No description available.

Recent evaluations have demonstrated the ability of the bacteria Rhodococcus rhodochrous DAP 96253 to inhibit the growth of molds associated with plant and animal diseases as well as post-harvest loss of fruits, vegetables and grains. Pre-pilot-scale fermentations (20-30L) of Rhodococcus rhodochrous DAP 96253 were employed as a research tool with the goal of producing a practical biological agent for field-scale application for the management of white-nose syndrome (WNS) in bats and post-harvest fungal losses in several fruit varieties. Several key parameters within the bioreactor were evaluated for the potential to increase production efficiency as well as activity of the biocatalyst. These parameters included elapsed fermentation time, dissolved Oxygen, and carbohydrate concentration of which increased carbohydrate concentration at the time of harvest was shown to have a negative impact on the catalyst activity. In addition, process improvements including utilization of a liquid inoculum, an autoinduction feed strategy, and increased glucose concentration in the feed medium increased fermentation yields to 100-150g/L, while the biocatalyst efficiency was increased from previous work. To increase production efficiency, a multi-bioreactor scheme was developed that used a seed bioreactor and subsequent production tank, which doubled run yields per production cycle. Amidase, cyanidase, urease, and alkene-monoxygenase activity were monitored throughout the study as potential indicators for the multi-faceted mechanism of fungal antagonism. Of these amidase, cyanidase, and urease were demonstrated to be more elevated in cells that showed antifungal activity than those that did not. This study represents the first example of a reproducible pre-pilot plant-scale biomanufacturing process for a contact-independent biological control agent for established and emerging fungal pathogens of plants and animals, and facilitates large-scale production for broad application.

O desenvolvimento sustentável é atualmente um dos maiores focos das pesquisas no mundo em virtude dos impactos ambientais, tais como aquecimento global, geração de resíduos e emissão de gases poluentes causados pelo uso de combustíveis fósseis. Deste modo, pesquisas têm sido concentradas em tecnologias que permitam a substituição de refinarias à base de petróleo por biorrefinarias que utilizam matérias-primas renováveis. Neste contexto, os fungos filamentosos surgem como um recurso promissor no desenvolvimento de novos produtos sustentáveis, entre os quais os fungos pertencentes ao filo Zigomiceto, contribuem significativamente para esse desenvolvimento e estão sendo extensivamente estudados para a aplicação em biorrefinarias, com destaque especial para os fungos do gênero Mucor. Esse gênero, particularmente da espécie Mucor circinelloides, é um potencial produtor da enzima lipase e de biomassa com quantidades significativas de lipídios (single cell oil) o que permite sua exploração no processo de produção de biodiesel. Desta forma, o objetivo deste trabalho foi explorar importantes aplicações biotecnológicas da linhagem de Mucor circinelloides URM 4182, tanto para produção de lipases quanto de lipídios. O trabalho experimental realizado consolidou dados da potencialidade do fungo para produção de lipase ligada ao micélio e sua utilização como biocatalisador para obtenção de biodiesel partir de óleos vegetais alternativos, como andiroba, coco, macaúba, palma e pinhão manso. Nas condições estudadas, foram obtidos rendimentos de transesterificação entre 88% a 97% e as conversões mais elevadas foram alcançadas para os óleos láuricos sugerindo seletividade da lipase para ácidos de cadeia curta. A potencialidade da linhagem Mucor circinelloides URM 4182 foi comprovada na obtenção de lipídios em cultivos efetuados em biorreator utilizando glicose e milhocina, respectivamente, como fontes de carbono e de nutrientes alternativos aos suplementos sintéticos. Nessas condições, valores médios de produtividade biomassa (3,10 ± 0,01 g/L/dia) contendo (31 ±0,01 % m/m) de lipídios, correspondendo produtividade lipídica de (0,97 ± 0,01 g/L.dia) foram alcançados. A composição do óleo microbiano revelou elevados teores dos ácidos graxos saturados palmítico (C16:0) e esteárico (C18:0) e dos insaturados oleico (C18:1) e linoleico (C18:2), que são os ácidos graxos semelhantes aos dos óleos vegetais convencionais utilizados para a produção de biodiesel. Além disso, foram verificados teores consideráveis do ácido gama-linolênico (GLA - C18:3), que tem relevante importância nas indústrias farmacêutica e de alimentos. A esterificação e transesterificação simultâneas do óleo microbiano ou diretamente da biomassa celular com etanol mediada por catalisadores heterogêneos bioquímicos e químicos forneceram amostras de biodiesel com elevados teores de ésteres de etila (> 97%) que atendem as normas de qualidade para uso como biocombustível. / Sustainable development is now the major research focus in the world because of the environmental impacts, such as global warming, waste generation and greenhouse gas emissions caused by the use of fossil fuels. Thus, research has been focused on technologies that enables the replacement of petroleum based refineries by biorefinery based on renewable raw materials. In this context, filamentous fungi emerge as a promising resource in the development of new sustainable products, including the fungi belonging to the phylum Zigomicete which contribute significantly to this development and are being extensively studied for using in biorefineries, with particular emphasis on the Mucor sp. fungus genus. This genus, particularly from the species of Mucor circinelloides, is a potential producer of the enzyme lipase and biomass having significant amounts of lipids (single cell oil) which allows its exploitation in the biodiesel production. Thus, the aim of this study was to explore important biotechnological applications of a Brazilian strain of Mucor circinelloides URM 4182 for both production of lipases and lipids. The experimental work consolidated this fungus capability to produce mycelium bound lipase and its use as biocatalysts for biodiesel production from alternative vegetable oils such as andiroba, coconut, macaw palm, palm and jatropha. The attained transesterification yields were in the range from 88 to 97%, and the highest conversions were achieved for lauric oils suggesting that M. circinelloides lipase has high selectivity for short chain fatty acids. The capability of this strain was also proven to produce lipids at cultivation conditions established in this work using glucose and corn steep liquor, respectively, as carbon sources and alternative nutrients to the mineral supplements. Under these conditions, average values for biomass productivity (3.10 ± 0.01 g/L/day) containing high lipis levels (31.2 ± 0.01% m/m) and lipid productivity of 0.97 ± 0.01 g/L.dia were achieved. The composition of microbial oil revealed high levels of saturated fatty acids such as palmitic acid (C16:0) and stearic (C18:0) and unsaturated oleic (C18:1) and linoleic (C18:2), which are fatty acids similar to conventional vegetable oils used for the production of biodiesel. In addition, considerable levels of gamma-linolenic acid (GLA-C18:3) were also verified which has relevant importance in the pharmaceutical and food industries. The simultaneous esterification and transesterification from microbial oil or direct fungal biomass with ethanol mediated by heterogeneous catalysts (biochemical and chemical) provided samples with high levels of ethyl esters (> 97%) that meet the quality standards for using as biofuel.

No description available.

O desenvolvimento sustentável é atualmente um dos maiores focos das pesquisas no mundo em virtude dos impactos ambientais, tais como aquecimento global, geração de resíduos e emissão de gases poluentes causados pelo uso de combustíveis fósseis. Deste modo, pesquisas têm sido concentradas em tecnologias que permitam a substituição de refinarias à base de petróleo por biorrefinarias que utilizam matérias-primas renováveis. Neste contexto, os fungos filamentosos surgem como um recurso promissor no desenvolvimento de novos produtos sustentáveis, entre os quais os fungos pertencentes ao filo Zigomiceto, contribuem significativamente para esse desenvolvimento e estão sendo extensivamente estudados para a aplicação em biorrefinarias, com destaque especial para os fungos do gênero Mucor. Esse gênero, particularmente da espécie Mucor circinelloides, é um potencial produtor da enzima lipase e de biomassa com quantidades significativas de lipídios (single cell oil) o que permite sua exploração no processo de produção de biodiesel. Desta forma, o objetivo deste trabalho foi explorar importantes aplicações biotecnológicas da linhagem de Mucor circinelloides URM 4182, tanto para produção de lipases quanto de lipídios. O trabalho experimental realizado consolidou dados da potencialidade do fungo para produção de lipase ligada ao micélio e sua utilização como biocatalisador para obtenção de biodiesel partir de óleos vegetais alternativos, como andiroba, coco, macaúba, palma e pinhão manso. Nas condições estudadas, foram obtidos rendimentos de transesterificação entre 88% a 97% e as conversões mais elevadas foram alcançadas para os óleos láuricos sugerindo seletividade da lipase para ácidos de cadeia curta. A potencialidade da linhagem Mucor circinelloides URM 4182 foi comprovada na obtenção de lipídios em cultivos efetuados em biorreator utilizando glicose e milhocina, respectivamente, como fontes de carbono e de nutrientes alternativos aos suplementos sintéticos. Nessas condições, valores médios de produtividade biomassa (3,10 ± 0,01 g/L/dia) contendo (31 ±0,01 % m/m) de lipídios, correspondendo produtividade lipídica de (0,97 ± 0,01 g/L.dia) foram alcançados. A composição do óleo microbiano revelou elevados teores dos ácidos graxos saturados palmítico (C16:0) e esteárico (C18:0) e dos insaturados oleico (C18:1) e linoleico (C18:2), que são os ácidos graxos semelhantes aos dos óleos vegetais convencionais utilizados para a produção de biodiesel. Além disso, foram verificados teores consideráveis do ácido gama-linolênico (GLA - C18:3), que tem relevante importância nas indústrias farmacêutica e de alimentos. A esterificação e transesterificação simultâneas do óleo microbiano ou diretamente da biomassa celular com etanol mediada por catalisadores heterogêneos bioquímicos e químicos forneceram amostras de biodiesel com elevados teores de ésteres de etila (> 97%) que atendem as normas de qualidade para uso como biocombustível. / Sustainable development is now the major research focus in the world because of the environmental impacts, such as global warming, waste generation and greenhouse gas emissions caused by the use of fossil fuels. Thus, research has been focused on technologies that enables the replacement of petroleum based refineries by biorefinery based on renewable raw materials. In this context, filamentous fungi emerge as a promising resource in the development of new sustainable products, including the fungi belonging to the phylum Zigomicete which contribute significantly to this development and are being extensively studied for using in biorefineries, with particular emphasis on the Mucor sp. fungus genus. This genus, particularly from the species of Mucor circinelloides, is a potential producer of the enzyme lipase and biomass having significant amounts of lipids (single cell oil) which allows its exploitation in the biodiesel production. Thus, the aim of this study was to explore important biotechnological applications of a Brazilian strain of Mucor circinelloides URM 4182 for both production of lipases and lipids. The experimental work consolidated this fungus capability to produce mycelium bound lipase and its use as biocatalysts for biodiesel production from alternative vegetable oils such as andiroba, coconut, macaw palm, palm and jatropha. The attained transesterification yields were in the range from 88 to 97%, and the highest conversions were achieved for lauric oils suggesting that M. circinelloides lipase has high selectivity for short chain fatty acids. The capability of this strain was also proven to produce lipids at cultivation conditions established in this work using glucose and corn steep liquor, respectively, as carbon sources and alternative nutrients to the mineral supplements. Under these conditions, average values for biomass productivity (3.10 ± 0.01 g/L/day) containing high lipis levels (31.2 ± 0.01% m/m) and lipid productivity of 0.97 ± 0.01 g/L.dia were achieved. The composition of microbial oil revealed high levels of saturated fatty acids such as palmitic acid (C16:0) and stearic (C18:0) and unsaturated oleic (C18:1) and linoleic (C18:2), which are fatty acids similar to conventional vegetable oils used for the production of biodiesel. In addition, considerable levels of gamma-linolenic acid (GLA-C18:3) were also verified which has relevant importance in the pharmaceutical and food industries. The simultaneous esterification and transesterification from microbial oil or direct fungal biomass with ethanol mediated by heterogeneous catalysts (biochemical and chemical) provided samples with high levels of ethyl esters (> 97%) that meet the quality standards for using as biofuel.

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2017-03-27T13:33:54Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertação Helena Reissig Soares Vitola.pdf: 1434457 bytes, checksum: 51af522eb00ca36c96559b364913e8d2 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-28T20:26:28Z (GMT) No. of bitstreams: 2 Dissertação Helena Reissig Soares Vitola.pdf: 1434457 bytes, checksum: 51af522eb00ca36c96559b364913e8d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-28T20:26:28Z (GMT). No. of bitstreams: 2 Dissertação Helena Reissig Soares Vitola.pdf: 1434457 bytes, checksum: 51af522eb00ca36c96559b364913e8d2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Bactérias ácido lácticas, com potencial probiótico, são consideradas de grande interesse para o mercado de alimentos funcionais, os quais estão em expansão, devido à contribuição desses alimentos à saúde dos indivíduos. Objetivou-se no presente estudo isolar, caracterizar bactérias acido lácticas com potencial probiótico e imobilizar em grãos de soja, a fim de manter sua viabilidade durante o armazenamento. Foram realizados testes para isolamento e caracterização (coloração de Gram, presença de catalase, propriedades tecnológicas, fermentação de carboidratos) dos isolados a partir de silagem de colostro bovino; avaliação dos parâmetros de segurança microbiológica (atividade das enzimas, hemolisina, DNase e gelatinase, susceptibilidade a antimicrobianos de uso clínico e perfil plasmidial), avaliação do potencial probiótico (simulação da tolerância ao trânsito gastrointestinal, capacidade de auto-agregação, co-agregação, hidrofobicidade, atividade da enzima β galactosidase e atividade antagonista), avaliação da produção de exopolissacarídeos, identificação molecular, imobilização celular utilizando como suporte grãos de soja e microscopia eletrônica de varredura do biocatalizador. Dos isolados caracterizados como bactérias ácido lácticas, seis foram eleitos em testes preliminares (potencial tecnológico, resistência a diferentes concentrações de bile e fermentação de açúcares), três apresentaram-se seguros microbiológicamente e um caracterizou-se como potencialmente probiótico. A bactéria selecionada para imobilização, SCL3, pertencente ao gênero Lactobacillus da espécie casei, produtora de exopolissacarídeo, passou por processo fermentativo que permitiu a adsorção de células na superfície dos grãos de soja. A imobilização possibilitou a viabilidade das células em concentração de 6,23 log UFC.g -1 e 6,71 log UFC.g -1 até o final do 300 dia de armazenamento após secagem (liofilização) e em refrigeração, respectivamente. A microscopia eletrônica de varredura permitiu a visualização das células aderidas a superfície dos grãos de soja. / Lactic acid bacteria, with probiotic potential, are considered of great interest for the functional food market, which are expanding due to the contribution of these foods to the health of individuals. The objective of this study was to isolate, characterize lactic acid bacteria with probiotic potential and immobilize them in soybean grains, in order to maintain their viability during storage. Isolation and characterization tests (Gram staining, presence of catalase, technological properties, carbohydrate fermentation) of the isolates were carried out from bovine colostrum silage; Evaluation of microbiological safety parameters (enzyme activity, hemolysin, DNase and gelatinase, susceptibility to antimicrobials of clinical use and plasmid profile), evaluation of probiotic potential (simulation of gastrointestinal transit tolerance, selfaggregation capacity, co-aggregation, Hydrophobicity, β-galactosidase enzyme activity and antagonist activity), evaluation of exopolysaccharide production, molecular identification, cellular immobilization using as support soybean grains and scanning electron microscopy of the biocatalyst. Of the isolates characterized as lactic acid bacteria, six were selected in preliminary tests (technological potential, resistance to different concentrations of bile and fermentation of sugars), three were microbiologically safe and one was characterized as potentially probiotic. The bacterium selected for immobilization, SCL3, belonging to the genus Lactobacillus of the casei species, exopolysaccharide producer, undergoes a fermentative process that allowed the adsorption of cells on the surface of the soybean grains. The immobilization made possible the viability of the cells at a concentration of 6.23 log CFU.g-1 and 6.71 log CFU.g-1 until the end of the 300 day of storage after drying (lyophilization) and in refrigeration, respectively. Scanning electron microscopy allowed the cells to adhere to the surface of the soybean grains.

Généralement, diagnostiquer une pathologie donnée à un stade de développement précoce favorise le pronostic vital du patient atteint. Une telle performance nécessite de détecter des marqueurs présents à des seuils de concentrations bas dans des fluides biologiques souvent complexes. Pour détecter ces concentrations extrêmement faibles en analyte donné, la stratégie employée au cours de ce travail est l’amplification moléculaire du signal. Pour cela, différentes approches sont possibles (i) amplifier le signal issu de l’évènement de reconnaissance cible/sonde, (ii et iii) amplifier le signal par régénération ou réplication de la cible. Les stratégies conçues au cours de ce travail de thèse se focalisent principalement sur la détection de petites molécules, telles que l’eau oxygénée ou encore l’anion fluorure, mais avec à terme l’idée de les étendre à la détection indirecte de biomarqueurs ou protéines d’intérêts. La première partie de cette thèse se focalise sur l’amplification moléculaire d’un signal par une catalyse allostérique en utilisant la réaction de reconstitution d’une apoenzyme donnée avec son cofacteur tandis que la seconde partie de cette thèse repose sur la mise en place de systèmes d’amplification catalytique et auto-catalytique pour la détection d’H2O2, grâce à des dérivés pro-quinoniques porteurs de groupement acide/ester boronique. La distinction entre les systèmes catalytique et auto-catalytique se fait selon qu’H2O2 est régénéré ou amplifié au cours de la réaction. / Generally, diagnosing a given pathology at an early stage of development promotes the patient's prognosis. Such a performance requires the detection of specific markers which are present in complex biological fluids at low concentration level. To detect these extremely low analyte concentrations, the strategy employed in this work is the molecular amplification of the signal. To this end, different approaches are possible (i) amplifying the signal resulting from the target / probe recognition event, (ii and iii) amplifying the signal by regeneration or replication of the target. The strategies conceived during this thesis work mainly focus on the detection of small molecules, such as hydrogen peroxide or fluoride anion, but with the idea of extending them to the indirect detection of biomarkers or proteins of interest. The first part of this thesis focuses on the molecular amplification of a signal by allosteric catalysis using the reconstitution reaction of a given apoenzyme with its cofactor. The second part of this thesis is based on the implementation of catalytic and auto-catalytic amplification systems for the detection of H2O2, thanks to pro-quinonic derivatives bearing boronic acid/ester group. The distinction between catalytic and auto-catalytic systems is based on whether H2O2 is regenerated or amplified during the reaction.

Optically active &amp / #945 / -hydroxy ketones are important subunits of many biologically active compounds and indispensable synthons for asymmetric synthesis. Benzaldehyde Lyase from Pseudomonas fluorescens Biovar I is a novel ThDP-dependent enzyme that catalyzes the synthesis of benzoin type chiral &amp / #945 / -hydroxy ketones starting from both benzaldehyde and racemic benzoin derivatives. Benzaldehyde Lyase is the first example of enzymes in the literature which leads to a chemical resolution of enantiomers of benzoin derivatives through a C-C bond cleavage reaction. Chiral 2-hydroxypropiophenone derivatives are formed by benzaldehyde lyase (BAL), catalyzing C-C bond formation after a selective C-C bond cleavage of a benzoin derivative accepted as a substrate. The enzyme uses only the (R)-benzoin derivatives as substrate for the formation of (R)-HPP derivatives and it is highly stereoselective. Thus, in the presence of the acetaldehyde as the acceptor aldehyde, the C-C bond cleavage of the benzoin molecule followed by the carboligation of the acetaldehyde to yield chiral 2-hydroxy propiophenone derivatives. Given the racemic benzoin to the enzyme as the substrate in the presence of acetaldehyde, both the racemic resolution of the substrate, revealing the unreacted (S)-Benzoin and the formation of the corresponding R-HPP occur.

An alpha-amino ester hydrolase (AEH) applicable to synthesis of semi-synthetic antibiotics was cloned from the genomic DNA of Xanthomonas campestris pv. campestris sp. strain ATCC 33913. AEHs catalyze the synthesis and hydrolysis of alpha-amino beta-lactam antibiotics. The enzyme was characterized for thermodynamic and kinetic parameters. The enzyme shows optimal ampicillin hydrolytic activity at 25C and pH 6.8. The AEH enzymes have been shown to have excellent synthetic capability. Additionally, we demonstrated the first fully aqueous enzymatic one-pot synthesis of ampicillin direct from the natural product penicillin G eliminating the isolation of the intermediate 6-APA. Lastly, to improve the thermostability of the AEH a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-factors from the available crystal structures of the known AEH from Xanthomonas citri. Our best variant, which is a quadruple mutant, E143H/A275P/N186D/V622I, which has a T_50_30, the temperature at which the half-life is 30 minutes, of 34C and 1.3-fold activity compared to wild-type. Overall, we have successfully improved the understanding of the AEH class of enzymes and applied a novel cascade application, demonstrating AEHs unique applicability in the synthesis of beta-lactam antibiotics. The improved thermostability will further improve the industrial relevance of AEHs.

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.