissolved substrates (such as phenolic compounds) in DES are extremely difficult to achieve without the addition of water. Studying DES-water mixtures (pH, thermodynamic activity of water, activity and stability of lipase, mixtures composition, etc ...) we were able to fine-tune the reaction conditions to optimize the performance of the lipasic catalysis. Thus, given the difficulties encountered when performing lipase-catalyzed reactions with substrates of two different polarities, it was still possible to synthesize high yields of a full range of lipophilic derivatives of ferulic and coumaric acids from C4 to C16 (aliphatic chain).

Xilitol é um açúcar-álcool com propriedades de interesse para as indústrias alimentícia, odontológica e farmacêutica. É tradicionalmente produzido em processo químico, sendo que a via fermentativa é a forma mais extensivamente estudada, entretanto, ainda possui limitações técnicas. Neste contexto, o presente trabalho teve como objetivo determinar condições de obtenção de xilitol por via enzimática utilizando a enzima xilose redutase de Candida guilliermondii FTI 20037. Numa primeira etapa, esta enzima foi produzida e pré-purificada e em seguida foi realizada a seleção do sistema de regeneração enzimática in situ de coenzima NADPH. Foram considerados sistemas hipotéticos com formato desidrogenase, glicose desidrogenase e álcool desidrogenase, sendo determinado o efeito dos possíveis substratos e produtos sobre a xilose redutase pré-purificada. O sistema de regeneração escolhido foi o que utilizou a enzima glicose desidrogenase, sendo o substrato glicose e o produto gluconato. Em seguida, foi realizada a avaliação e seleção de variáveis do processo enzimático segundo planejamento fatorial fracionado 25-1. Foi avaliada a influência da concentração de xilose, de NADPH e de glicose, a carga de xilose redutase e de glicose desidrogenase sendo que a variável resposta foi considerada a produtividade volumétrica em xilitol. As duas variáveis selecionadas para otimização foram a concentração de xilose e de NADPH. Para a otimização do processo de produção de xilitol em meio sintético sob regime de batelada empregou-se um planejamento composto central rotacional (estrela) 22. A partir dos resultados pode-se construir um modelo quadrático que relacionou a produtividade com os fatores na região de estudo. De acordo com este modelo, a melhor condição operacional resulta em alto valor de produtividade e eficiência em xilitol, 1,68 g.l-1.h-1 e 100 %, respectivamente. Visando a viabilidade econômica do processo foram avaliadas membranas de ultra e nanofiltração para retenção das enzimas e coenzimas no sistema reacional. Foi constatado que a membrana de tamanho de poro de 1 kDa permitiu a retenção de 99 % da coenzima. Adicionalmente, foi avaliado o desempenho da enzima obtida a partir de hidrolisado hemicelulósico de bagaço de cana-de-açúcar, comparando-se os resultados obtidos com aquela obtida pelo cultivo da levedura em meio baseado em xilose comercial. Foi comprovado que a fonte de carbono não teve efeito sob a produção enzimática de xilitol. Finalmente, foram realizados testes preliminares com a produção enzimática de xilitol em meio de hidrolisado de bagaço de canade- açúcar. Foi observado que a produção de xilitol não se alterou com meio contendo 20 e 40 % v.v-1 de hidrolisado. Em função dos resultados, foi concluído que a produção enzimática de xilitol é tecnicamente viável e que possui grande potencial como bioprocesso para aproveitamento de bagaço de cana-de-açúcar. / Xylitol is a sugar-alcohol with proven interesting properties for food, odontological and pharmaceutical industries. It is traditionally produced in chemical process and the fermentative way, the most extensively studied alternative, nevertheless, still has disadvantages. In this context, the present work has as objective to determinate optimal conditions for xylitol attainment by enzymatic way using xylose redutase from Candida guilliermondii FTI 20037. Firstly, xylose redutase was produced, pre-purified and then the selection of an in situ enzymatic regeneration of coenzyme NADPH was carried out. Hypothetical regeneration systems were considered: formate dehydrogenase, glucose dehydrogenase and alcohol dehydrogenase, being determined the effect of the possible substrates and products under pre-purified xylose redutase. The glucose dehydrogenase regeneration system, being glucose the substrate and gluconate the product. Afterwards, it was carried out the screening and evaluation of the enzymatic process variables according to a fractioned factorial design 25-1. It was evaluated the influence of xylose, NADPH and glucose concentrations, xylose reductase and glucose dehydrogenase loads using xylitol volumetric productivity as response. Xylose and NADPH concentrations were selected for further optimization. A rotational central composite design (star) 22 was used for optimization of xylitol enzymatic process in synthetic media under batch regime. From the results, a quadratic model could be elaborated which relates the productivity with the factors in the studied region. According to this model, the best operational condition resulted in high productivity and efficiency values, 1,68 g.l-1.h-1 and 100 %, respectively. Aiming economical viability of the process, ultra and nanomembranes were studied for coenzyme and enzymes retention. It was verified that the 1 kDa cut off membrane allowed 99 % retention of the coenzyme. Additionally, it was evaluated the enzyme performance produced from sugarcane bagasse hemicellulosic hydrolysate, comparing the results attained with the enzyme produced from synthetic media. It was evidenced that the carbon source did not affected xylitol enzymatic production. Finally, xylitol enzymatic production preliminary assays were carried out using media containing sugarcane bagasse hydrolysate. It was observed that xylitol enzymatic production was not altered when compared to the control, in the experiments media containing 20 and 40 % v.v-1 hydrolysate. Based on the results, it was concluded that xylitol enzymatic production is technically viable and has great potential as a bioprocess for sugarcane bagasse use as raw material.

Os objetivos do presente trabalho foram: i) aprimorar o procedimento alternativo de mediação por íons metálicos da alcoólise da ligação peptidil-resina com vistas à obtenção de ésteres metílicos de peptídeos protegidos (Nα-acil-peptídeo protegido-OMe) em condição reacional branda e com alta eficiência; ii) investigar a aplicabilidade do procedimento para a preparação de Nα-acil-peptídeo protegido-SR e de Nα-acil-aminoácido-OR; iii) verificar se os Nα-acil-peptídeo-OMe obtidos atuariam como doadores de acila em reações de formação de ligação peptídica catalisadas por lipases. Para tanto, na busca da melhor condição de metanólise e comparação com os procedimentos usuais de alcoólise de ligação peptidil-resina, foram usados: o fragmento 22-24 da colecistocinina-33 humana (tripeptídeo modelo), Ca2+, Zn+2, Co+2 e Cu+2 (mediadores), as resinas oxima de Kaiser (KOR), p-hidroximetilfenil acetamidometil, ácido p-hidroximetilbenzóico e álcool p-benziloxibenzil (suportes poliméricos), misturas de MeOH com DCM, DMSO, NMP, THF ou DMF (solventes) e 25, 37, 50 ou 60°C. A condição ótima encontrada [KOR, Ca+2 (1 eq./eq. de peptídeo), 50% MeOH/DMF, 50°C] foi empregada com sucesso na preparação do Nα--acil-heptapeptídeo protegido-OMe, fragmento do peptídeo quimiotático M de Vespa mandarinia. Variações dessa condição foram usadas com sucesso nas preparações dos Nα-acil-tripeptídeo protegido-S(CH2)2COOEt e Nα-acil-Ala-OR (R: Me; Bzl), pois eles foram gerados com boas qualidades e rendimentos similares ou superiores aos obtidos via procedimentos usuais. Após desproteção de cadeias laterais, os Nα-acil-tripeptídeo-OMe e Nα-acil-heptapeptídeo-OMe foram usados em reações de acoplamento com Gly-NH2 em presença de preparações lipásicas comerciais. Estes ensaios inéditos também foram bem sucedidos, pois após adequação das condições reacionais, os Nα-acil-tetrapeptídeo-NH2 e Nα-acil-octapeptídeo-NH2 foram obtidos com boas qualidades e rendimentos de 65% (1 h) e 55% (24 h), respectivamente. / The present work aimed to: i) improve the alternative procedure based on mediation by metal ions of peptide-resin linkage alcoholysis to obtain fully protected peptide methyl esters (Nα-acyl-protected peptide-OMe) under mild reaction condition and with high efficiency; ii) investigate the usefulness of the alternative procedure for preparing Nα-acyl-protected peptide-SR and Nα-acyl-amino acid-OR; iii) verify whether the resulting Nα-acyl-peptide-OMe would act as acyl donors in peptide bond formation catalyzed by lipases. Thus, in the search for the best methanolysis condition and comparison with the usual procedures for that, we used: fragment 22-24 of human cholecystokinin-33 (model tripeptide), Ca+2, Zn+2, Co+2 and Cu+2 (mediators), Kaiser oxime resin (KOR), p-hydroxymethylphenylacetamido methyl resin, p-hydroxymethylbenzoic acid resin and p-benzyloxy benzyl alcohol resin (polymeric supports), mixtures of MeOH and DCM, DMSO, NMP, THF or DMF (solvents) and 25, 37, 50 or 60°C. The optimal condition found [KOR, Ca+2 (1 eq./eq. of peptide), 50% MeOH/DMF, 50°C] was used successfully for preparing Nα-acyl-protected heptapeptide-OMe, fragment 1-7 of the chemotactic peptide M produced by Vespa mandarinia. Variations of this condition were employed successfully for preparing Nα-acyl-protected tripeptide-SR and Nα-acyl-Ala-OR (R: Me, Bzl): indeed, these compounds were obtained in good quality and with similar or superior yields than those provided by usual procedures. After side chain deprotections, the Nα-acyl-tripeptide and Nα-acyl-heptapeptide methyl esters obtained were used in coupling reactions with Gly-NH2 in the presence of commercial lipase preparations. Those pioneer reactions were also successful, since after optimizing the conditions, Nalfa-acyl-tetrapeptide-NH2 and Nα-acyl-octapeptide-NH2 were obtained in good qualities with yields of 65% (1 h) and 55% (24 h), respectively.

Neste trabalho foi avaliado a seletividade de reações biocatalisadas por fungos basidiomicetos frente a compostos bifuncionalizados com os grupos cetona e seleneto (1-2) ou cetona e sulfeto (3-4). Os compostos 1-4 foram sintetizados de acordo com metodologias descritas na literatura. Na síntese dos padrões racêmicos dos β-hidróxi-selenetos 1a-2a e β-hidróxi-sulfetos 3a-4a observou-se que a redução química utilizando NaBH4 levou a formação preferencial dos estereoisômeros cis-(1a-4a). Enquanto que a redução promovida na utilização dos fungos basidiomicetos levou a formação preferencial do estereoisômero trans-(1a-4a). Desta forma duas metodologias complementares para a preparação de β-hidróxi-selenetos e β-hidróxi-sulfetos foram estabelecidas. Cinco linhagens destes fungos (Irpex lacteus CCB 196, Trametes rigida CCB 285, Pycnoporus sanguineus CCB 501, Trametes byssogenum CCB 203, Trametes versicolor CCB 202 ) foram testadas visando à obtenção de β-hidróxi-selenetos (1a-2a) e β-hidróxi-sulfetos (3a-4a) na forma enantiomericamente pura ou enriquecida. Um estudo qualitativo indicou que o fungo Trametes rigida CCB 285 apresentou alta seletividade frente aos compostos utilizados levando aos produtos cis-(1a-4a) e trans-(1a-4a) com elevados valores de excessos enantioméricos (e.e. 99 %). Posteriormente, um estudo quantitativo permitiu o isolamento dos produtos, a determinação dos rendimentos isolados e também a configuração absoluta para os compostos isolados. Na segunda parte deste trabalho avaliou-se a aplicação de fungos Aspegillus terreus em reações de desmetilação/desalquilação de aminas terciárias. Os resultados obtidos indicam que os estes fungos apresentam grande potencial para reações de desmetilação de aminas terciárias aromáticas, sendo que as reações conduzidas em meio neutro (pH = 7) levaram aos produtos desmetilados com bons valores de conversão. No entanto, não foram observadas reações de desalquilação com grupos etila nas condições utilizadas. / In this work was considered the selectivity of reactions biocatalysted by basidiomycetes fungi with bifuncionalized compounds with ketone and selenide groups (1-2) or ketone and thio groups (3-4). The compounds 1-4 were prepared in according with methodologies from literature. During the synthesis of standard racemic β-hydroxyselenides 1a-4a and β-hydroxysulfides 3a-4a we noticed that chemical reduction using NaBH4 led to the preferential formation of the cis-(1a-4a) isomers. On the other hand, the bioreduction promoted by basidiomycetes fungi led to the preferential formation of trans-(1a-4a) isomers. Therefore, two complementary methodologies for preparation of β-hydroxyselenides and β-hydroxysulfides were established. Five strains of basidiomycetes fungi (Irpex lacteus CCB 196, Trametes rigida CCB 285, Pycnoporus sanguineus CCB 501, Trametes byssogenum CCB 203, Trametes versicolor CCB 202) were examined to aim at the preparation of β-hydroxyselenides and β-hydroxysulfides on the enantiomerically pure form. A qualitative study indicated that cells of T. rigida CCB 285 showed high selectivity led to the products cis-(1a-4a) and trans-(1a-4a) in high enantiomeric excess (ca 99 %). After that, a quantitative study allowed us to determine the isolated yields and the absolute configuration for the compounds cis-(1a-4a) and trans-(1a-4a). In the second part of this work was considered the application of Aspergillus terreus fungi in demethylation/dealkylation reactions by tertiary amines. The results indicated that those fungi showed a big potencial for demethylation reaction of aromatic tertiary amines. The reaction done on the pH = 7 led to the demethylated products with good values of conversion. Dealkylation reactions were not observed.

Neste trabalho foram realizadas reações de resolução enzimática de ciano-hidrinas [(±)-mandelonitrila 1a, (±)-2-(4-clorofenil)-2-hidroxiacetonitrila 2a, (±)-2-hidroxi-2-(4- hidroxifenil)acetonitrila 3a, (±)-2-hidroxibutanonitrila 4a, (±)-2-(4- fluorofenil)acetonitrila 5a, (±)-2-hidroxi-2-(4-metoxifenil)acetonitrila 6a, (±)-2-hidroxi- 2-(3-fenoxifenil)acetonitrila 7a e (±)-(E)-2-hidroxi-4-fenilbut-3-enonitrila 8a] e de álcoois organofluorados [(±)-2,2,2-trifluoro-1-feniletanol ±9a, ±±)-1-(2,4,5- trifluorofenil)etanol 10a, (±)-1-(3-bromofenil)-2,2,2-trifluoroetanol 11a, (±)-1-(4- bromofenil)-2,2,2-trifluoroetanol 12a e (±)-1-(2-trifluorometil)feniletanol 13a] utilizando a lipase imobilizada de Candida antarctica (CALB). As reações foram realizadas em agitador orbital por um período de tempo que variou entre 24-168 h de reação apresentando diferentes conversões e excessos enantioméricos: [(R)-álcool 1a (c = 51%, ee = 51%), (S)-acetato 1b (c = 49%, ee = 98%); (R)-álcool 2a (c = 42%, ee > 99%), (S)-acetato 2b (c = 58%, ee = 94%); (R)-álcool 3a (c = 34%), (S)-acetato 3b (c = 32%, ee = 28%); (R)-álcool 4a (c = 82%), (S)-acetato 4b (c = 18%, ee = 25%); (R)-álcool 5a (c = 5%), (S)-acetato 5b (c = 55%, ee = 97%); (R)-álcool 6a (c = 44%), (S)-acetato 6b (c = 56%, ee = 99%); (R)-álcool 7a (c = 53%), (S)-acetato 7b (c = 47%, ee = 92%); (R)-álcool 8a (c = 40%), (S)-acetato 8b (c = 60%, ee = 80%); (R)-álcool 9a (c = 51%, ee = 62%), (S)- acetato 9b (c = 49%, ee > 99%); (S)-álcool 10a (c = 50%, ee > 99%), (R)-acetato 10b (c = 50%, ee > 99%); (R)-álcool 11a (c = 49%, ee = 61%), (S)-acetato 11b (c = 51%, ee = 82%); (R)-álcool 12a (c = 51%, ee = 72%), (S)-acetato 12b (c = 49%, ee > 99%); (S)-álcool 13a (c = 88%), (R)-acetato 13b (c = 12%, ee > 99). Os resultados por irradiação micro-ondas para os compostos obtidos apresentaram menores tempos de reação (1-14 h) comprovando a sua eficiência na resolução quimio-enzimática de compostos organofluorados e ciano-hidrinas: [(R)-álcool 1a (c = 60%, ee = 89%), (S)-acetato 1b (c = 40%, ee = 92%); (R)-álcool 2a (c = 47%, ee = 82%), (S)-acetato 2b (c = 53%, ee = 90%); (R)-álcool 3a (c = 34%), (S)-acetato 3b (c = 17%, ee = 59%); (R)-álcool 5a (c = 4%, ee = 88%), (S)-acetato 5b (c = 50%, ee = 92%); (R)-álcool 6a (c = 44%, ee = 73%), (S)-acetato 6b (c = 56%, ee = 90%); (R)-álcool 7a (c = 50%, ee = 84%), (S)-acetato 7b (c = 50%, ee = 84%); (R)-álcool 8a (c = 41%, ee = 91%), (S)-acetato 8b (c = 59%, ee = 74%); (S)-álcool 9a (c = 95%), (R)-acetato 9b (c = 5%, ee > 99%); (R)-álcool 10a (c = 50%, ee >99%), (S)- acetato 10b (c = 50%, ee >99%); (R)-álcool 11a (c = 58%, ee = 43%), (S)-acetato 11b (c = 42%, ee = 78%); (S)-álcool 12a (c = 51%, ee = 70%), (R)-acetato 12b (c = 49%, ee = 98%); (S)-álcool 13a (c = 85%), (R)-acetato 13b (c = 15%, ee > 99)]. Em especial destaca-se, neste trabalho o uso de células microbianas utilizando a irradiação micro-ondas na redução de fluorocetonas. Sendo assim, foi realizada reações de biorredução da (±)- 2,2,2-trifluoroacetofenona 3 em agitador orbital e irradiação micro-ondas pelo fungo marinho Mucor racemosus CBMAI 847 nas concentrações de (2,9; 5,7; 8,5 e 14 mmol/L) em pH 8 e na concentração de 14 mmol/L em pH 5. Após 6 h de reação obtiveram-se conversões entre 39-100% e excessos enantioméricos entre 74-96% em agitador orbital e por irradiação micro-ondas obteve-se uma variação de 28-64% de conversão e excesso enantiomérico entre 73-96%. Também foram realizadas reações de biorredução com as bactérias termofílicas SPZSP005, SPZSP088, SPZSP051 e SPZSP055 para cetonas organofluoradas obtendo-se elevadas enantiosseletividades (>99%) e conversões (>99%). Esse estudo relata a primeira investigação da literatura frente ao uso de fungo e bactérias termofílicas por irradiação micro-ondas aplicada em biocatálise. Foram também realizadas reações de adição de aza-Michael entre a benzilamina e cetonas ?,β-insaturadas (ciclo-hexenona, 3-metil-2-ciclo-hexen-1-ona e a 2,5-dimetil-para-benzoquinona) utilizando a CALB em diferentes solventes orgânicos (EtOAc, CH2Cl2, n-hexano, MeOH, tolueno, éter etílico e THF) em agitador orbital e por irradiação micro-ondas. Através das reações de adição de aza-Michael foi obtido por adição-1,2 e adição-1,4 como adutos iminas, os quais foram caracterizadas por espectrometria de massas. Finalmente neste trabalho aplicou a irradiação micro-ondas em biocatálise via resolução cinética, redução de cetonas e adição de Michael. / In this study, enzymatic kinetic resolutions of cyanohydrins [(±)-mandelonitrile 1a, (±)-2-(4-chlorophenyl)-2-hydroxyacetonitrile 2a, (±)-2-hydroxy-2-(4- hydroxyphenyl)acetonitrile 3a, (±)-2-hydroxybutanenitrile 4a, (±)-2-(4- fluorophenyl)acetonitrile 5a, (±)-2-hydroxy-2-(4-metoxiphenyl)acetonitrile 6a, (±)-2- hydroxy-2-(3-fenoxyphenyl)acetonitrile 7a and (±)-(E)-2-hydroxy-4-phenylbut-3- enonitrile 8a], and organofluorine alcohols [(±)-2,2,2-trifluoro-1-phenylethanol 9a, (±)-1-(2,4,5-trifluorophenyl)ethanol 10a, (±)-1-(3-bromophenyl)-2,2,2-trifluoroethanol 11a, (±)-1-(4-bromophenyl)-2,2,2-trifluoroethanol 12a and (±)-1-(2- trifluoromethyl)phenylethanol 13a] were performed using immobilized lipase from Candida Antarctica (CALB). The reactions were performed on an orbital shaking for a period ranging from 24 to 168 h with different conversions and enantiomeric excesses. [(R)- alcohol 1a (c = 51%, ee = 51%), (S)-acetate 1b (c = 49%, ee = 98%); (R)- alcohol 2a (c = 42%, ee > 99%), (S)-acetate 2b (c = 58%, ee = 94%); (R)-alcohol 3a (c = 34%), (S)-acetate 3b (c = 32%, ee = 28%); (R)-alcohol 4a (c = 82%), (S)-acetate 4b (c = 18%, ee = 25%); R)-alcohol 5a (c = 5%), (S)-acetate 5b (c = 55%, ee = 97%); (R)-alcohol 6a (c = 44%), (S)-acetate 6b (c = 56%, ee = 99%); (R)-alcohol 7a (c = 53%), (S)-acetate 7b (c = 47%, ee = 92%); (R)-alcohol 8a (c = 40%), (S)-acetate 8b (c = 60%, ee = 80%); (R)- alcohol 9a (c = 51%, ee = 62%), (S)-acetate 9b (c = 49%, ee > 99%); (S)-alcohol 10a (c = 50%, ee > 99%), (R)-acetate 10b (c = 50%, ee > 99%); (R)-alcohol 11a (c = 49%, ee = 61%), (S)-acetate 11b (c = 51%, ee = 82%); (R)-alcohol 12a (c = 51%, ee = 72%), (S)- acetate 12b (c = 49%, ee > 99%); (S)-alcohol 13a (c = 88%), (R)-acetate 13b (c = 12%, ee > 99). The results obtained by microwave irradiation for the substrates showed shorter reaction times (1 to 14 h) demonstrating its efficiency in chemoenzymatic esterifications of organofluorine compounds and cyanohydrins [(R)-alcohol 1a (c = 60%, ee = 89%), (S)-acetate 1b (c = 40%, ee = 92%); (R)-alcohol 2a (c = 47%, ee = 82%), (S)-acetate 2b (c = 53%, ee = 90%); (R)-alcohol 3a (c = 34%), (S)-acetate 3b (c = 17%, ee = 59%); (R)-alcohol 5a (c = 4%, ee = 88%), (S)-acetate 5b (c = 50%, ee = 92%); (R)-alcohol 6a (c = 44%, ee = 73%), (S)-acetate 6b (c = 56%, ee = 90%); (R)-alcohol 7a (c = 50%, ee = 84%), (S)-acetate 7b (c = 50%, ee = 84%); (R)-alcohol 8a (c = 41%, ee = 91%), (S)-acetate 8b (c = 59%, ee = 74%); (S)-alcohol 9a (c = 95%), (R)-acetate 9b (c = 5%, ee > 99%); (R)- alcohol 10a (c = 50%, ee >99%), (S)-acetate 10b (c = 50%, ee >99%); (R)-alcohol 11a (c = 58%, ee = 43%), (S)-acetate 11b (c = 42%, ee= 78%); (S)-alcohol 12a (c = 51%, ee = 70%), (R)-acetate 12b (c = 49%, ee = 98%); (S)-alcohol 13a (c = 85%), (R)-acetate 13b (c = 15%, ee > 99)]. In particular, this thesis show the use of microbial cells in reduction of fluoroketones by microwave irradiation. Thus, bioreduction reactions of (±)-2,2,2- trifluoroacetophenone 3 was performed in orbital shaking and microwave irradiation by marine fungus Mucor racemosus CBMAI 847 in different concentrations (2.9, 5.7, 8.5 and 14 mmol/L) at pH 8 and in the concentration of 14 mmol/L at pH 5. In the reactions after 6 h were obtained a conversion of 39 to 100% and enantiomeric excess of 74-96%, in orbital shaking. The reaction on microwave irradiation gave an increase conversion of 28-64% and enantiomeric excess of 73-96%. Bioreduction reactions were also performed with the thermophilic bacteria SPZSP005, SPZSP088, SPZSP051 and SPZSP055 for organofluorine ketones obtaining high enantioselectivities (> 99%) and conversions (> 99%). This study describes the first investigation on the literature regarding the use of thermophilic bacteria and fungus by microwave irradiation applied to biocatalysis. Were also carried out reactions of aza-Michael addition of benzylamine and ?, β-unsaturated cyclohexenones (cyclo- hexenone, 3-methyl-2-cyclo-hexen-1-one and 2,5-dimethyl-para-benzoquinone) were investigated, using CALB in different organic solvents (EtOAc, CH2Cl2, n-hexane, MeOH, toluene, ethylic ether and THF) in orbital shaking and microwave irradiation. From aza-Michael addition reactions was possible to obtain by 1,2- and 1,4-adition the adduct imines, which were characterized by mass spectrometry. Finally this thesis applied the microwave irradiation in biocatalysis via kinetic resolution, reduction of ketones and aza-Michael addition.

Die Kohlenmonoxid-Dehydrogenase (CODH) ist ein Schlüsselenzym des reduktiven Acetyl-CoA Wegs, und katalysiert in diesem die Reduktion von CO2 zu CO mit Raten von bis zu 12 s 1. Die Rückreaktion, die Oxidation von CO, katalysiert die CODH mit Raten von bis zu 31000 s-1. Beide Reaktionen finden am aktiven Zentrum des Enzyms, einem [NiFe4S4OHx]-Cluster (C Cluster), statt. Das Genom des hydrogenogenen Bakteriums C. hydrogenoformans beinhaltet fünf putative CODHs, welche vermutlich unterschiedliche Funktionen im Organismus ausüben. Diese Arbeit charakterisiert CODH-II und CODH-IV strukturell und biochemisch. In CODH-II wurden dafür Seitenketten der 1. und 2. Koordinationssphäre des C-Clusters ausgetauscht und einer strukturellen und kinetischen Analyse unterzogen. Der zweite Teil der Arbeit analysiert den Einfluss von O2 auf CODH-II und CODH-IV. In Lösung zeigte CODH-II bei Inkubation mit O2 einen Verlust der CO-Oxidationsaktivität. Analog dazu konnte in CODH-II Kristallen die Zerstörung des C Clusters durch O2 verfolgt werden. Elektrochemisch wurde das Verhalten der CODH-II in der Gegenwart von O2 mit der CODH-IV, für welche eine Rolle in der oxidativen Stressantwort von C. hydrogenoformans diskutiert wird, verglichen. CODH-IV ist sauerstofftoleranter als CODH-II und katalysiert die Oxidation von CO hocheffizient nahe des CO-Diffussionslimits. Die Aufklärung der Struktur von CODH-IV erlaubte die Identifikation einer möglichen Ursache der höheren O2-Toleranz. Die Strukturen beider CODHs ähneln sich stark. Allerdings weist CODH IV an der Rückseite des C-Clusters eine dichtere Packung der Seitenketten auf, wodurch der Cluster von einem Angriff durch O2 abgeschirmt werden könnte. / CO-Dehydrogenase (CODH) is a key enzyme of the reductive acetyl-CoA pathway, in which it catalyses the reduction of CO2 to CO with rates up to 12 s-1. CODH catalyses the reverse reaction, the oxidation of CO with rates up to 31000 s-1. Both reactions take place at the active site of the enzyme, a [NiFe4S4OHx] cluster (cluster C). The genome of the hydrogenogenic bacterium C. hydrogenoformans contains five putative CODHs which might serve distinctive functions within the organism. This study characterises CODH-II and CODH-IV structurally and biochemically. Residues of the first and second coordination sphere of cluster C from CODH-II were exchanged and analysed concerning their structural and biochemical properties. The second part of this study analyses the influence of O2 on CODH-II and CODH-IV. CODH-II showed in solution a loss in CO-oxidation activity upon incubation with O2. In an analogous experiment, the destruction of cluster C by O2 was followed crystallographically. A role for CODH-IV in the oxidative stress response of C. hydrogenoformans has been discussed previously. Therefore, the behaviour of CODH-II and CODH-IV was analysed electrochemically in the presence of O2. CODH IV is more O2-tolerant than CODH-II and catalyses the oxidation of CO with high efficiency close to the diffusion limit of CO. Reasons for the enhanced O2-tolerance of CODH-IV could be deduced by elucidating its structure. Generally, both CODHs show high structural similarity. However, at the backside of cluster C CODH-IV shows a tighter packing of residues by which the cluster C might be shielded from O2.