Thesis (PhD (Biochemistry))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: In the last decade a need arose to find a theoretical framework capable of gaining a quantitative understanding of ecosystems. Control analysis was proposed as a suitable candidate for the analysis of ecosystems with various theoretical applications being developed, i.e. trophic control analysis (TCA) and ecological control analysis (ECA). We set out to test the latter approach through experimental means by applying techniques akin to enzyme kinetics of biochemistry on a simple ecosystem between Saccharomyces cerevisiae and Gluconobacter oxydans. However, this exercise was far more complex than we originally expected due to the extra metabolic activities presented by both organisms. Nevertheless, we derived suitable kinetic equations to describe the metabolic behaviour of both organisms, with regards to the activities of interest to us, from pure culture experiments. We developed new techniques to determine ethanol and oxygen sensitivity of G. oxydans based on its obligately aerobic nature. These parameters were then used to build a simple kinetic model and a more complex model incorporating oxygen limited metabolism we observed at higher cell densities of G. oxydans. Our models could predict both situations satisfactorily for pure cultures and especially the more complex model could describe the lack of linearity observed between metabolic activity and cell density at higher cell densities of G. oxydans. Mixed populations of S. cerevisiae and G. oxydans reached quasi-steady states in terms of ethanol concentration and acetate flux, which was a positive indication for the application of control analysis on the ecosystem. However, the theoretical models based on parameters derived from pure culture experiments did not predict mixed culture steady states accurately. Careful analysis showed that these parameters were mostly under-estimated for G. oxydans and overestimated for S. cerevisiae. Hence, we calculated the kinetic parameters for mixed population assays directly from the experimental data obtained from mixed cultures. We could calculate the control coefficients directly from the experimental data of mixed population studies and compare it with those from theoretical models based on 3 different parameter sets. Our analysis showed that the yeast had all the control over the acetate flux while control over the steady-state ethanol was shared. The strength of our approach lies in designing our experiments with a control analysis approach in mind, but we have also shown that even for simple ecosystems this approach is non-trivial. Despite the various experimental challenges, this approach was very rewarding due to the extra information obtained especially regarding control structure with regards to the steady-state ethanol concentration. / AFRIKAANSE OPSOMMING: In die afgelope dekade het daar ’n behoefte ontstaan na ‘n teoretiese raamwerk om tot ‘n kwantitatiewe begrip van ekosisteme te kom. As kandidaat vir so tipe raamwerk is kontrole analise voorgestel gepaardgaande met die ontwikkeling van verskeie teoretiese toepassings, i.e. trofiese kontrole analise en ekologiese kontrole analise. In hierdie tesis het ons laasgenoemde aanslag eksperimenteel ondersoek op ‘n eenvoudige ekosisteem, tussen Saccharomyces cerevisiae en Gluconobacter oxydans, deur gebruik te maak van tegnieke vanuit ensiemkinetika van biochemie. Hierdie strategie was egter baie meer kompleks as wat oorspronklik verwag is as gevolg van verdere metabolise aktiwiteite aanwesig in beide organismes. Ons het egter steeds daarin geslaag om kinetiese vergelykings af te lei, vanuit suiwer kulture, wat die metaboliese gedrag van beide organismes beskryf vir die aktiwiteite van belang vir ons studie. Ons het nuwe tegnieke, gebaseer op die aerobiese natuur van G. oxydans, ontwikkel om die sensitiwiteit van G. oxydans vir etanol en suurstof te bepaal. Hierdie parameters is gebruik om eers ’n eenvoudige model en toe ‘n meer gevorderde model, wat die suurstof-beperkte metabolisme van G. oxydans by hoër biomassa te beskryf, op te stel. Beide modelle was baie effektief in die voorspelling van die situasies waarvoor hulle ontwikkel is vir die suiwer kulture waar veral die meer gevorderde model die gebrek aan ‘n linieêre verband tussen die metabolisme van G. oxydans en biomassa by hoër biomassa kon beskryf. ’n Bemoedigende aanduiding dat kontrole analise toegepas kon word op die ekosisteem was dat mengkulture van S. cerevisiae en G. oxydans het quasi-bestendige toestande bereik het in terme van etanol konsentrasies en asetaat-fluksie. Die teoretiese modelle gebaseer op die parameters afgelei vanaf suiwer kulture kon egter nie die bestendige toestande in mengkulture akkuraat voorspel nie. Nadere ondersoek het aangedui dat die parameters meesal onderskat is vir G. oxydans en oorskat is vir S. cerevisiae. Gevolglik het ons die kinetiese parameters vir mengkulture direk van eksperimentele data van die mengkulture bereken. Verder kon ons die kontrole koeffisiente ook direk vanaf die eksperimentele data van mengkulture bereken en vergelyk met dié bereken vanuit die teoretiese modelle gebaseer op drie verskillende paremeter-stelle. Ons analise het gewys dat die gis alle beheer op die asetaat-fluksie uitoefen en dat die beheer oor die etanol-konsnetrasie gedeel is tussen die twee organismes. Die krag van ons aanslag lê daarin dat die eksperimente ontwerp is met ‘n kontrole analise in gedagte, maar ons het ook bewys dat hierdie aanslag selfs vir eenvoudige ekosisteme nie triviaal is nie. Ten spyte van die eksperimentele uitdagings, was die aanslag baie waardevol as gevolg van die ekstra inligting verkry met spesifieke klem op die kontrole-struktuur met betrekking tot die etanol konsentrasie by bestendige toestand.

Por sua capacidade de produzir e secretar uma mistura de enzimas durante seu crescimento em material lignocelulósico os fungos filamentosos são reconhecidos como importantes organismos no processo de degradação de biomassa. Porém, o processo demonstra-se limitado, uma vez que o polímero de lignina dificulta o acesso das enzimas celulolíticas a estrutura da celulose. Nesse contexto, o objetivo do presente estudo foi identificar fungos filamentosos capazes de produzir enzimas oxidorredutases e avaliar sua utilização em complemento a enzimas celulases de Trichoderma reesei no processo de sacarificação do bagaço de cana-de-açúcar. Cepas de fungos foram avaliadas quanto a sua capacidade em degradar compostos modelo de lignina, permitindo a seleção e identificação do fungo Pestalotiopsis sp., um ascomiceto com grande importância em biotecnologia. O fungo foi avaliado quanto a sua capacidade de produzir enzimas oxidorredutases e celulases. Os resultados mostraram uma maior atividade da enzima lacase (7,18 U/mg ao 6° dia de cultivo) em relação as outras enzimas estudadas. Os sobrenadantes de T. reesei e Pestalotiopsis sp. foram concentrados 5x e utilizados no ensaio final de sacarificação do bagaço de cana-de-açúcar. O coquetel enzimático produzido a partir da mistura do sobrenadante de T. reesei como o sobrenadante de Pestalotiopsis sp. produziu maior quantidade de açúcares redutores, gerando 15,52 g/L contra 12,29 g/L da amostra controle após 24 horas. Além disso, foi avaliada a atividade enzimática durante o processo de sacarificação. Constatou-se que no coquetel enzimático ocorre um aumento na atividade de celulases em relação ao controle durante as primeiras 6 horas de reação, podendo estar relacionado a despolimerização da lignina pelas enzimas oxidorredutases. Concluiu-se que para a produção de coquetéis enzimáticos mais eficientes para a degradação da biomassa vegetal, podem ser combinados diferentes grupos de enzimas provenientes de diferentes fungos. Assim, são necessários mais estudos acerca do papel de diferentes enzimas durante o processo e a identificação de novos microrganismos e seus perfis de secreção. / Due to its capacity to produce and secrete a mixture of enzymes during its growth in lignocellulosic material the filamentous fungi are recognized as important organisms in the process of degradation of biomass. However, the process is shown to be limited, since the lignin polymer hinders the access of cellulolytic enzymes to the cellulose structure. In this context, the objective of the present study was to identify filamentous fungi capable of producing oxidoreductase enzymes and to evaluate its use in complement to the enzymes cellulases of Trichoderma reesei in the saccharification process of sugarcane bagasse. Fungi strains were evaluated for their ability to degrade lignin model compounds, allowing the selection and identification of the fungus Pestalotiopsis sp., an ascomycete with great importance in biotechnology. The fungus was evaluated for its ability to produce oxidoreductase enzymes and cellulases. The results showed a higher activity of the enzyme laccase (7.18 U / mg) at the 6th day of culture in relation to the other enzymes studied. Supernatants of T. reesei and Pestalotiopsis sp., were concentrated 5x and used in the final saccharification test of sugarcane bagasse. The enzymatic cocktail produced from the mixture of T. reesei and Pestalotiopsis sp. supernatants produced a higher amount of reducing sugars, generating 15.52 g / L against 12.29 g / L of the control sample after 24 hours. In addition, the enzymatic activity during the saccharification process was evaluated. It was observed that in the enzymatic cocktail there is an increase in cellulase activity in relation to the control during the first 6 hours of reaction, and may be related to depolymerization of lignin by oxidoreductases enzymes. It was concluded that for the production of more efficient enzymatic cocktails for the degradation of the vegetal biomass, different groups of enzymes from different fungi can be combined. Thus, further studies are needed on the role of different enzymes during the process and the identification of new microorganisms and their secretion profiles.

Aliando reações enzimáticas a compostos orgânicos de silício, objetivou-se explorar o potencial destes substratos em reações biocatalisadas. A intenção era ampliar o escopo de substratos e desbravar novas transformações. Inicialmente foram abordados os trabalhos relativos ao uso de hidrolases em reações envolvendo organossilanos, cujo uso de lipases foi o foco da nossa contribuição. Nela, a resolução cinética enzimática (RCE) de alcoóis quirais benzílicos contendo silício e outros heteroátomos (fósforo e estanho) foi explorada e transesterificações enantiosseletivas eficientes foram alcançadas, em que tanto os produtos acetilados e os alcoóis remanescentes foram obtidos em excelentes excessos enantioméricos (e.e. >99% em todos os casos). Considerações sobre a relação estrutura/atividade das reações catalisadas por lipase foram feitas, e foi possível perceber que os compostos contendo silício reagiram mais rapidamente que aqueles contedo fósforo e estanho. Em seguida, numa extensão natural da RCE, buscou-se realizar a resolução cinética dinâmica (RCD). Diversos experimentos de RCD foram realizados utilizando lipase e dois tipos de catalisadores de racemização diferentes: complexos de rutênio e uma resina de troca catiônica. Embora tenham sido encontrados indícios de que a racemização utilizando os catalisadores de rutênio estava acontecendo no meio reacional, a inativação do catalisador durante o processo foi uma dificuldade que, nos estudos realizados, não foi possível contornar. Foi então que uma resina de troca catiônica foi utilizada como alternativa de racemização, e dependendo do substrato utilizado foi possível realizar eficientes RCDs (rendimento até 93% e e.e. até 96%) através de uma esterificação enzimática empregando um acilante de cadeia longa. O último trabalho empregando hidrolases foi na acilação de silanóis. A partir dos resultados interessantes envolvendo a acilação de um silanol arílico (conversão de 75% para o acetoxissilano derivado usando a CAL-B), tentou-se acilar um silanol benzílico racêmico e, embora o substrato tenha sido acetilado enzimaticamente (conversão de até 47% para o acetoxissilano derivado nas condições estudadas), a reação se deu sem enantiosseletividade. Nas reações envolvendo oxidorredutases, tanto mono-oxigenases quanto enzimas provenientes da bactéria Arthrobacter sp., foram empregadas como biocatalisadores. Na tentativa de se realizar a oxidação da ligação C-Si utilizando BVMOs (Baeyer-Villiger mono-oxigenases), foi possível concluir que a instabilidade dos silanos e alcoxissilanos nas reações em meio aquoso poderia configurar um entrave no desenvolvimento da metodologia. Por outro lado, evidências de oxidação enzimática da ligação Si-H foram observadas em dois substratos arílicos, que podem servir de direcionamento para futuros projetos envolvendo este tema. Por fim, células íntegras da bactéria Arthrobacter sp. foram utilizadas em reações de desracemização aeróbica (R)-seletiva de alcoóis e redução anaeróbica (S)-seletiva de cetonas, ambas utilizando substratos contendo silício, fósforo, estanho e boro. Transformações com elevada enantiosseletividade foram encontradas, provando a versatilidade da Arthrobacter sp. em mediar reações enantiocomplementares. / By combining both enzymatic reactions and organosilicon compounds, we aimed to explore the potential of these substrates in biocatalytic reactions. The main goal was to expand the scope of substrates and breakthrough new transformations. Initially the study was based on the use of hydrolases in reactions involving organosilanes, in which lipases were the focus of our contribution. Thus, enzymatic kinetic resolution (EKR) of chiral benzylic alcohols containing silicon and other heteroatoms (phosphorus and tin) was explored and efficient enantioselective transesterifications were achieved, in which both acetylated products and remaining alcohols were obtained in excellent enantiomeric excesses (e.e. > 99% in all cases). Considerations about the structure/activity relationship of lipase-catalyzed reactions were done, and it was found out that silicon-containing compounds can react faster than those phosphorus- or tin-containing analogues. Then, an extension of EKR was the dynamic kinetic resolution (DKR). Several experiments were performed using lipase and different racemization catalysts: ruthenium complexes and a cation exchange resin. Although racemization by ruthenium catalysts have been found, in our studies inactivation of the catalyst during the process was a problem that was not possible to be solved. A cation exchange resin was used in racemization, and depending on the substrate it was possible to perform efficient DKRs (yield up to 93% and e.e. up to 96%) via an enzymatic esterification using an acylating agent with long chain. Another work with hydrolases was the enzymatic acylation of silanols. From the interesting results involving the acylation of an aryl-silanol (75% conversion to the acetoxy-silane derivative, by CAL-B), the acylation of a racemic benzyl-silanol was performed and although the substrate has been successfuly acetylated by a series of lipases (up to 47% conversion to the acetoxy-silane derivative under the conditions studied), the reaction occurred without any enantioselectivity. In reactions involving oxidoreductases, both mono-oxygenases and enzymes from the bacterium Arthrobacter sp., were used as biocatalysts. In an attempt to carry out the oxidation of the C-Si bond using BVMOs, it was found out that the instability of the substrates in aqueous media could set an obstacle in the development of the methodology. Moreover, evidence of enzymatic oxidation of Si-H bond were observed for two aryl substrates, which can serve as guidance for future projects involving the topic. Finally, whole cells of the bacterium Arthrobacter sp. were used for (R)- selective deracemization of alcohols and (S)-selective reduction of ketones, both using silicon-, phosphorus-, tin- and boron-containing substrates. Transformations with high enantioselectivity were achieved, showing the versatility of Arthrobacter sp. in mediating enantiocomplementary reactions.

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.

Aliando reações enzimáticas a compostos orgânicos de silício, objetivou-se explorar o potencial destes substratos em reações biocatalisadas. A intenção era ampliar o escopo de substratos e desbravar novas transformações. Inicialmente foram abordados os trabalhos relativos ao uso de hidrolases em reações envolvendo organossilanos, cujo uso de lipases foi o foco da nossa contribuição. Nela, a resolução cinética enzimática (RCE) de alcoóis quirais benzílicos contendo silício e outros heteroátomos (fósforo e estanho) foi explorada e transesterificações enantiosseletivas eficientes foram alcançadas, em que tanto os produtos acetilados e os alcoóis remanescentes foram obtidos em excelentes excessos enantioméricos (e.e. >99% em todos os casos). Considerações sobre a relação estrutura/atividade das reações catalisadas por lipase foram feitas, e foi possível perceber que os compostos contendo silício reagiram mais rapidamente que aqueles contedo fósforo e estanho. Em seguida, numa extensão natural da RCE, buscou-se realizar a resolução cinética dinâmica (RCD). Diversos experimentos de RCD foram realizados utilizando lipase e dois tipos de catalisadores de racemização diferentes: complexos de rutênio e uma resina de troca catiônica. Embora tenham sido encontrados indícios de que a racemização utilizando os catalisadores de rutênio estava acontecendo no meio reacional, a inativação do catalisador durante o processo foi uma dificuldade que, nos estudos realizados, não foi possível contornar. Foi então que uma resina de troca catiônica foi utilizada como alternativa de racemização, e dependendo do substrato utilizado foi possível realizar eficientes RCDs (rendimento até 93% e e.e. até 96%) através de uma esterificação enzimática empregando um acilante de cadeia longa. O último trabalho empregando hidrolases foi na acilação de silanóis. A partir dos resultados interessantes envolvendo a acilação de um silanol arílico (conversão de 75% para o acetoxissilano derivado usando a CAL-B), tentou-se acilar um silanol benzílico racêmico e, embora o substrato tenha sido acetilado enzimaticamente (conversão de até 47% para o acetoxissilano derivado nas condições estudadas), a reação se deu sem enantiosseletividade. Nas reações envolvendo oxidorredutases, tanto mono-oxigenases quanto enzimas provenientes da bactéria Arthrobacter sp., foram empregadas como biocatalisadores. Na tentativa de se realizar a oxidação da ligação C-Si utilizando BVMOs (Baeyer-Villiger mono-oxigenases), foi possível concluir que a instabilidade dos silanos e alcoxissilanos nas reações em meio aquoso poderia configurar um entrave no desenvolvimento da metodologia. Por outro lado, evidências de oxidação enzimática da ligação Si-H foram observadas em dois substratos arílicos, que podem servir de direcionamento para futuros projetos envolvendo este tema. Por fim, células íntegras da bactéria Arthrobacter sp. foram utilizadas em reações de desracemização aeróbica (R)-seletiva de alcoóis e redução anaeróbica (S)-seletiva de cetonas, ambas utilizando substratos contendo silício, fósforo, estanho e boro. Transformações com elevada enantiosseletividade foram encontradas, provando a versatilidade da Arthrobacter sp. em mediar reações enantiocomplementares. / By combining both enzymatic reactions and organosilicon compounds, we aimed to explore the potential of these substrates in biocatalytic reactions. The main goal was to expand the scope of substrates and breakthrough new transformations. Initially the study was based on the use of hydrolases in reactions involving organosilanes, in which lipases were the focus of our contribution. Thus, enzymatic kinetic resolution (EKR) of chiral benzylic alcohols containing silicon and other heteroatoms (phosphorus and tin) was explored and efficient enantioselective transesterifications were achieved, in which both acetylated products and remaining alcohols were obtained in excellent enantiomeric excesses (e.e. > 99% in all cases). Considerations about the structure/activity relationship of lipase-catalyzed reactions were done, and it was found out that silicon-containing compounds can react faster than those phosphorus- or tin-containing analogues. Then, an extension of EKR was the dynamic kinetic resolution (DKR). Several experiments were performed using lipase and different racemization catalysts: ruthenium complexes and a cation exchange resin. Although racemization by ruthenium catalysts have been found, in our studies inactivation of the catalyst during the process was a problem that was not possible to be solved. A cation exchange resin was used in racemization, and depending on the substrate it was possible to perform efficient DKRs (yield up to 93% and e.e. up to 96%) via an enzymatic esterification using an acylating agent with long chain. Another work with hydrolases was the enzymatic acylation of silanols. From the interesting results involving the acylation of an aryl-silanol (75% conversion to the acetoxy-silane derivative, by CAL-B), the acylation of a racemic benzyl-silanol was performed and although the substrate has been successfuly acetylated by a series of lipases (up to 47% conversion to the acetoxy-silane derivative under the conditions studied), the reaction occurred without any enantioselectivity. In reactions involving oxidoreductases, both mono-oxygenases and enzymes from the bacterium Arthrobacter sp., were used as biocatalysts. In an attempt to carry out the oxidation of the C-Si bond using BVMOs, it was found out that the instability of the substrates in aqueous media could set an obstacle in the development of the methodology. Moreover, evidence of enzymatic oxidation of Si-H bond were observed for two aryl substrates, which can serve as guidance for future projects involving the topic. Finally, whole cells of the bacterium Arthrobacter sp. were used for (R)- selective deracemization of alcohols and (S)-selective reduction of ketones, both using silicon-, phosphorus-, tin- and boron-containing substrates. Transformations with high enantioselectivity were achieved, showing the versatility of Arthrobacter sp. in mediating enantiocomplementary reactions.

Orientadores: Beatriz Gomes Guimarães, Jose Antonio Brum / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Fisica Gleb Wataghin / Made available in DSpace on 2018-09-27T18:06:10Z (GMT). No. of bitstreams: 1 Rinaldi_FabioCupri_D.pdf: 8466921 bytes, checksum: 8a88bf7cf4ccef10efbca8ec0412db74 (MD5) Previous issue date: 2008 / Resumo: As oxidoredutases de pontes dissulfeto da família DsbA são responsáveis pela catálise da formação de pontes dissulfeto em proteínas secretadas para o periplasma, participando do processo de enovelamento de fatores de virulência de diversos organismos. É a proteína com maior potencial de oxidação atualmente caracterizada e tal propriedade é associada às interações eletrostáticas envolvendo resíduos de seu sítio ativo, que apresenta um arranjo Cys-Pro-His-Cys altamente conservado. A bactéria fitopatogênica Xylella fastidiosa possui dois genes adjacentes que codificam duas oxidoredutases pertencentes à família das DsbAs (XfDbsA e XfDbsA2). Embora a XfDbsA conserve o arranjo CPHC, a XfDbsA2 possui a substituição do resíduo histidina, descrito como essencial à atividade da enzima, por alanina (CPAC). Visando a caracterização estrutural e funcional destas proteínas, a estrutura cristalográfica da XfDsbA foi determinada a 1,9 Å de resolução e um modelo por homologia da XfDsbA2 foi construído. Além disso os potenciais de oxidação das enzimas foram determinados por medidas de fluorescência. A estrutura da XfDsbA revelou a presença de um peptídeo ligado próximo a região do sítio ativo em um dos monômeros mostrando, pela primeira vez em uma estrutura a alta resolução, o provável modo de interação da DsbA com um substrato. Os ensaios funcionais revelaram que as DsbAs de X. fastidiosa apresentam potenciais redox similares e ligeiramente superiores ao da homóloga de Escherichia coli. Embora trabalhos sobre a importância do arranjo CPHC têm associado o alto potencial redox das DsbAs à presença do resíduo histidina no sítio ativo, os resultados obtidos para a XfDsbA2 mostraram que a substituição do resíduo de histidina por alanina não afeta seu potencial redox. A análise das interações envolvendo resíduos do sítio ativo mostrou diferenças importantes entre XfDsbA, XfDsbA2 e suas homólogas de E. coli e Vibrio cholerae. Ensaios funcionais com mutantes foram realizados em busca da identificação dos resíduos que possam compensar a ausência da histidina em XfDsbA2. Os resultados obtidos fornecem novas informações sobre o mecanismo molecular dessa família de enzimas / Abstract: Disulfide oxidoreductase DsbA catalyzes disulfide-bond formation in proteins secreted to the periplasm and has been related to the folding process of virulence factors in many organisms. It is the most oxidizing of the thioredoxin-like proteins and DsbA redox power is understood in terms of the electrostatic interactions involving the active site motif CPHC. The plant pathogen Xylella fastidiosa has two chromosomal genes encoding two oxidoreductases belonging to the DsbA family and, in one of them, the canonical motif CPHC is replaced by CPAC. Aiming at the structural and functional characterization of X. fastidiosa DsbAs, the crystal structure of XfDsbA was solved at 1.9 Å resolution and the XfDsbA2 homology model was calculated. We also determined the redox potential of both enzymes by means of fluorescence experiments. The crystal structure of the XfDsbA revealed an electron density corresponding to an 8-mer peptide interacting with the hydrophobic groove on the surface of the monomer C next to the active site. This modeled peptide shows at first time in a high-resolution crystal structure the probable mode of interaction between DsbA and a substrate. Furthermore, the results presented in this work surprisingly show that, despite the absence of the active site histidine in XfDsbA2, both proteins have similar redox potentials. In addition, the structure of XfDsbA revealed critical differences in the interactions involving the active site residues. Biochemical assays with XfDsbA mutants were performed in order to investigate the residues which may be responsible for compensate for the lack of the conserved histidine in XfDsbA2. The results presented contribute to the understanding of DsbA molecular mechanism / Doutorado / Física da Matéria Condensada / Doutor em Ciências

Wong, Kin Sing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 86-98). / Abstract also in Chinese. / THESIS COMMITTEE --- p.i / ABSTRACT (ENGLISH) --- p.ii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGEMENTS --- p.v / DECLARATION --- p.vi / ABBREVIATIONS --- p.vii / TABLE OF CONTENTS --- p.x / LIST OF TABLES --- p.xiv / LIST OF FIGURES --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Antibiotics market and β-lactam antibiotics --- p.1 / Chapter 1.2. --- Semi-synthetic cephems --- p.1 / Chapter 1.3. --- Conversion of CPC to 7-ACA --- p.3 / Chapter 1.4. --- Chemical production versus enzymatic bioconversion --- p.5 / Chapter 1.5. --- Industrial two-step bioconversion of CPC --- p.11 / Chapter 1.6. --- Phylogenetics and physiological roles of DAAO --- p.15 / Chapter 1.7. --- Yeast DAAOs are suitable candidates for enzymatic bioconversion --- p.17 / Chapter 1.8. --- Structural and mechanistic studies of DAAOs --- p.18 / Chapter 1.9. --- "Modifications of pkDAAO, RgDAAO and TvDAAO" --- p.25 / Chapter 1.10. --- Objectives of the study --- p.26 / Chapter CHAPTER 2 --- HOMOLOGY MODELLING / Chapter 2.1. --- Introduction --- p.27 / Chapter 2.2. --- Methods / Chapter 2.2.1. --- Sequence alignment and selection of homologs --- p.28 / Chapter 2.2.2. --- Generation of three-dimensional TvDAAO model --- p.28 / Chapter 2.3. --- Results --- p.29 / Chapter 2.4. --- Discussion --- p.33 / Chapter CHAPTER 3 --- "MUTAGENESIS, EXPRESSION, PURIFICATION AND SCREENING OF MUTANTS" / Chapter 3.1. --- Introduction --- p.38 / Chapter 3.2. --- Materials and methods / Chapter 3.2.1. --- Cloning of TvDAAO mutants / Chapter 3.2.1.1. --- Preparation of competent E. coli --- p.39 / Chapter 3.2.1.2. --- Transformation of E. coli --- p.40 / Chapter 3.2.1.3. --- Agarose gel electrophoresis and gel-purification --- p.41 / Chapter 3.2.1.4. --- Plasmid extraction --- p.42 / Chapter 3.2.1.5. --- Site-directed mutagenesis of TvDAAO --- p.42 / Chapter 3.2.2. --- Heterologous expression and purification of mutants / Chapter 3.2.2.1 --- Shake flask fermentation --- p.45 / Chapter 3.2.2.2. --- Cell harvest and disruption --- p.45 / Chapter 3.2.2.3. --- Purification of WT and mutants --- p.47 / Chapter 3.2.2.4. --- Determination of protein concentration --- p.47 / Chapter 3.2.2.5. --- SDS-PAGE --- p.48 / Chapter 3.2.3. --- Screening of mutants --- p.48 / Chapter 3.3. --- Results / Chapter 3.3.1. --- Preparation of purified TvDAAO mutants --- p.50 / Chapter 3.3.2. --- Evaluation of activity and thermostability --- p.50 / Chapter 3.4. --- Discussion --- p.53 / Chapter CHAPTER 4 --- ENZYME KINETICS / Chapter 4.1. --- Introduction --- p.57 / Chapter 4.2. --- Materials and methods / Chapter 4.2.1. --- Standard assay --- p.58 / Chapter 4.2.2. --- Determination of kinetic parameters --- p.59 / Chapter 4.2.3. --- Inhibitory studies --- p.59 / Chapter 4.2.4. --- Effects of pH --- p.60 / Chapter 4.2.5. --- Heat treatments --- p.60 / Chapter 4.2.6. --- CD measurements --- p.61 / Chapter 4.3. --- Results / Chapter 4.3.1. --- Time progress curve analysis --- p.61 / Chapter 4.3.2. --- Kinetics of WT and mutants --- p.62 / Chapter 4.3.3. --- Temperature-dependent and time-dependent thermostability --- p.67 / Chapter 4.3.4. --- Secondary structure measurements --- p.71 / Chapter 4.4. --- Discussion --- p.71 / Chapter CHAPTER 5 --- CONCLUSIONS AND PERSEPECTIVES --- p.83 / BIBLIOGRAPHY --- p.86

Asymmetric synthesis with biocatalyst has become an increasingly interesting and cost effective manufacturing process in fine chemicals, pharmaceuticals, and agrochemical intermediates. Enoate reductases from the Old Yellow Enzyme family offer high substrate efficiency, region, stereo-, and enantioselectivity in the catalyzed biotransformations. Asymmetric reduction of activated C=C bond is one of the most widely applied synthetic tools for the potential to generate up to two stereogenic centers in one step reaction. The thesis contributed to the development and characterization of the Old Yellow Enzyme family members including NRSal from Salmonella typhimurium, YersER from Yersinia bercoviei, KYE1 from Kluyveromyces lactis, and XenA from Pseudomonas putida. We explored the possible new chemistry, gathered further understanding of enzymes functionality and biochemistry, evaluated parameters such as enzyme stability, productivity, and selectivity, and improved enzyme specificity through computational guided protein engineering method. In overall, the increasing knowledge about this Old Yellow Enzyme family together with recent advances in biotechnology renders the enoate reductases a tool of choice for industrial applications.

Thesis (Ph. D.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains x, 121 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 84-118).

Pyrithiamine-induced thiamine deficiency in rats is a well-established animal model of Wernicke's Encephalopathy (WE). This thesis project, submitted as four articles, presents an examination of metabolic events that contribute to the selective neuronal lesions observed in the medial thalamus (MT) of thiamine-deficient (TD) rat. In addition, the phenomenon of glucose-precipitated worsening of neurological status in WE patients (Wallis et al., 1978; Watson et al., 1981) is explored. / Lactate accumulation is known to occur selectively in regions of the TD brain, which ultimately express neuronal cell death (McCandless, 1982; Munujos et al., 1996). In Article 1, the metabolic origin and cellular localization of region-selective lactate accumulation in the MT of TD rats was studied using combined 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Parallel studies were performed to examine the effects of glucose loading on regional brain lactate synthesis in TD animals. Thiamine deficiency caused focal increases in the de novo synthesis of lactate via elevated glycolytic flux in the MT, while contribution via pyruvate recycling and the periphery remained nominal. Lactate levels remained unaltered in the frontal cortex (FC), a brain region that is spared in thiamine deficiency. Administration of a glucose load intensified the selective increases in lactate de novo synthesis and accumulation in the MT of TD rats, positing a role for lactic acidosis in the glucose-precipitated worsening of neurological status in TD patients. Accordingly, Article 2 addresses the effect of glucose loading on local cerebral pH in the vulnerable MT, compared to the FC, of TD rats. Administration of a glucose load resulted in detrimental decreases in regional pH selectively in the MT, implying that alterations of brain pH contribute to the pathogenesis of thalamic neuronal damage and consequent cerebral dysfunction in WE. / Region-specific alterations in the steady state levels of cerebral amino acid neurotransmitters have been well-documented in experimental animal models of thiamine deficiency (Butterworth et al., 1979; Butterworth & Heroux, 1989; Gaitonde et al., 1975; Plaitakis et al., 1979); however, the dynamics of these changes have never been systematically explored. In Article 3, we examined the metabolic fluxes through thiamine-dependent pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) using multinuclear NMR spectroscopy. Furthermore the cellular localization of the metabolic changes in relation to regional vulnerability to thiamine deficiency was addressed. Our studies clearly demonstrate that early decreases m metabolic flux through alpha-KGDH result in commensurate declines in aspartate concentrations in the MT of TD rats. Impairments to PDH flux manifest secondarily to the metabolic block at alpha-KGDH, likely due to depleted oxaloacetate pools. As a result of impaired pyruvate oxidation, declines in the de novo synthesis of glutamate and GABA ensue. The present findings also suggest that inhibition of flux through alpha-KGDH in TD brain occurs primarily in the neurons, while astrocytes possess compensatory mechanisms, i.e. the anaplerotic pathway, to replenish oxaloacetate concentrations via metabolic pathways that do not involve thiamine-dependent enzymes. / In Article 4, we investigated the regional effects of thiamine deficiency on the activity of thiamine-dependent branched-chain alpha-ketoacid dehydrogenase (BCKDH) and the resultant effects on regional cerebral branched-chain amino acid (BCAA) oxidation. Thiamine deficiency resulted in significant impairments in BCKDH activity; while parallel studies on enzyme distribution confirmed a lower oxidative capacity for BCAAs in the MT compared with the Fe. / The data presented in these four articles confirm and extend findings for the region-selective impairments in thiamine-dependent metabolic processes as the foundation of vulnerability of the MT to thiamine deficiency. In addition, glucose loading of TD rats exacerbates both lactic acidosis and impaired pyruvate oxidation in this vulnerable brain region, positing a role for these processes in the glucose-precipitated worsening of neurological status in TD patients. Impaired oxidative metabolism of glucose and BCAAs in the MT leads to the accumulation of potentially harmful metabolic intermediates, contributing to the mitochondrial dysfunction, cellular energy failure and ultimately neuronal cell death observed in thiamine deficiency.