Orientador: Adriana Zerlotti Mercadante / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-16T15:01:11Z (GMT). No. of bitstreams: 1 Mandelli_Fernanda_M.pdf: 677502 bytes, checksum: 40f71e5be548712e1d9c40d24476ba1b (MD5) Previous issue date: 2010 / Resumo: Os carotenoides pertencem a um grupo de pigmentos naturais amplamente distribuídos na natureza, com grande diversidade de estruturas e funções. O interesse pelos carotenoides tem aumentado consideravelmente nos últimos anos pela evidência dos seus já conhecidos benefícios à saúde humana, devido às suas propriedades antioxidantes, anticarcinogênicas e imunomodulatórias. As principais fontes industriais de carotenoides são a síntese química e a extração a partir de plantas. Porém, são poucos os carotenoides que possuem métodos economicamente eficientes de produção. Uma alternativa para a obtenção natural destes pigmentos é a sua produção por micro-organismos, sendo uma das fontes as bactérias termófilas que, em sua maioria, possuem carotenoides com estrutura específica para fortalecimento da membrana celular. Assim sendo, o presente estudo tem como objetivos: identificar os carotenoides da linhagem Thermus filiformis ATCC 43280, avaliar o crescimento de massa celular, produção de carotenóides e porcentagem de proteção contra o oxigênio singlete através de diferentes condições de cultivo e verificar a atividade antioxidante do extrato rico em carotenoides na desativação do radical ABTS¿+ (2,2¿- azinobis-(3-etilbenzotiazolina-6-ácido sulfônico)) e do peróxido de hidrogênio. Para atingir os objetivos, os carotenoides foram caracterizados por cromatografia líquida de alta eficiência conectada aos detectores de arranjo de diodos e espectrômetro de massas (HPLCDAD-MS/MS), e as condições de cultivo e a interação entre as variáveis foram estudadas através de planejamento estatístico fatorial. Foram identificados nove carotenoides sintetizados pela bactéria Thermus filiformis, todos apresentando o mesmo comprimento de onda máximo a 449 ± 1 nm, que é um indicativo de um cromóforo de 11 ligações duplas conjugadas, sendo duas em anel ?. O carotenoide majoritário identificado em T. filiformis foi termozeaxantina-15 que consiste do carotenoide zeaxantina ligada a uma glicose acilada com ácido graxo ramificado de 15 carbonos ((3R,3'R)-3'-[(6-O-13-metiltetradecanoil-ß-Dglucopiranosil) oxi]-ß,ß-caroten-3-ol) o que representa 35,3 % do conteúdo total de carotenóides. Foram também identificados os carotenoides: zeaxantina (14,4 %), zeaxantina monoglicosilada (4,7 %) e termozeaxantinas com ácidos graxos ramificados, com cadeia de cadeia de 11 (2,3 %), 12 (5,2 %), 13 (25,4 %) e 14 (9,0 %) carbonos, além de derivados dos carotenoides termozeaxantina-13 (0,8 %) e termozeaxantina-15 (2,8 %). Dentre as variáveis estudadas no planejamento experimental (temperatura, pH, quantidade de triptona, extrato de levedura e elementos traços de Nitsch¿s), a temperatura foi a que mais influenciou a quantidade de biomassa, carotenoides totais e proteção proporcionada pelos carotenoides contra oxigênio singlete. Com a utilização desta ferramenta foi possível atingir valores de 658 mg/L de biomassa seca, 1517 µg/g de carotenoides totais e porcentagem de proteção conferida ao DMA (dimetilantraceno) frente a oxidação por oxigênio singlete na ordem de 85 %. O extrato rico em carotenoides de T. filfiormis apresentou capacidade de desativar o radical ABTS¿+ com valor de capacidade antioxidante equivalente a Trolox (TEAC) de 2,87, valor este superior aos relatados para outros carotenoides como ß-caroteno (1,9) e zeaxantina (1,4). / Abstract: Carotenoids are a group of natural pigments widely distributed in the nature with great diversity of structures and functions. The interest on carotenoids has considerably increased in recent years by the evidence of its known benefits to human health, due to its antioxidant, anticarcinogenic and immunomodulatory properties. The main industrial sources of carotenoids are chemical synthesis and extraction from plants. However, there are few carotenoids which production is economically feasible. An alternative to obtain these natural pigments is their production by microorganisms, and one of the sources is the thermophilic bacterium that produces carotenoids with specific structure for the strength of cell membrane. Therefore the aims of this work were to evaluate the biomass, carotenoid production and quenching of singlet oxygen by the strain Thermus filiformis ATCC 43280 through different cultivation conditions and to verify the antioxidant activity by scavenging of the ABTS¿+ (2,2'-azino-bis(3-ethylbenzthiazoline-6-Sulfonic Acid)) radical and hydroxy peroxide deactivation. In order to achieve these aims, the carotenoids were characterized by high-performance liquid chromatography connected to a photodiode array and mass spectrometry detectors (HPLC-DAD-MS/MS), and the cultivation conditions and the interactions between the variables were studied using a fractionated factorial design. Nine carotenoids synthesized by Thermus filiformis, were identified, all showing the same maximum absorbance wavelength, indicating the presence of a chromophore of 11 conjugated double bounds, being two in the ?-ring. The major carotenoid identified in T. filiformis was thermozeaxanthin-15 which is the carotenoid zeaxanthin linked to a glucose, acylated with a branched fatty acid of 15 carbons ((3R, 3'R) -3'- [(6-O-13- methyltetradecanoyl-ß-D-gluycopiranosyl) oxy]-ß,ß-carotene-3-ol) what represents 35.3 % of the total content of carotenoides. Zeaxanthin (14.4 %), zeaxanthin monoglucoside (4.7 %) and thermozeaxanthins with branched fatty acids chain with 11 (2.3 %), 12 (5.2 %), 13 (25.4 %) and 14 (90 %) carbons, besides thermozeaxanthin-13 (0,8 %) and thermozeaxanthina-15 (2,8 %) derivatives were also identified. Among the studied variables in the experimental design (temperature, pH, tryptone, yeast extract and Nitsch¿s trace elements) the temperature was the one that most influenced the biomass amount, total carotenoids and percentage of protection against singlet oxygen. Using this tool, it was possible to achieve values of 658 mg/L of dry biomass, 1517 µg/g of total carotenoids and percentage of protection of DMA (dimethylanthracene) against the oxidation by singlet oxygen about 85 %. The extract rich in carotenoids from T. filiformis showed capacity of scavenging ABTS¿+ radical with Trolox equivalent antioxidant capacity (TEAC) of 2.87, being this value higher than those reported in the literature for ß-carotene (1.9) and zeaxanthin (1.4) / Mestrado / Mestre em Ciência de Alimentos

Group II Introns are small segments of DNA that reside in the chromosome of bacteria or the organelles of primitive eukaryotes. These elements have some very interesting properties. First, they are retrotransposons that can move from one location to a new location in DNA via a reverse transcription mechanism. Second, they form a large ribozyme that mediates self-splicing of the intron from pre-mRNA. A Group II Intron type protein with similarity to reverse transcriptase was discovered in the thermophilic bacterium Geobacillus stearothermophilus strain 10 (Vellore et al., 2004, Appl. Environ. Microbiol. 70: 7140-7147). Numerous copies of the intron, designated Gs. Int1, are present in the chromosome of strain 10 but absent from a related strain ATCC 12980. Experiments to detect the in vivo splicing of intron Gs.Int1 from G. stearothermophilus cells did not work. Plasmids to that will over-express the Gs. Int1 intron to detest splicing in vivo in Escherichia coli have been constructed.

Thesis (Ph. D. (Microbiology)) -- University of Limpopo, 2020 / The use of lignocellulosic biomass for bioethanol production is largely dependent on cost effective production of cellulase enzymes and most importantly, the availability of cellulases with sufficient β-glucosidase activity for complete hydrolysis of cellulose to glucose. Commercial cellulase preparations are often inefficient in the complete hydrolysis of cellulose to glucose. The addition of β-glucosidases to commercial cellulase preparations may enhance cellulolytic activity in the saccharification of cellulose to fermentable sugars. A β-glucosidase producing thermophilic bacterium, Anoxybacillus sp. KTC2 was isolated from a hot geyser in the Zambezi Valley, Zimbabwe. The bacterium identified through biochemical tests and 16S rDNA sequencing, had an optimal growth temperature and pH of 60˚C and pH 8, respectively. The β-glucosidase enzyme had an optimal temperature of 60˚C and a broad pH range for activity, between 4.5 and 7.5 with an optimum at pH 7. The β-glucosidase enzyme retained almost 100% activity after 24 hours’ incubation at 50˚C. The Anoxybacillus sp. KTC2 β-glucosidase was partially purified and a partial amino acid sequence obtained through MALDI-TOF analysis. The whole genome of Anoxybacillus sp KTC2 β-glucosidase was sequenced and a β-glucosidase gene identified. The deduced amino acid sequence corresponded to the peptide sequences obtained through MALDI-TOF, confirming the presence of the a β glucosidase on the genome of Anoxybacillus sp KTC2. Analysis of the deduced amino acid sequence revealed that the β-glucosidase enzyme belongs to the GH family 1. The β-glucosidase gene was isolated by PCR and successfully cloned into an E. coli expression system. The saccharification efficiency of the β-glucosidase enzyme was evaluated through the creation of enzyme cocktails with the commercial cellulase preparation, CelluclastTM. CelluclastTM with the Anoxybacillus sp KTC2 β-glucosidase were used to hydrolyse pure Avicel cellulose, at 50˚C over a 96 hour reaction time. The Anoxybacillus sp KTC2 β-glucosidase enabled a 25% decrease in the total cellulose loading without a decrease in the amount of glucose released. / University of Limpopo staff development programme and VLIR