Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1971. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Thesis (M. S.)--University of North Carolina at Greensboro, 2006. / Title from PDF title page screen. Advisor: Jason J. Reddick ; submitted to the Dept. of Chemistry. Includes bibliographical references ( p. 71-77)

Thesis (M.S.)--University of North Carolina at Greensboro, 2007. / Title from PDF title page screen. Advisor: Jason J. Reddick; submitted to the Dept. of Chemistry. Includes bibliographical references (p. 87-91).

There is increasing interest in producing biofuels; biofuels are preferable to fossil fuels as the biomass from which they are derived is seen as a renewable source, as opposed to fossil fuels which are a finite resource. “First Generation” biofuels are derived from food crops such as grains and sugar cane. The use of food crops is not sustainable in this age of increasing food insecurity. A promising alternative appears to be what is termed “Second Generation” feedstocks, such as energy crops like Miscanthus spp., and agricultural by-products. The problem with the use of second generation feedstocks is firstly that the sugars are locked up in the cell wall polymers (CWP), which need to be released by physio-chemical pre-treatments, that are costly and time consuming. The second problem is that not all the sugars that are released from CWP are able to be utilised by wild type product-forming organisms. However, model chassis organisms can be genetically modified to utilise these sugars and /or produce enzymes to degrade biomass which reduces the time and costs involved in the process. While engineering these organisms to utilise a range of monosaccharides has already been successful, engineering them to produce degradation enzymes is proving to be problematic. A potentially more effective system is to use co-cultures of both cellulose-degrading and product-forming organisms. Since this is a novel approach it is not known whether the two organisms are able to live together without any adverse effects. The aims of this study were firstly to determine whether mixed cultures of both cellulose-degrading and potential product-forming organisms could survive in the presence of one another, secondly whether the cellulose-degrading organisms could degrade potential feedstock down into their monosaccharide building blocks and thirdly whether the potential product-forming organisms could survive and utilise these monosaccharides for growth and potential fermentation. It was discovered that C. hutchinsonii can degrade both paper and Triticum aestivum straw polymers into their monosaccharide components and that B. subtilis can survive on the sugars released by C. hutchinsonii. It was also discovered that C. hutchinsonii and B. subtilis 168 can only tolerate an ethanol concentration of up to 2% (v/v) and that this is below the baseline for a biofuel system to be economically viable. Likewise, C. hutchinsonii and B. subtilis 168 have an even poorer tolerance for butanol; growth is inhibited by < 1% butanol in its growth media. A series of physio-chemical pre-treatments were developed in order to make the monosaccharides present in the cell wall polymers more accessible to microbial saccharification. Sequential pre-treatments, both physical milling and chemical hydrolysis in tandem, had the greatest effect on the bio chemistry of the biomass, but that these physio-chemical pre-treatments produced inhibitory compounds in the medium that retarded microbial growth. Attempts were made to genetically modified Bacillus subtilis 168 to produce lactic acid and ethanol by over expressing the native ldh gene under the highly-expressed promoter of the cspD gene and by integrating the fused pdc:adh gene from Z. mobilis under the same promoter. Transformation of B. subtilis to over express LDH was successful, with PCR confirmation of the correct insertion and enzyme activity for the ldh both in vitro and in vivo, with the latter producing more lactic acid aerobically than the wild type. Transformation of B. subtilis to express pdc:adh and subsequent production of ethanol was not successful.

Enzimas industriais movimentam um mercado mundial estimado em sete bilhões de dólares anualmente. Entre essas enzimas, incluem-se as queratinases, enzimas capazes de catalisar a hidrólise de queratina e com aplicação potencial na indústria coureira. A queratinase KerS14, produzida pelo Bacillus subtilis S14 é capaz de depilar o couro sem causar danos ao colágeno. Embora tenha esta capacidade de grande interesse biotecnológico, sua baixa termoestabilidade a 50 °C é uma característica desvantajosa para a aplicação industrial da enzima. Visando aumentar a produção de queratinases pelo B. subtilis S14 e a termoestabilidade da KerS14, duas estratégias foram adotadas: mudanças nas condições de cultura do microrganismo selvagem e obtenção de enzimas mutantes recombinantes. Na primeira estratégia, B. subtilis S14 foi cultivado em meio farinha de pena 1,5% e CaCl2 1%. O sobrenadante da cultura foi caracterizado e 60% da atividade queratinolítica permaneceu depois de armazenada por nove dias a temperatura de 50 °C. Em paralelo, a ORF que codifica a KerS14 foi amplificada e clonada em vetor de expressão. Os resíduos de aminoácidos G61, S98 e P239 da KerS14 foram modificados por mutagênese sítio dirigida , as enzimas mutantes foram expressas in Escherichia coli e purificadas. A enzima recombinante (rKerS14) foi mais termoestável do que a enzima selvagem. Além disso, foi verificado que o fibrinogênio e fibrina são hidrolisados pela rKerS14, mostrando que a enzima também tem potencial para desenvolver drogas para o tratamento de doenças cardiovasculares. / Industrial enzymes have a world market of more than seven billion dollars per year. Keratinases are among these enzymes. They have a potential for use in tannery. The keratinase KerS14, produced by Bacillus subtilis S14, differ from other keratinases because it can depilate leather without damaging collagen. Despite this great biotechnological potential, its low thermal stability at 50 °C is an undesirable property for industrial application. In order to increase keratinases production by B. subtilis S14 and KerS14 thermal stability, two strategies were adopted: changes in wild–type microorganism growth conditions and producing recombinant mutant enzymes. In first strategy, B. subtilis S14 were grown in feather meal 1.5% and CaCl2 1 %. The supernatant obtained after fermentation was characterized and its keratinolytic activity remained in 60% after nine days of storage at 50 °C. In parallel, the KerS14 ORF was amplified and cloned into an expression vector. The KerS14 amino acid residues G61, S98 and P239 were modified and mutant enzymes were expressed in Escherichia coli and purified. It was verified that recombinant enzyme (rKerS14) is more thermal stable than the wild–type enzyme. In addition, it is able to hydrolyze fibrinogen and fibrin which is useful to develop drugs for the treatment of cardiovascular diseases.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Engenharia Química, Florianópolis, 2015. / Made available in DSpace on 2016-03-15T04:01:39Z (GMT). No. of bitstreams: 1 337597.pdf: 2403339 bytes, checksum: c506d45d38180aab9f1ebe6de23d3502 (MD5) Previous issue date: 2015 / Biossurfactantes são compostos anfifílicos de origem biológica que possuem capacidade de reduzir a tensão superficial e interfacial. Dentre os mais efetivos estão os lipopeptídeos produzidos por bactérias do gênero Bacillus, especialmente a surfactina. Devido aos seus altos custos de produção sua aplicação ainda é limitada, tornando-se necessário processos que viabilizam economicamente sua produção. A produção de surfactina a partir de processos anaeróbios pode servir como uma alternativa na redução dos custos operacionais e estruturais. Além disso, microrganismos com capacidade de sintetizar surfactina em anaerobiose são de interesse da indústria petroquímica, pois ambos, surfactina e microrganismo podem ser empregados na extração do petróleo a partir de poços esgotados. Este trabalho teve como objetivo realizar o estudo cinético da produção do biossurfactante surfactina por Bacillus subtilis ATCC 21332 em condição anaeróbia. Todos os ensaios foram realizados em biorreator operado de forma descontínua, em meio salino baseado na composição da água do mar (meio MM), acrescido de glicose e de NaNO3 como fontes de carbono e nitrogênio, respectivamente, além de outros micronutrientes. Inicialmente, para efeito comparativo, B. subtilis foi cultivado na presença e na ausência de oxigênio, em meio MM contendo 10 g.L-1 de glicose e 4,25 g.L-1 de NaNO3. Os resultados mostraram que a presença de oxigênio favoreceu a síntese de surfactina, resultando em maior produtividade, 4,92 mg.L-1.h-1, em comparação ao anaeróbio, 1,84 mg.L-1.h-1. Os cultivos com diferentes relações carbono/nitrogênio (C/N) no meio MM, foram realizados com 10 g.L-1 de glicose e diferentes concentrações de NaNO3. Das condições testadas, a que se mostrou mais adequada foi a relação C/N 3,6, resultando em produção de 49,7 mg.L-1 e produtividade de 2,49 mg.L-1.h-1 em surfactina. A partir desta foram realizados dois cultivos no meio MM aumentando-se proporcionalmente as fontes de carbono e nitrogênio. A produtividade média de 1,75 mg.L-1.h-1 de surfactina, obtida nos cultivos contendo 15 e 20 g.L-1 de glicose foi inferior à produtividade alcançada com 10 g.L-1 de glicose, mostrando que houve efeito negativo com o aumento das concentrações. Avaliou-se também a produtividade em surfactina quando B. subtilis foi cultivado no meio mineral adicionado de micronutrientes (MM1) e de micronutrientes suplementado com 1 g.L-1 de extrato de levedura(MM2), acrescido de 20 g.L-1 de glicose. A maior produção de 101,8 mg.L-1 e a maior produtividade de 3,39 mg.L-1.h-1 em surfactina foram obtidas utilizando o meio MM2. Quando se avaliou a influência do aumento proporcional das fontes de carbono e de nitrogênio no MM2, os resultados mostraram efeito negativo sobre a produtividade emsurfactina a partir do aumento na concentração de glicose. A produtividade foi de 3,18 mg.L-1.h-1 e 1,63 mg.L-1.h-1 de surfactina, nos cultivos contendo 30 e 40 g.L-1 de glicose, respectivamente. Os resultados mostraram que a maior produtividade em surfactina (3,39 mg.L-1.h-1) foi obtida cultivando B. subtilis ATCC 21332 em biorreator anaeróbio no meio MM2 contendo 20 g.L-1 de glicose. As velocidades específicas de crescimento (µX), de consumo de substrato (µS) e de produção de biossurfactante (µP) correlacionaram-se bem, podendo-se então afirmar que a surfactina produzida por Bacillus subtilis ATCC 21332 está associada ao crescimento celular. A análise de espectroscopia de infravermelho por transformada de Fourier (FTIR) confirmou que o biossurfactante produzido é um lipopeptídeo do tipo surfactina. A surfactina semipurificada se mostrou efetiva e eficaz, pois apresentou CMC de 10 mg.L-1 e foi capaz de reduzir a tensão superficial da água de 72,0 para 29,9 mN.m-1. A solução aquosa de surfactina exibiu excelente capacidade emulsificante e estabilidade das emulsões formadas e, apresentou estabilidade na faixa de pH entre 6 e 8, salinidade de até 10 % de NaCl e tempo de exposição de 140 min a 100 °C.<br> / Abstract : Biosurfactants are amphiphilic compounds that have the ability to lower the surface and interfacial tension. Among the most effective biosurfactants are the lipopeptides produced by Bacillus, especially the surfactin. Due to their high production costs, its implementation is still limited, making necessary processes that economically allow its production. The production of surfactin from anaerobic processes can serve as an alternative in reduction of operational and structural costs. In addition, microorganisms capable of synthesizing surfactin anaerobically are interesting for petrochemical industry since both, surfactin and microorganism can be employed in the extraction of oil from depleted wells. This study aimed to perform a kinetic study of the production of surfactin by Bacillus subtilis ATCC 21332 in anaerobic conditions. All assays were performed in a bioreactor operated discontinuously, in saline medium based on the composition seawater (MM medium) supplemented with NaNO3 and glucose as carbon and nitrogen sources, respectively, and other micronutrients. Initially, for comparative purposes, B. subtilis was cultivated with and without oxygen, in MM medium containing 10 g.L-1 of glucose and 4.25 g.L-1 of NaNO3. Results showed that the presence of oxygen favored the surfactin synthesis, resulting in higher productivity, 4.92 mg.L-1.h-1, compared to the anaerobic process that was 1.84 mg.L-1.h-1. Cultures with different carbon/nitrogen (C/N) ratio in MM medium were performed at 10 g.L-1 glucose and different concentrations of NaNO3. Among the tested conditions, the C/N 3.6 had the best result, resulting in surfactin production and productivity of 49.7 mg.L-1 and 2.49 mg L-1.h-1, respectively. Based on these results, two cultures were performed in MM medium by increasing proportionally carbon and nitrogen concentrations. Average productivity in surfactin of 1.75 mg.L-1.h-1, that was obtained in the culture containing 15 and 20 g.L-1 glucose was lower than the productivity achieved with 10 g.L-1 of glucose, showing that there was a negative effect of increasing concentrations. It also evaluated the productivity of surfactin whenB. subtilis was grown on mineral medium supplemented with micronutrients (MM1) and added micronutrients and supplemented with 1 g.L-1 yeast extract (MM2) plus 20 g.L-1 glucose. The highest production of surfactin of 101.8 mg.L-1 and the highest productivity of 3.39 mg.L-1.h-1 were obtained usingMM2 medium. When it was evaluate the influence of the proportional increase of carbon and nitrogen sources in MM2, results showed negative effect on the surfactin productivity. The surfactin productivity was 3.18 mg.L-1.h-1 and 1.63 mg.L-1.h-1, in cultures containing 30 and 40 g.L-1 glucose, respectively. The results showed that the greater surfactin productivity (3.39 mg.L-1.h-1) was obtained by cultivating B. subtilis ATCC 21332 in an anaerobic bioreactor using MM2 as medium containing 20 g.L-1 glucose. From the specific growth rates (µX), substrate consumption (µS) and biosurfactant production (µP) data, it can be said that the surfactin produced by Bacillus subtilis ATCC 21332 its associated with cell growth. Analysis of Fourier transform infrared spectroscopy (FTIR) confirmed that the biosurfactant produced in this work is surfactin. The semi purified surfactin was effective and efficient, since it presented a CMC of 10 mg.L-1 and was able to reduce the surface tension of water from 72.0 to 29.9 mN.m-1. The aqueous solution of surfactin showed excellent emulsifying capacity and stability of emulsions formed and was stable in the pH range between 6 and 8, salinity up to 10 % NaCl and exposure time of 140 min at 100 °C.

Enzimas industriais movimentam um mercado mundial estimado em sete bilhões de dólares anualmente. Entre essas enzimas, incluem-se as queratinases, enzimas capazes de catalisar a hidrólise de queratina e com aplicação potencial na indústria coureira. A queratinase KerS14, produzida pelo Bacillus subtilis S14 é capaz de depilar o couro sem causar danos ao colágeno. Embora tenha esta capacidade de grande interesse biotecnológico, sua baixa termoestabilidade a 50 °C é uma característica desvantajosa para a aplicação industrial da enzima. Visando aumentar a produção de queratinases pelo B. subtilis S14 e a termoestabilidade da KerS14, duas estratégias foram adotadas: mudanças nas condições de cultura do microrganismo selvagem e obtenção de enzimas mutantes recombinantes. Na primeira estratégia, B. subtilis S14 foi cultivado em meio farinha de pena 1,5% e CaCl2 1%. O sobrenadante da cultura foi caracterizado e 60% da atividade queratinolítica permaneceu depois de armazenada por nove dias a temperatura de 50 °C. Em paralelo, a ORF que codifica a KerS14 foi amplificada e clonada em vetor de expressão. Os resíduos de aminoácidos G61, S98 e P239 da KerS14 foram modificados por mutagênese sítio dirigida , as enzimas mutantes foram expressas in Escherichia coli e purificadas. A enzima recombinante (rKerS14) foi mais termoestável do que a enzima selvagem. Além disso, foi verificado que o fibrinogênio e fibrina são hidrolisados pela rKerS14, mostrando que a enzima também tem potencial para desenvolver drogas para o tratamento de doenças cardiovasculares. / Industrial enzymes have a world market of more than seven billion dollars per year. Keratinases are among these enzymes. They have a potential for use in tannery. The keratinase KerS14, produced by Bacillus subtilis S14, differ from other keratinases because it can depilate leather without damaging collagen. Despite this great biotechnological potential, its low thermal stability at 50 °C is an undesirable property for industrial application. In order to increase keratinases production by B. subtilis S14 and KerS14 thermal stability, two strategies were adopted: changes in wild–type microorganism growth conditions and producing recombinant mutant enzymes. In first strategy, B. subtilis S14 were grown in feather meal 1.5% and CaCl2 1 %. The supernatant obtained after fermentation was characterized and its keratinolytic activity remained in 60% after nine days of storage at 50 °C. In parallel, the KerS14 ORF was amplified and cloned into an expression vector. The KerS14 amino acid residues G61, S98 and P239 were modified and mutant enzymes were expressed in Escherichia coli and purified. It was verified that recombinant enzyme (rKerS14) is more thermal stable than the wild–type enzyme. In addition, it is able to hydrolyze fibrinogen and fibrin which is useful to develop drugs for the treatment of cardiovascular diseases.

Enzimas industriais movimentam um mercado mundial estimado em sete bilhões de dólares anualmente. Entre essas enzimas, incluem-se as queratinases, enzimas capazes de catalisar a hidrólise de queratina e com aplicação potencial na indústria coureira. A queratinase KerS14, produzida pelo Bacillus subtilis S14 é capaz de depilar o couro sem causar danos ao colágeno. Embora tenha esta capacidade de grande interesse biotecnológico, sua baixa termoestabilidade a 50 °C é uma característica desvantajosa para a aplicação industrial da enzima. Visando aumentar a produção de queratinases pelo B. subtilis S14 e a termoestabilidade da KerS14, duas estratégias foram adotadas: mudanças nas condições de cultura do microrganismo selvagem e obtenção de enzimas mutantes recombinantes. Na primeira estratégia, B. subtilis S14 foi cultivado em meio farinha de pena 1,5% e CaCl2 1%. O sobrenadante da cultura foi caracterizado e 60% da atividade queratinolítica permaneceu depois de armazenada por nove dias a temperatura de 50 °C. Em paralelo, a ORF que codifica a KerS14 foi amplificada e clonada em vetor de expressão. Os resíduos de aminoácidos G61, S98 e P239 da KerS14 foram modificados por mutagênese sítio dirigida , as enzimas mutantes foram expressas in Escherichia coli e purificadas. A enzima recombinante (rKerS14) foi mais termoestável do que a enzima selvagem. Além disso, foi verificado que o fibrinogênio e fibrina são hidrolisados pela rKerS14, mostrando que a enzima também tem potencial para desenvolver drogas para o tratamento de doenças cardiovasculares. / Industrial enzymes have a world market of more than seven billion dollars per year. Keratinases are among these enzymes. They have a potential for use in tannery. The keratinase KerS14, produced by Bacillus subtilis S14, differ from other keratinases because it can depilate leather without damaging collagen. Despite this great biotechnological potential, its low thermal stability at 50 °C is an undesirable property for industrial application. In order to increase keratinases production by B. subtilis S14 and KerS14 thermal stability, two strategies were adopted: changes in wild–type microorganism growth conditions and producing recombinant mutant enzymes. In first strategy, B. subtilis S14 were grown in feather meal 1.5% and CaCl2 1 %. The supernatant obtained after fermentation was characterized and its keratinolytic activity remained in 60% after nine days of storage at 50 °C. In parallel, the KerS14 ORF was amplified and cloned into an expression vector. The KerS14 amino acid residues G61, S98 and P239 were modified and mutant enzymes were expressed in Escherichia coli and purified. It was verified that recombinant enzyme (rKerS14) is more thermal stable than the wild–type enzyme. In addition, it is able to hydrolyze fibrinogen and fibrin which is useful to develop drugs for the treatment of cardiovascular diseases.