Diversos biomateriais podem ser aplicados como suportes na imobilização de células totais de fungos filamentosos ou enzimas isoladas, visando a manutenção e o prolongamento da atividade enzimática em processos biocatalíticos. Exemplos promissores de biomateriais são a fibroína da seda e o alginato de sódio. A fibroína é um material protéico com alta estabilidade térmica, elasticidade, resistência à tensão, não sofre ataque microbiano, baixo custo de purificação e alta tenacidade, o alginato é um biopolímero versátil, devido a suas propriedades gelificantes em soluções aquosas. Assim, neste trabalho empregou-se micélios do fungo derivado de ambiente marinho, Penicillium citrinum CBMAI 1186, livres e imobilizados em biopolímeros (fibra de algodão, fibra de fibroína da seda e fibra de paina) na biorredução quimiosseletiva, regiosseletiva e enantiosseletiva da ligação α,β-C=C de enonas α,β-, α,β,γ,δ- e di-α,β-insaturadas previamente sintetizados pela a reação de condensação aldólica. Foi possível a utilização do fungo P. citrinum CBMAI 1186 na redução quimiosseletiva, regiosseletiva e enantiosseletiva da ligação dupla carbono-carbono de sistemas α,β-insaturados. A imobilização do fungo P. citrinum CBMAI 1186 em biopolímeros (algodão, fibroína da seda, paina e quitosana) permitiu a prolongamento da atividade celular do fungo. O protocolo desenvolvido foi capaz de obter compostos até então descritos apenas por síntese clássica. Também foi realizado reações de resolução enzimática de derivados de haloidrinas por diferentes lipases microbianas de: Pseudomonas fluorescens, Candida cylindracea, Rhizopus niveus e Aspergillus niger. A lipase de P. fluorescens foi imobilizada em esferas de fibroína do bicho da seda (método 1, via adsorção) e em blenda com alginato de cálcio (método 2, via encapsulação) em diferentes condições, tais como, variação de solvente, variação da quantidade de enzima imobilizada e tempo de reação. As condições otimizadas foram empregadas em diferentes haloidrinas, rendendo elevados excessos enantioméricos (ee > 99%) e alta razão enanantiomérica (E > 200) para os produtos acetilados. Foi possível desenvolver um protocolo simples, barato e prático para a síntese enantiosseletiva de haloidrina reforçando a versatilidade da fibroína e do alginato como suportes de imobilização para catalisadores heterogêneos. Também foi possível utilizar a lipase imobilizada (método 2) na reação de transesterificação para obtenção do biodiesel etílico. As melhores condições para o bom funcionamento do biocatalisador foram: 30% do biocatalisador, 20% de n-hexano, relação óleo e etanol de 1:4 a 32 ºC por 48 h em agitação magnética (400 rpm). Essas condições permitiram a formação de 42% de rendimento do biodiesel etílico. O biocatalisador apresentou algumas limitações reacionais, tais como, fragilidade frente a elevadas temperaturas (> 32 ºC) e prolongado tempo de agitação magnética. Porém, permaneceu apto no meio por 4 ciclos consecutivas. Conclui-se que os biomateriais (fibroína, alginato e quitosana) podem ser utilizados como alternativas versáteis na imobilização de micélios de fungos filamentoso e de enzimas isoladas para aplicações em biocatalíticas. / Various biomaterials have been used as matrices for immobilization of whole filamentous fungi or isolated enzymes in order to maintain and extend enzyme activity in biocatalytic processes. Promising examples of biomaterials include silk fibroin and sodium alginate. Fibroin is a protein with high thermal stability, elasticity, and tensile strength; the ability to resist microbial attacks; and low purification costs. Owing to its gelling properties in aqueous solutions, alginate is a versatile biopolymer. In this work, we used the mycelia of a marine fungus, Penicillium citrinum CBMAI 1186, in the free form as well as immobilized in biopolymers (cotton fiber, silk fibroin, and kapok fiber) for the chemoselective, regioselective, and enantioselective bioreduction of the α,β-C=C bond of unsaturated α,β-, α,β,γ,δ-, and di-α,β-enones previously synthesized by aldol condensation. P. citrinum CBMAI 1186 successfully performed chemoselective, regioselective, and enantioselective reduction of the double carbon-carbon bond in unsaturated α,β-systems. Immobilization of P. citrinum CBMAI 1186 in biopolymers (cotton, silk fibroin, kapok fiber, and chitosan) prolonged the biocatalytic activity of the fungus. This protocol could be used to obtain compounds that have until now only been produced using classic synthesis methods. Enzymatic resolution of derivatives of halohydrins by lipases from Pseudomonas fluorescens, Candida cylindracea, Rhizopus niveus, and Aspergillus niger was also carried out. P. fluorescens lipase was immobilized in silk fibroin spheres (method 1, via adsorption) and in a calcium alginate blend (method 2, via encapsulation) under different conditions, i.e., different solvents, quantity of immobilized enzyme, and reaction time. The optimized conditions were used to resolve different halohydrins, and the acetylated products were obtained at high enantiomeric excess (ee > 99%) and high enantiomeric ratio (E > 200). A simple, inexpensive, and convenient protocol was developed for the enantioselective synthesis of halohydrins. The results highlight the versatility of fibroin and alginate as immobilization matrices for heterogeneous catalysts. The lipase immobilized by method 2 was also used in a transesterification reaction to obtain ethyl biodiesel. The optimum conditions for biocatalysis were: 30% biocatalyst, 20% n-hexane, and oil-to-ethanol ratio of 1:4 at 32°C for 48 h on a magnetic stirrer (400 rpm). Under these conditions, the yield of ethyl biodiesel reached 42%. Biocatalysts have some limitations, such as low stability at temperatures > 32°C and prolonged time of magnetic stirring. However, the catalysts remained active for 4 consecutive cycles. In conclusion, biomaterials (fibroin, alginate, and chitosan) can be used as versatile alternatives for immobilization of fungal mycelium and isolated enzymes for use in biocatalysis.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O presente trabalho relata a utilizaÃÃo do caldo de cana e suas enzimas imobilizadas como biocatalisadores em reaÃÃes de reduÃÃo e acetilaÃÃo. Numa primeira parte, uma sÃrie de reaÃÃes de reduÃÃo utilizando como catalisador o sistema enzimÃtico contido no caldo de cana (CC) foi realizada, tendo como substratos: acetofenona, 3-metoxi-acetofenona, benzaldeÃdo, anisaldeÃdo, m-anisaldeÃdo, vanilina, cinamaldeido, -metilcinamaldeÃdo, furfural, citronelal, 3-hexanona, ciclopentanona, ciclohexanona, pulegona, carvona, acetoacetato de etila, benzoato de etÃla, butirato de etÃla, benzonitrila, benzamida, isoniazida e nitrobenzeno. A metodologia aplicada consistiu na utilizaÃÃo de proporÃÃes definidas dos substratos e do sistema biocatalisador. Para vÃrios dos compostos citados, os alcoÃis foram obtidos com excelentes rendimentos, em alguns casos as reaÃÃes procederam com quimiosseletividade e, em outros, com enantiosseletividade. A segunda parte do trabalho consistiu na utilizaÃÃo do sistema enzimÃtico do caldo de cana em reaÃÃes de esterificaÃÃo. Nestas reaÃÃes foram utlizados como biocatalisador as enzimas imobilizadas do caldo de cana (EICC), aplicando metodologia adaptada da literatura. O Ãlcool anÃsico, selecionado como substrato padrÃo para os testes iniciais, em mistura com anidrido acÃtico e em presenÃa de EICC originou o produto identificado como acetato de anisila. Em seguida, apÃs vÃrios experimentos, os parÃmetros reacionais: quantidade de EICC, quantidade de substrato, tempo reacional, solvente, velocidade de agitaÃÃo, temperatura e reutilizaÃÃo do sistema enzimÃtico foram determinados. As condiÃÃes que permitiram a obtenÃÃo do produto esterificado com melhor rendimento foram: 150 mg de EICC, 200 mg de substrato, 20 mL de hexano, 150 rotaÃÃes por minuto (rpm), 60 horas de reaÃÃo a 30ÂC e 24 h a 70ÂC. Estas condiÃÃes foram utilizadas nas reaÃÃes de acetilaÃÃo de outros compostos, obtendo-se bons rendimentos de conversÃo. Assim, as reaÃÃes com os alcoÃis: anÃsico, benzÃlico, 3- metoxi-benzÃlico, cinÃmico, furfurÃlico, 1-fenil-etan-1-ol e 3-metoxi-1-fenil-etan-1-ol e alcoÃis alifÃticos: amÃlico, octÃlico, decanÃico, citronelol, alÃlico, ciclopentanol, ciclohexanol, 3-hidroxi-butirato de etila, -terpineol e com um composto fenÃlico o carvacrol foram testadas, demonstrando a eficiÃncia de EICC nestas reaÃÃes. Os produtos, nas misturas reacionais, foram analisados atravÃs de CCD, CG-EM e RMN 1H. / The present work reports the use of integral cells and immobilized enzymes of the sugar cane (Saccharum officinarum) juice as biocatalyst applied in reactions of reduction and acetylation reactions. In a first part, a set of reduction reactions was carried out using sugar cane juice (CC) as catalyst and, as substrates: acetophenone, 3-methoxyacetophenone,benzaldehyde, anisaldehyde, m-anisaldehyde, vanillin, cinnamaldehyde, -methyl-cinnamaldehyde, furfuryl, citronellal, 3-hexanone, cyclopentanone, cyclohexanone, pulegone, carvone, -keto ethyl butyrate, ethyl benzoate, ethyl butyrate, benzonitrile, benzamide, isoniazide, nitrobenzene. For several of the mentioned compounds, were obtained alcohols with excellent yields, in some cases the reactions proceded with chemoselectivity and, in other, with enantioselectivity. The second part of this work it was used immobilized enzymes of cane juice in esterification reactions of alcohols. In this sense, the alcohols: anisyc, benzylic, 3-methoxy-benzylic, cinnamyc, furfurylic, 1-phenylethan-1-ol, 3-methoxy-1-phenyl-ethan-1-ol, amylic, octylic, decanoic, citronellol, allylic, cyclopentanol, cyclohexanol, 3-hydroxy-ethyl-butyrate, -terpineol and carvacrol were tested. In the esterifications reactions were using as biocatalisador the immobilized enzymes of the cane juice (EICC), using adapted methodology of the literature. The alcohol anisyc, selected as substrate for the tests initials, in mixture with acetic anhydride and in presence of EICC it afforded an identified product as anisyl acetate. After several experiments, reactional parameters: amount of EICC, amount of substrate, time reacional, solvent, agitation speed, temperature and reuse of the enzymatic system were determined. The conditions that allowed the obtaining of the esterified derivative with better yields were: 150 mg of EICC, 200 mg substrate, 20 mL hexane, 150 rpm, 60 hours of reaction to 30ÂC and 24 h to 70ÂC. So, acetylation reactions with other alcohols were processed, being obtained good yelds. Consequently, the immobilized enzymes of Saccharum officinarum constitute a promising biocatalysts in acetylation reactions, although, other tests need to be made in the sense of optimizing the potential of the biocatalysts. The products, in the reactional mixtures were analyzed by TLC, GC-MS and 1H NMR.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / O estudo quÃmico do basidiomiceto Lentinus strigellus foi realizado atravÃs da investigaÃÃo da produÃÃo de metabÃlitos secundÃrios em diferentes meios de culturas, alÃm da sua utilizaÃÃo em processos de biorreduÃÃo de compostos carbonÃlicos prÃquirais (cetona e -cetoÃster). A partir de L. strigellus cultivado em meio de peptona foi possÃvel isolar os benzopiranos 2,2-dimetil-6-metoxicroman-4-ona, 4-hidroxi-2,2-dimetil-6-metoxicromano e (3R,4S)-3,4-dihidroxi-2,2-dimetil-6-metoxicromano do meio lÃquido. Do micÃlio, foram isolados o alcalÃide indÃlico echinulina e a antraquinona fisciona, ambos inÃditos para o gÃnero Lentinus. Do microrganismo cultivado em Czapek, enriquecido com caldo de batata, foram isolados do meio lÃquido os mesmos benzopiranos produzidos em peptona, alÃm de (3S,4S)-3,4-dihidroxi-2,2-dimetil-6-metoxicromano. Quando Czapek foi utilizado como meio de cultivo, foram isoladas a panepoxidona e a isopanepoxidona. CÃlulas em crescimento de L. strigellus em meio de batata-dextrose foram investigadas, pela primeira vez, na biorreduÃÃo estereoseletiva da acetofenona e nove derivados aromÃticos, alÃm das cetonas alifÃticas ciclo-hexilmetilcetona, octan-2-ona, undecan-2-ona e do -cetoÃster 4-cloroacetoacetato de metila. A maioria das cetonas aromÃticas foi convertida ao respectivo Ãlcool de configuraÃÃo S, em excessos enantiomÃricos superiores a 99%. Exceto para a undecan-2-ona, as cetonas alifÃticas foram reduzidas enzimaticamente ao Ãlcool de configuraÃÃo S em elevadas taxas de conversÃo e excessos enantiomÃricos. O -cetoÃster 4- cloroacetoacetato de metila foi quimiosseletivamente reduzido ao Ãlcool correspondente de configuraÃÃo R, mas com excesso enantiomÃrico moderado. / The chemical study of the basidiomicete Lentinus strigellus was done by the investigation of its secondary metabolites production in varied culture media, besides its utilization in the bioreduction of prochiral carbonyl compounds (ketone and - ketoester). From the liquid medium of L. strigellus grown in peptone broth, it was isolated the benzopyranes 2,2-dimethyl-6-methoxycroman-4-one, 4-hydroxy-2,2-dimethyl-6-methoxycromane and (3R,4S)-3,4-dihydroxy-2,2-dimethyl-6-methoxycromane . From the mycelium, the indol alkaloid echinuline and the antraquinone fiscione were isolated, both compounds reported for the first time in Lentinus. The same benzopyranes isolated from L. strigellus grown in peptone, besides (3S,4S)-3,4-dihydroxy-2,2-dimethyl-6-methoxycromane were isolated from the liquid medium of the microorganism grown in Czapek medium enriched with potato broth. When only Czapek was used as culture medium, panepoxidone and isopanepoxidone were isolated. Growing cells of L. strigellus in potato-dextrose medium were investigated, for the first time, in the stereoselective reduction of acetophenone and nine aromatic derivatives, besides the aliphatic ketones cyclohexylmethylketone, octan-2-one and undecan-2-one, and the -ketoester methyl 4-chloroacetoacetate. Most of the aromatic ketones were converted into the respective alcohols with S configuration in high enantiomeric excesses (> 99%). Except for undecan-2-one, the aliphatic ketones were enzimatically reduced to the alcohols with S configurations in high conversion ratios and enantiomeric excesses. -ketoester methyl 4-chloroacetoacetate was chemoselectivelly reduced to its corresponding alcohol with R configuration but with moderate ee.

A primeira parte desta tese consiste na preparação de compostos aromáticos contendo átomos de selênio e de telúrio. Quatro metodologias foram utilizadas para essa finalidade, a saber: orto-metalação de compostos contendo oxigênio, troca metal-halogênio em haletos aromáticos substituídos, reação de substituição eletrofílica aromática entre tetracloreto de telúrio e compostos aromáticos ativados e reação de sais de diazônio com disselenetos e diteluretos orgânicos. Alguns dos teluretos preparados foram usados em reações de acoplamento com alcinos terminais catalisada por Paládio, em reações de troca telúrio-lítio e em reações de oxidação de Te (II) a Te(IV). A segunda parte da tese consiste no uso de calcogenetos de arila em reações biocatalisadas. Inicialmente foram estudadas biotransformações em substratos não contendo átomo de selênio e de telúrio. Reações de redução de carbonila e de desracemização de álcoois foram observadas por ação de fungos e de raízes de plantas. Meta e para organosseleno acetofenonas foram reduzidas aos organosseleno feniletanóis correspondentes por ação de fermento de pão, células íntegras de fungos e por Daucus carota com conversões e excessos enantioméricos que chegaram a >99%. Orto organosseleno e metiltio feniletanóis foram resolvidos em seus isômeros R e S por ação de lipase imobilizada (Novozyme 435) em presença de acetato de vinila com excessos enantioméricos acima de 99% . / The first part of this thesis shows the preparation of organic compounds containing selenium or tellurium. For this purpose, four methodologies were applied: ortho-metallation of 1-phenylethanol, metal-halogen exchange involving aromatic halides, electrophilic aromatic substitution using tellurium tetrachloride and reactions with aryldiazonium salts and diselenides or ditellurides. Next, the aryl tellurides were applied in Palladium catalyzed coupling reactions with terminal alkynes, tellurium-lithium exchange reactions and oxidation of Te(II) to Te(IV). The second part of this thesis consists in the inverstigation of biocatalyzed reactions. Biotransformations of substrates without Se or Te atoms were initially investigated. Carbonyl reduction reactions and deracemization of secondary alcohols were observed by means of whole fungal cells and plants. Meta and para organoseleno acetophenones were then reduced with baker´s yeast, whole fungal cells and Daucus carota, yielding the corresponding organoseleno phenylethanols optically pure with enantiomeric excess up to >99%. Ortho organoseleno e methylthio phenylethanols were resolved in both enantiomeric forms by reacting them with immobilized lipase (Novozyme 435) and vinyl acetate in hexane. High values of enantiomeric excess (>99%) were obtained.

Neste trabalho foi realizada a síntese de amidas, bem como de ácidos e ésteres borônicos, derivados dos aminoácidos L-cistina e L-cisteína, através de rota sintética simples, curta e de baixo custo, com o intuito de busca e a identificação de novo(s) inibidor(es) do proteassomo 20S. Esta classe de compostos possui estrutura que permite a inserção de diversos grupos funcionais, o que confere versatilidade e a construção de biblioteca de compostos que contém partes hidrofílicas e hidrofóbicas importantes para posterior avaliação inibitória. Para tanto, empregou-se rota sintética química convencional e rota biocatalisada para a formação da ligação amida. Os compostos derivados de L-cisteína foram obtidos via síntese clássica de peptídeos a qual forneceu os compostos desejados em rendimentos de até 85%. Por outro lado, tentativas de obtenção das amidas via biocatálise não se mostraram efetivas. Já amidas derivadas de L-cistina foram obtidas em rendimentos de até 79%, via síntese tradicional e até 100% de conversão através de rota biocatalítica. A inserção do átomo de boro nas estruturas se deu utilizando-se metodologias sintéticas já bem estabelecidas na literatura. Os ésteres borônicos derivados de L-cisteína foram obtidos em bons rendimentos (até 78%), enquanto que não foi possível obter-se compostos de boro derivados de L-cistina. Por sua vez, os compostos contendo ácido borônico na estrutura foram sintetizados via reação de hidrólise dos respectivos ésteres borônicos, em rendimentos moderados (até 34%). Após a obtenção dos compostos contendo grupamentos organoboro realizou-se avaliação inibitória dos mesmos frente ao proteassomo 20S. Valores de IC50 iguais a 52 µM foram obtidos para composto derivado de L-cisteína contendo grupamento éster borônico, que se mostraram inibidores moderados e reversíveis. Ácidos borônicos se mostraram sem capacidade de inibir o proteassomo 20S. Adicionalmente, realizaram-se estudos de modelagem molecular com a finalidade de elucidar os resultados obtidos experimentalmente. Inicialmente realizaram-se cálculos de modelagem molecular através da realização de docking de alguns compostos e após geração de modelo farmacofórico. De maneira geral observou-se que os inibidores derivados da L-cisteína não ocupam a mesma cavidade que o fármaco bortezomibe, o que pode explicar a diferença na atividade dos compostos frente à inibição do proteassomo 20S. Também se observou que, tendo-se a interação dos inibidores com a enzima, a vizinhança do átomo de boro tem grande influência na capacidade inibitória, uma vez que estes grupamentos determinam qual a região da cavidade do proteassomo 20S será ocupada pelo inibidor. / In our study, amides, boronic acids and esters derivatives from L-cysteine and L-cysteine were synthesized by simple, short and inexpensive synthetic route, in order to search for new inhibitor(s) of the 20S proteasome. This class of compounds has a structure that allows inclusion of various functional groups, giving it versatility and allowing the construction of library compounds containing hydrophilic and hydrophobic moieties, important for further evaluation. To this end, we used conventional chemical synthetic route and biocatalysis for peptide bond formation. The compounds derived from L-cysteine were obtained by classical synthesis of peptides which provided the desired compounds up to 85% yields. On the other hand, attempts to obtain the amides via biocatalysis were not effective. However, amides derived from L-cystine were obtained with up to 79% yields via chemical synthesis and conversion up to 100% using biocatalytic route. The insertion of the boron atom in the structures was possible using synthetic methodologies well established in literature. Boronic esters derived from L-cysteine were obtained in good yields (up to 78%), whereas it was not possible to obtain boron compounds derived from L-cystine. In turn, compounds containing boronic acids in the structure were synthesized by hydrolysis reaction of the respective boronic esters in moderate yields (up to 34%). With organoboron compounds in hand, we turned our attention to inhibitory assessment against the 20S proteasome. IC50 up to 52 µM were obtained when L-cysteine boronic ester derivatives were evaluated. These compounds are moderate and reversible inhibitors. L-cysteine boronic acids derivatives have shown not ability to inhibit the 20S proteasome. Additionally, molecular modeling studies were carried out in order to elucidate the results obtained experimentally. Initially molecular modeling calculations were carried out by performing docking experiments of some compounds. Generation of pharmacophoric model calculations was also executed. In general, it was observed that inhibitors derived from L-cysteine do not occupy the same cavity that drug bortezomib, which may explain the difference in the activity of compounds against the inhibition of 20S proteasome. We also observed that, with the interaction of inhibitors and enzyme, the side chains around boron atom has a great influence on inhibitory capacity, since these groups determine which region of the 20S proteasome cavity is occupied by the inhibitor.

Conjugates of soybean trypsin inhibitor (SBTI) and potato serine protease inhibitor (PSPI) immobilized on metal oxide particles of ~100nm diameter were prepared. Inhibition of trypsin hydrolysis of BAPA by these conjugates was measured and enzyme kinetics constants kcat, KM, kcat/KM and ki were determined. Metal oxide particles presented an inhibitory effect similar to that of a competitive inhibitor, noticed through the increase value of the K M constant. Furthermore, PSPI conjugates had the highest inhibition of trypsin, illustrated by the significantly higher value of KM relative to the value for particles only.

Biocatalysis involves the use of enzymes to perform stereo- and enantio-specific reactions. One of the reactions where biocatalysis is a valuable technology is oxidation of naphthalene to 1-naphthol using Toluene ortho-Monooxygenase (TOM) variant TmoA3 V106A, also known as TOM-Green. Whole-cell biocatalysis in a water-organic solvent biphasic system was used to minimize naphthalene and 1-naphthol toxicity, and to increase substrate loading. Recombinant Escherichia coli TG1 cells expressing TOM-Green were used for biphasic biocatalysis and lauryl acetate gave best results among the solvents tested. On a constant volume basis, 8 - fold improvement in 1-naphthol production was achieved using biphasic systems compared to biotransformation in aqueous medium. The organic phase was optimized by studying the effects of organic phase ratio and naphthalene concentration in the organic phase. The efficiency of biocatalysis was further improved by application of a solvent tolerant strain Pseudomonas putida S12. P. putida S12 is solvent tolerant owing to its two adaptive mechanisms: outer membrane modification and solvent extrusion using solvent resistant pump srpABC. P. putida S12, in addition to its tolerance to various organic solvents, showed better tolerance to naphthalene compared to E. coli TG1 strain expressing TOM-Green. Application of solvent tolerant P. putida S12 further improved 1-naphthol productivity by approximately 42%. Solvent tolerance of P. putida S12 was further analyzed by transferring its tolerance to a solvent sensitive E. coli strain by transfer of solvent resistant pump srpABC genes. Engineered E. coli strain bearing srpABC genes either in low-copy number plasmid or high-copy number plasmid grew in the presence of a saturated toluene concentration. Engineered E. coli strains were also more tolerant to toxic solvents, e. g., decanol and hexane, compared to the control E. coli strain without srpABC genes. The expression of solvent resistant pump genes was confirmed by Reverse Transcriptase PCR analysis. The main drawbacks of biocatalysis for production of chemicals were addressed and approaches to minimize the drawbacks have been presented. The production of 1-naphthol was significantly improved using biocatalysis in liquid-liquid biphasic systems.

The employment of enzymes and whole cells has been important in many industries for centuries. However, it is only in the last 30 years that the use of enzymes for the synthesis of high-value fine chemicals has enjoyed increasing popularity. In fact, esterases and lipases are used almost routinely these days to provide optically active building blocks for the construction of imaginative new routes to chiral target molecules. The major topic of this work describes the utilization of enzymes (namely lipases) in the synthesis and modification of natural and non-natural compounds. Chapter 1 outlines the strengths and weaknesses of the most widely used enzyme systems and a description of a brief summary on the state of the art of biotransformations with special emphasis on the general applicability and reliability of various reaction types is described. Chapter 2 describes the enzymatic resolution of various 3-acetoxy-4-aryl-substituted azetidin-2-ones. Following screening of enzymes, such as Novozym-435, PS-30, PPL and AYS the best conditions were a phosphate buffer with PS-30 as the enzyme. The resulting products were the (3S, 4R)-3-hydroxy-4-aryl-substituted azetidin-2-ones and the unreacted (3R, 4S)-3-acetoxy-4-aryl-substituted azetidin-2-ones. Reactions generally occurred with high conversion and high selectivity. In Chapter 3, the regioselective transesterifications and hydrolysis of peracylated sophorolipid (SL) derivatives catalyzed by lipases was investigated. It was confirmed from the detailed spectral analysis of the products that transesterification failed to furnish any free hydroxyls on the sophorose ring. Instead, transesterification took place on the methyl ester located at the carboxylic end of the 17-hydroxyoctadecenoic acid chain attached to the C-1' position of the sophorose ring. In Chapter 4, the chemo-enzymatic syntheses of enantiomerically pure R and S imperanene from vanillin are described. The key step entails the asymmetrization of a prochiral diol using lipase PS-30. The resulting monoacetate has enantiomeric excesses of >97%. Biocatalysts represent a new class of chiral catalysts useful for a broad range of selective organic transformations. It is stating the obvious to say that biocatalysis is not a panacea for synthetic organic chemistry. However, advances over the past thirty years mean that it would be a serious mistake not to consider the employment of a biocatalyst, in, perhaps, the key step in a sequence of transformations that turn a cheap starting material into an expensive fine chemical.

Encapsulation of biomolecules is of great interest to research advances related to biology, physiology, immunology, and biochemistry, as well as industrial and biomedical applications such as drug delivery, biocatalysis, biofuel, food and cosmetics. Encapsulation provides functional characteristics that are not fulfilled by free biomolecules and stabilizes the fragile biomolecules. In terms of biocatalysis, solid support can often enhance the stability of enzymes, as well as facilitate separation and recovery for reuse while maintaining activity and selectivity. Various kinds of materials have been used for encapsulation of biomolecules, among which, porous materials are an important group. Metal-organic frameworks (MOFs) have attracted much attention and emerged as a new generation of highly porous functional materials with potential in a variety of fields such as gas separation and storage, catalysis, sensors and biomedical applications. Their structural versatility and amenability to be designed with specific functionality, together with their extra-large surface areas confer them a special place amongst traditional porous materials. In particular, because ligands can be designed with particular organic functional groups for specific interactions with biomolecules, they are attractive in the stabilization and retention of enzyme/proteins for biomedical or biocatalysis applications. With enlarged pore sizes, mesoporous (pore sizes in the range of 2 to 50 nm) MOFs are of great interest in the encapsulation of proteins. In this dissertation, I am focusing on the encapsulation of biomolecules into mesoporous MOFs (mesoMOFs) to estabilish the biomolecules@mesoMOF platform, including synthesis, characterization and mechanistic studies of a series of novel biomolecules@mesoMOF materials, and to develop the biomolecule@mesoMOFs platform for various applications.

Bioelectrochemical systems couple the oxidation of an electron donor at the anode with the reduction of an electron acceptor at the cathode, using microorganisms to catalyse one or both reactions. When the overall reaction is exergonic, a power output is generated and the system is referred to as microbial fuel cell (MFC); when power is added to the system and hydrogen is produced at the cathode through electrolysis of water, the system is referred to as microbial electrolysis cell (MEC). This PhD thesis is principally focused on the microbial fuel cells technology. Microbial fuel cells are regarded as a sustainable technology for electric energy generation from the oxidation of organic substrates contained in wastewater. The rising need for renewable energy sources and sanitation has encouraged intense research in this novel technology. Nevertheless, up untill now the interest has been primarily focused on the anodic oxidation of organic matter contained in wastewater. However, in addition to organics, wastewater also contains other pollutants, such as soluble nitrogen compounds, for which specific treatment is required. In conventional wastewater treatment systems, the organics available in the wastewater are typically used as electron donor during denitrification. However, a considerable fraction (>50%) of the chemical oxygen demand (COD) is still oxidized aerobically due to the large recirculation flows from the nitrification to the denitrification stages required in anoxic/aerobic configurations to allow for low nitrate levels in the final effluent. This increased COD demand is normally fulfilled by supplementary COD addition, with consequent increase of treatment costs. Alternatively, microorganisms can use inorganic carbon substrates and inorganic electron donors such as hydrogen for denitrification. However, the use of compressed hydrogen is hampered by its low solubility. As a solution, electrochemical hydrogen production permits in situ delivery of the electron donor and is advantaged by simplified control and dissolution of H2. The energy requirements to provide reducing power for denitrification can be decreased if bacteria use the electrode directly as electron donor without intermediate hydrogen production in bioelectrochemical systems. However, fundamental knowledge on bioelectrochemical denitrification is still lacking, therefore, this PhD thesis aims to fill some of these knowledge gaps and to solve some of the bottlenecks of the use of biocathodes. In particular, the goals of this work are: (i) to produce a suitable microbial community able to use the cathode as the sole electron donor during denitrification; (ii) to engineer a bioelectrochemical system able to couple the cathodic denitrification with the oxidation of organics at the anode; (iii) to characterize and quantify the electron losses during anodic and cathodic processes; (iv) to develop a bioelectrochemical system that maximises the nitrogen removal by integrating the nitrification stage into the cathode; finally, (v) to provide an insight into the structural properties of the biofilm performing nitrogen removal at the cathode. The results reveal that microbes can effectively utilize the electrode as electron donor for nitrate reduction to gaseous nitrogen at a redox potential that excludes intermediate production of hydrogen. Measurements revealed that acetoclastic methanogenesis and bacterial growth were responsible for causing the major electron losses at the anode. Adjusting the anodic potential did not achieve a significant overall reduction of the electron losses. At the cathode, the charge transfer efficiencies were instead very high, with the losses only due to the generation of nitrous oxide. Moreover, adjustments of the cathode potential resulted in higher efficiency. High carbon and nitrogen removal was obtained with a COD demand for denitrification as low as 2.4 g per g nitrogen denitrified, which is much lower than typically observed in heterotrophic–based nitrogen removal technologies (>7 g g 1). Nitrogen was removed at rates up to 0.256 kg N m-3 d-1, which is comparable to other autotrophic denitrification processes. Simultaneous nitrification and denitrification was observed in a combined system with cathodic aeration, at bulk dissolved oxygen (DO) levels up to 5 mg L-1, which is considerably higher than normally considered feasible for the process. Confocal laser scanning microscope analysis revealed the existence of a structured biofilm where putative nitrifying organisms occupied the outer layers in contact with the aerated bulk liquid, and putative denitrifying organisms occupy the layers closer to the electrode. These findings are significant in the field of bioelectrochemical systems as they help to unravel some of the complex questions relating to biocathodes. Additionally, the system provides an attractive option to achieve a very high level of nitrogen removal from wastewater with low COD/N ratios due to the selective utilisation of the COD for the denitrification reaction via the electrical transfer of reducing equivalents from the anode to the cathode. However, this research creates new questions, particularly regarding the mechanisms of electron transfer at the cathode. Also a number of practical design and optimisation challenges need to be overcome before wider applications can be considered.