The goals of this research were 1) to produce hydrogen in high yields from cellulosic materials and water by synthetic pathway biotranformation (SyPaB), and 2) to increase the hydrogen production rate to a level comparable to microbe-based methods (~ 5 mmol H2/L/h). Cell-free SyPaB is a new biocatalysis technology that integrates a number of enzymatic reactions from four different metabolic pathways, e.g. glucan phosphorylation, pentose phosphate pathway, gluconeogenesis, and hydrogenase-catalyzed hydrogen production, so as to release 12 mol hydrogen per mol glucose equivalent. To ensure the artificial enzymatic pathway would work for hydrogen production, thermodynamic analysis was firstly conducted, suggesting that the artificial enzymatic pathway would spontaneously release hydrogen from cellulosic materials. A kinetic model was constructed to assess the rate-limited step(s) through metabolic control analysis. Three phosphorylases, i.e. α-glucan phosphorylase, cellobiose phosphorylase, and cellodextrin phosphorylase, were cloned from a thermophile Clostridium thermocellum, and heterologously expressed in Escherichia coli, purified and characterized in detail. Finally, up to 93% of hydrogen was produced from cellulosic materials (11.2 mol H2/mol glucose equivalent). A nearly 20-fold enhancement in hydrogen production rates has been achieved by increasing the rate-limiting hydrogenase concentration, increasing the substrate loading, and elevating the reaction temperature slightly from 30 to 32°C. The hydrogen production rates were higher than those of photobiological systems and comparable to the rates reported in dark fermentations. Now the hydrogen production is limited by the low stabilities and low activities of various phosphorylases. Therefore, non-biologically based methods have been applied to prolong the stability of α-glucan phosphorylases. The catalytic potential of cellodextrin phosphorylase has been improved to degrade insoluble cellulose by fusion of a carbohydrate-binding module (CBM) family 9 from Thermotoga maritima Xyn10A. The inactivation halftime of C. thermocellum cellobiose phosphorylase has been enhanced by three-fold at 70°C via a combination of rational design and directed evolution. The phosphorylases with improved properties would work as building blocks for SyPaB and enabled large-scale enzymatic production of cellulosic hydrogen. / Ph. D.

Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes that microbes cannot do. One of the largest challenges is the high cost and instability of enzymes and cofactors. To overcome this obstacle, strong motivations have driven intensive efforts in discovering, engineering, and producing thermostable enzymes. In this project, ribose-5-phosphate isomerase (RpiB), one of the most important enzymes in the pentose phosphate pathway, was cloned from a thermophile Thermotoga maritima, and heterologously expressed in Escherichia coli, purified and characterized. High-purity RpiB was obtained by heat pretreatment through its optimization in buffer choice, buffer pH, as well as temperature and duration of pretreatment. This enzyme had the maximum activity at 80°C and pH 6.5-8.0. It had a half lifetime of 71 h at 60°C, resulting in its turn-over number of more than 2 x108 mol of product per mol of enzyme. Another two thermostable enzymes glucose-6-phosphate dehydrogenase (G6PDH) and diaphorase (DI) and their fusion proteins G6PDH-DI and DI-G6PDH were cloned from Geobacillus stearothermophilus, heterologouely expressed in E. coli and purified through its His-tag. The individual proteins G6PDH and DI have good thermostability and reactivity. However, the presence of DI in fusion proteins drastically decreased G6DPH activity. However, a mixture of G6PDH and a fusion protein G6PDH-DI not only restored G6PDH activity through the formation of heteromultimeric network but also facilitated substrate channeling between DI and G6PDH, especially at low enzyme concentrations. My researches would provide important building blocks for the on-going projects: high-yield hydrogen production through cell-free enzymatic pathways and electrical energy production through enzymatic fuel cells. / Master of Science

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No. of bitstreams: 2 Dissertação - Kelly Carolina Frauzino Araujo - 2014.pdf: 2783382 bytes, checksum: f8ddacd33ce41be9f223a9570381c87b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The "Microbial Models of Mammalian Metabolism" represents an alternative to use of animals on metabolism studies. Introduced in the 70s this model, also called biotranformation, has several advantages for their application as low cost, reduction of animals for experimentation and and higher number and range of metabolites produced. 1-(4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazin-1-yl)ethanone (LQFM030) and 4-((1-(4-chlorophenyl)-1H-pyrazol-4-il)methyl)piperazin-1-ethyl carboxylate (LQFM018) compounds were synthesized by molecular simplification of a series of compounds with chronic myeloid leukemia antiproliferative activity already described, the Nutlins prototypes. This study aims to produce a probable human metabolites of LQFM 030 and LQFM 018 by microbial biotranformation with filamentous fungi. To do so, analytical methodologies were developed by thin layer chromatography and high performance liquid chromatography in order to monitor metabolites production. After perform a screening of tem microorganisms Mortierella isabellina NRRL 1757 strain was selected to obtain metabolites on a larger scale. Incubations were carried out with 100 mL of glucose culture medium in each flask . At the end of incubation (96 h) extraction and purification of possible metabolites was performed . In an independent assay with LQFM 030, ketoconazole (10, 20 and 30 mg) was added to inhibit P450 cytochrome . In another test 1 mL of ethanol was added every 24 hours to induce cytochrome totaling 96 hours of incubation. To evaluate the best time to finalize incubation , an experiment was conducted in 168 hours with different concentrations of the substrate, 0.25 mg / mL and 50 mg / mL. A possible role of CYP 3A was evidenced by the inhibition caused by ketoconazole addition, that can inhibit the formation of N-oxide metabolite in LQFM 030 biotransformation. Ethanol addition does not induce LQFM 030-N-oxide production, but was able to induce other metabolites formation. The best time to end LQFM 030 incubation was defined as 168 hours and 0.25 mg/mL concentration. / O “Modelo Microbiano do Metabolismo Animal”, pode representar uma alternativa ao uso de animais nos estudos de metabolismo de novos candidatos a fármacos. Introduzido nos anos 70, este modelo, também denominado de biotransformação, apresenta várias vantagens para a sua aplicação como o baixo custo, a redução da utilização de animais de experimentação e maior quantidade e variedade de metabólitos produzidos. Os compostos 1-(4-((1-(4-clorofenil)-1H-pirazol-4-il)metil)piperazin-1-il)etanona (LQFM 030) e 4-((1-(4-clorofenil)-1H-pirazol-4-il)metil)piperazin-1-carboxilato de etila (LQFM 018) foram sintetizados a partir da simplificação molecular de uma série de compostos com atividade antiproliferativa frente à leucemia mielóide crônica já descrita, os protótipos Nutlins. O presente estudo objetivou a produção de prováveis metabólitos humanos do LQFM 030 e LQFM 018 através de biotransformação microbiana com fungos filamentosos. Para isso, foram desenvolvidas metodologias analíticas por cromatografia em camada delgada e cromatografia líquida de alta eficiência, a fim de se monitorar a produção dos metabólitos. Após seleção dentre dez microrganismos a cepa Mortierella isabellina NRRL 1757 foi escolhida para obtenção de metabólitos em maior escala. A incubação foi realizada com 100 mL de meio de cultura de glicose, em cada Erlenmeyer. Ao término da incubação (96 horas) foi realizada a extração e purificação dos possíveis metabólitos. Em um ensaio independente foi adicionado cetoconazol (10, 20 e 30 mg) ao meio contendo LQFM 030 com o objetivo de inibir o citocromo P450. Em outra etapa foi adicionado 1 mL de etanol a cada 24 horas para induzir o citocromo, totalizando 96 horas de incubação. Para avaliar a melhor cinética reacional, um experimento foi conduzido em 168 horas, com diferentes concentrações do substrato, 0,25 mg/mL e 50 mg/mL. A biotransformação do LQFM 030 e do LQFM 018 produziu seus respectivos N-óxidos, caracterizados por RMN 1H e 13C e EM. Uma provável participação da CYP 3A foi evidenciada através da inibição causada pela adição de cetoconazol capaz de inibir a formação do metabólito N-óxido na biotransformação do LQFM 030. A adição de etanol não induziu a produção do LQFM 030-N-óxido, contudo foi capaz de induzir a formação de outros metabólitos. O melhor tempo para término da incubação do LQFM 030 foi definido como 168 hrs e 0,25 mg/mL de concentração.