Orientador: Eleonora Cano Carmona / Banca: Rosa dos Prazeres Melo Furriel / Banca: Rubens Monti / Banca: João Atilio Jorge / Banca: Michel Brienzo / Resumo: Xilanases são enzimas que possuem uma gama de aplicações biotecnológicas, como hidrólise enzimática da biomassa lignocelulósica para produção de etanol, branqueamento de polpa de papel Kraft, na ração animal, na indústria de alimentos como aditivo para melhorar a qualidade de pães, na clarificação de sucos, entre outras. Essa variedade de aplicações deve-se a atuação das xilanases sobre os resíduos de xilose da cadeia principal do xilano, o segundo principal componente da parede celular vegetal, liberando xilooligossacarídeos. A amplitude de aplicações confere às xilanases posição de destaque econômico e grande exploração científica, principalmente na busca por xilanases estáveis ou tolerantes a condições físico-químicas mais extremas de temperatura, pH e salinidade. Este estudo mostra, pela primeira vez, a produção, purificação e aplicação de xilanases de Aspergillus hortae, um fungo isolado de lagoas salinas da região do Pantanal do Mato Grosso do Sul, Brasil. A produção de xilanases foi realizada utilizando sabugo de milho como substrato. Após a realização de um planejamento experimental, as condições ótimas de cultura foram pH 5,36 e 35 ºC. As xilanases caracterizadas no filtrado de cultura apresentaram pH e temperatura ótimos de 6,5 e 55 ºC bem como estabilidade em pH 3,0 a 8,0 e cerca de 70% da atividade ainda estava presente após 5 h de incubação do filtrado a 50 ºC. A aplicação do filtrado de cultura na produção de pães, resultou na redução da firmeza do miolo, provocando um efeito anti-endurecimento, o que é um fator positivo quando se considera a vida de prateleira do pão. O filtrado de cultura foi submetido aos testes de purificação em trocador catiônico e aniônico bem como cromatografias de exclusão molecular, resultando na purificação de três xilanases, ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Xylanases are enzymes that have a range of biotechnological applications, such as enzymatic hydrolysis of lignocellulosic biomass for ethanol production, bleaching of Kraft paper pulp, in animal feed, in the food industry as an additive to improve the quality of breads, clarification of juices, among others. This variety of applications is due to the action of xylanases on the xylose residues of the xylan main chain, the second main component of the plant cell wall, releasing xylooligosaccharides. The range of applications gives xylanases an outstanding economic position and great scientific exploration, mainly in the search for stable or tolerant xylanases to extreme physicochemical conditions, such as high temperature, pH or salinity. This study shows, for the first time, the production, purification and application of xylanases of Aspergillus hortae, a fungus isolated from saline lagoons in the Pantanal region of Mato Grosso do Sul, Brazil. The xylanases production was carried out using corn cobs as a substrate. After an experimental design, optimum culture conditions were pH 5.36 and 35 ºC. The xylanases characterized in the culture filtrate presented optimum pH and temperature of 6.5 and 55 ºC as well as stability at pH 3.0 to 8.0 and almost 70% of the activity was still present after 5 h incubation of the filtrate at 50 °C. The application of the culture filtrate in the bread production resulted in a reduction of the firmness of the crumb, causing an anti-stalling effect, which is a positive factor when considering the shelf life of the bread. The culture filtrate produced under the conditions described above was subjected to purification tests, which included chromatographic steps in cationic and anionic exchangers as well as exclusion chromatography, resulting in the purification of three xylanases, Xyl I, Xyl II and Xyl III, ... (Complete abstract click electronic access below) / Doutor

This study investigates the postlarval development of Penaeus indicus. Particular emphasis is given to characterisation of developmental morphology, growth, ontogenetic change in digestive enzymes, and assessment of energy requirements for postlarval substages. The morphology of the Penaeus indicus postlarvae (PL) changes continuously as consecutive substages (PL1-14) were reached by daily moults. After 22 ecdyses (typically 35 days) the PL22 substage is succeeded by the adult form. Most of the morphological differentiation is observed after 2 weeks at substage PL14, but the branchiae only reach full development from substage PL16. The rostrum teeth, telson spines and ratio of body segments are important characters for identification of Penaeus indicus PL stage. Growth of PL1 postlarvae was significantly slower when fed artificial diets rather than Artemia nauplii. Similarly 14-day postlarvae which were slow developers (PL9) also grew slowly on artificial diets whereas postlarvae of the same age (substage PL14) did not show this suppression. Trypsin and amylase digestive activities increased with PL development (P<0.001) but did not change significantly (P>0.05) with diet fed. Trypsin was low during early PL stages of development and a sharp increase in tryptic activity was only observed at substage PL20 (24 mm). Amylase increased from PL1 and exceeded that of trypsin for 2-3 weeks after metamorphosis. It appears that during early stages of development postlarvae are unable to efficiently digest artificial diets due to low digestive activity. For smaller 14-day postlarvae, poor performance is possible related to a genetic regulated constraint and not to digestive capacity since enzyme levels were similar to those in larger PL14. PL1 fed for 15 days on commercially dried low-hatch decapsulated Artemia cysts showed comparable growth and survival to that of PLs fed on Artemia nauplii, but significantly higher (P<0.05) than that supported by commercial granulated and flake diets and low-hatch decapsulated cysts processed into a granulated diet or dried at 90°C. Leaching of soluble protein and carbohydrates was high for all artificial diets but low-hatch decapsulated cysts were highly stable in water. Commercially dried low-hatch cysts retain a living membrane capable of osmoregulation and retaining highly digestible nutrients important for fast growth and development of postlarvae. Survival of postlarvae was negatively correlated (P<0.05) with leaching of soluble protein, but no correlation was observed for loss of soluble carbohydrates. Daily food ingestion and routine metabolism of postlarvae increased with PL development. Food metabolism (SDA) was low for early PL stages, but increased steadily up to stage PL16 and remained the same from this substage onwards. Assimilation efficiency decreased for early PL substages and remained low up to PL13, and then increased steadily. Different energy strategies seem to be adopted during postlarval development to cope with ontogenetic modifications after metamorphosis. During early development little energy is lost in metabolism, and so more energy is converted to growth to support fast development, with increase in predatory behaviour and development of digestive system. Later more energy is lost in metabolism and committed for maintenance. The ontogenetic changes in digestive activity, energy trend and assimilation efficiency latter in PL development seems to reflect the adaptation to benthic carnivorous existence and migration of postlarvae and juveniles form inshore nursery to deeper waters. Stocking density above 20 PLs 1- reduced growth and survival but increased size variability above the inherent range, for postlarvae PL1-18 days old. However, Penaeus indicus postlarvae showed low agonistic behaviour and tolerated relatively high densities similar to that of other penaeid species, which further enhances the potential and advantages of the white prawn for culture.

Field and laboratory studies on the morphology, gut contents, ingestion rates and digestion efficiency of Pollicipes pollicipes were combined to obtain estimates of the likely range and quality of materials required to sustain this species . Orientation of P. pollicipes on the shores of south; west Portugal appeared to be determined by microtopography. Animals generally faced into the wave backwash. Orientation could be temporarily altered by torsion of the peduncle in response to changes in flow direction, permitting more efficient filter feeding. Cirral and mouthpart morphology suggested that P. pollicipes, Capitulum mitella and Lepas anatifera were omnivores. Size-related changes in cirral morphology made small juveniles better equipped than adults to feed on small particles. Cirral activity of P. pollicipes was investigated. Very slow rhythmic cirral extension (or 'beating') was observed in all P. pollicipes, J but only in relatively still water; once flow rates exceeded 14 cm s all barnacles exhibited prolonged cirral extension. The 'beat' rate was temperature dependent in most animals and larger animals exhibited a lower extension frequency than juveniles. It was concluded that 'beating' was primarily respiratory in function and not a feeding mechanism. The gut contents of wild P. pollicipes included animal and algal material but little inorganic matter, -.,. Small organic material predominated in small juveniles while large organic material predominated in adults. The rates of faecal production and growth were much higher in barnacles feeding on zooplankton than on algae and although algal cells were ingested in high numbers, the energy intake was so low that animals barely maintained their body weight. Digestive efficiencies varied with diet but little with barnacle size. A wide range of digestive enzymes were identified in P. pollicipes and L. anatifera suggesting that a variety of foods may be digested. Specific enzyme activity was low, characteristic of more carnivorous animals.

No description available.

Made available in DSpace on 2016-02-05T18:29:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-11-07. Added 1 bitstream(s) on 2016-02-05T18:33:55Z : No. of bitstreams: 1 000856555_20161231.pdf: 775185 bytes, checksum: 33a10066608b881bcb1a54b01ef02bf3 (MD5) Bitstreams deleted on 2017-01-02T15:03:52Z: 000856555_20161231.pdf,. Added 1 bitstream(s) on 2017-01-02T15:05:09Z : No. of bitstreams: 1 000856555.pdf: 4072099 bytes, checksum: bf31ef2e26f6adc6f49a96b1e2bbecfd (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O presente estudo foi desenvolvido sobre aplicações tecnológicas da dextrana produzida pela bactéria Leuconostoc mesenteroides FT045B. Para que fosse possível trabalhar com a enzima, desenvolveu-se metolodogia para purificação com polietilenoglicol (PEG), seguida de filtração em gel cromatográfico. Após análise por SDS-PAGE, foi constatado que a enzima dextranasacarase FT045B apresenta massa molar de aproximadamente 136 kDa. Resultados obtidos pela análise de variância (ANOVA) mostraram que o experimento com 10% de PEG 1500 e 20% de PEG 4000 apresentou melhor purificação da enzima dextranasacarase. Foi confirmado que a enzima dextranasacarase purificada é uma glicosiltransferase e não uma frutosiltransferase, pela análise de atividade em gel de acrilamida sob condições não denaturantes. O ácido ascórbico α-glicosídeo foi sintetizado, pela reação de transglicosilação catalisada pela dextranasacarase previamente purificada, e estruturalmente caracterizado após purificação, por espectrofotômetro de massas. Também com a dextranasacarase purificada, foi realizado um delineamento composto central para otimizar as condições de temperatura, concentração de sacarose e atividade enzimática para a produção de dextrana de massa molar controlada, sendo que o ponto ótimo obtido foi estabelecido em 4,27% de Tween 80 e 1,83g de agitação. O composto, Bromo-dextrana, foi sintetizado pela reação de acetobromação de dextrana, seguido de purificação com etanol, secagem e identificação / Development on technological applications of dextran produced by the bacterium Leuconostoc mesenteroides FT045B. Enzyme dextransucrase was purified with polyethyleneglycol (PEG), followed by gel filtration chromatography. After analysis by SDS-PAGE revealed that the enzyme presents dextransucrase FT045B molar mass of approximately 136 kDa. Results obtained by analysis of variance (ANOVA) showed that the experiment with 10% PEG 1500 and 20% PEG 4000 showed better dextransucarase enzyme purification. It was confirmed that the purified enzyme is a glycosyltransferase, but not a fructosyltransferase by activity on acrylamide gel under non denaturants conditions. The α-glycoside ascorbic acid was synthesized by transglycosylation reaction catalyzed by dextransucarase previously purified, and structurally characterized after purification by mass spectrometer ESI-MS. A central compositio design was also purified with dextransucarase performed to optimize the conditions of temperature, concentration and enzyme activity of sucrose to produce dextran of molecular weight controlled, and the optimal point obtained was set at 4.27% Tween 80 and 1,83g of agitation. The compound Bromo-dextran was synthesized by the reaction of acetylation followed by bromation of dextran, and purification with ethanol, drying and identification

Made available in DSpace on 2016-02-05T18:30:04Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-04-11. Added 1 bitstream(s) on 2016-02-05T18:34:10Z : No. of bitstreams: 1 000857393.pdf: 1233316 bytes, checksum: df86cf7d2b7b3a47fa18b9bd274e26e2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / As amilases pertencem à classe das hidrolases e são enzimas de grande importância para aplicações industriais representando de 25 a 33% do mercado mundial de enzimas. Há uma gama extensiva de aplicações e um enorme interesse na descoberta de enzimas com melhores propriedades na degradação do amido. O presente trabalho visou otimizar a tecnologia de produção de amilases por Rhizopus microsporus var. oligosporus em fermentação em estado sólido com farelo de trigo. Em uma segunda etapa o extrato enzimático foi parcialmente caracterizado e utilizado para produção de xarope de glicose. Foi determinada a concentração ideal de sais para suplementação do meio de cultivo, a influência da umidade e inóculo na produção amilolítica utilizando-se um delineamento central composto rotacional (DCCR). Estabeleceu-se o protocolo para extração enzimática e verificou-se a produção ao longo do tempo em diferentes temperaturas de fermentação. A atividade enzimática foi determinada a partir da quantificação dos açúcares redutores liberados durante reação de hidrólise de amido. A menor concentração otimizada foi de 1,25% (m/m) de sulfato de amônio, 0,25% de fosfato de potássio e 1,25% de ureia. O melhor teor de umidade para a produção amilolítica foi de 50% e a extração mais eficiente ocorreu com 10 mL de água destilada por grama de substrato. A máxima produção enzimática ocorreu nos cultivos a 30 °C durante 120 h. A melhor temperatura para atividade amilolítica foi de 60 a 65 °C. A melhor estabilidade térmica (80%) foi obtida até 50 °C por 1 hora de incubação. A enzima produzida apresentou maior atividade em uma faixa de pH entre 4,0 e 5,0. A estabilidade foi superior a 80% após 1 hora de contato em pH de 3,0 a 7,0. O extrato enzimático bruto foi capaz de realizar a conversão total de amido em açúcar redutor após 6 horas de reação a 55 °C. Para a farinha de trigo tipo II foi obtido um rendimento... / The amylases belong to the class of the hydrolases and they are enzymes of great importance to industrial applications, representing from 25% to 33% of the worldwide enzymes business. There is a wide range of applications and a huge interest in the discovery of enzymes with better properties in the starch degradation. The present work aims to optimize the technology of production of amylases by Rhizopus microsporus var. oligosporus in solid state fermentation with wheat bran. In second stage the enzymatic extract was partially characterized and used in glucose syrup production. The ideal concentration of salts to supplement the culture medium was determined, as well as the influence of moisture and inoculum on amylase production using a central composite rotational design (DCCR). The best protocol for enzymatic extraction and profiles of amylase production at different temperatures of fermentation were determined. The enzymatic activity was measured by the quantification of reducing sugars released during hydrolysis reaction of starch. The lowest optimal concentration was 1.25% (w/w) ammonium sulfate, 0.25% potassium phosphate and 1.25% urea. The best moisture level for amylolytic production was 50%, and the most efficient extraction was with 10 ml of distilled water per gram of substrate. The maximum enzyme production in the cultures was at 30 °C for 120 h. The best temperature for amylolytic activity was 60 to 65 °C, and better stability (80%) was obtained up to 50 °C for 1 hour of incubation. The enzyme produced showed greater activity in a pH range 4.0-5.0, and more than 80% of stability after 1 hour of contact in a pH range 3.0-7.0. The crude enzymatic extract was able to perform the total conversion of starch to reducing sugar in 6 hours of reaction at 55 °C. For type II wheat flour was obtained a yield of 83% in 6.5 hours, and for wheat bran, 62% in 7 hours. The product of the catalytic reaction was concentrated to obtain 20% ...

Made available in DSpace on 2014-11-10T11:09:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-05-28Bitstream added on 2014-11-10T11:58:42Z : No. of bitstreams: 1 000789693.pdf: 3140266 bytes, checksum: 8020e0cbe99572a2e4a68115eacf1d3e (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Lipases são hidrolases (triacilglicerol acil hidrolases, EC 3.1.1.3) capazes de hidrolisar ésteres carboxílicos de cadeia longa liberando monoacilgliceróis, diacilgliceróis, ácidos graxos e glicerol. Em adição, as lipases são eficientes em reações de síntese como esterificação, glicerólise, aminólise e transesterificação, essa capacidade relacionada ao deslocamento do equilíbrio da reação no sentido de hidrólise ou síntese dependendo da concentração de água presente no meio, característica que as tornam biocatalizadores muito versáteis, com importantes aplicações biotecnológicas, como em indústrias de alimentos, detergentes, farmacêutica, couro, têxtil, cosméticos e papel. O presente trabalho objetivou identificar as seis espécies de fungos lipolíticos, otimizar a produção dessa enzima pela linhagem selecionada, bem como purificar e caracterizar parcialmente a lipase produzida em condição otimizada. A atividade lipase foi determinada após cultivos submersos, objetivando o aumento da produção da enzima. Os fatores avaliados no processo de otimização da produção de lipase foram: meios minerais, tempo de cultivo, fontes de carbono e nitrogênio e suas concentrações, pH e inóculo. A avaliação das melhores condições de cultivo foi realizada pela atividade lipase, determinada pelo íon ρ-nitrofenol (pNP) liberado na hidrólise do substrato sintético ρ-nitrofenil palmitato a 37 °C. O uso de marcadores moleculares permitiu identificar os seis fungos lipolíticos até espécie, exceto para a linhagem Fusarium sp. CBMAI 1227. A metodologia de triagem dos fungos para produção de lipase em meio neutro permitiu a seleção do isolado Trichodemra harzianum CBMAI 1229 como melhor produtor. A melhor condição para produção de lipase (231,58 ± 35,59 U mL-1) foi alcançada após 120 horas de cultivo a 25 °C e 150 rpm de agitação utilizando meio mineral Olson e Johnson (1948), suplementado com óleo... / Lipases are hydrolases (triacylglycerol acylhydrolases EC 3.1.1.3) capable of hydrolyzing carboxyl esters of long-chain acylglycerol release mono- or diacyglycerol, free glycerol and fatty acid. In addition, the lipases are effective for synthesis reactions such as esterification, glycerolysis, transesterification and aminolysis, this capability related to the displacement of the equilibrium of the reaction towards synthesis or hydrolysis inherent in the concentration of water present in the medium, the characteristic that makes biocatalysts very versatile, with important biotechnological applications. Such as food, detergents, pharmaceutical, textile, leather, paper and cosmetics industries. This work aimed to identify six species of fungi that produce lipase, optimize the production of this enzyme by lineage selected and purify and characterize partially the lipase produced in optimal condition. Enzyme activity was determined after submerged cultures, aiming to increase enzyme production. The factors measured in this optimization process of lipase production were: mineral components, time of cultivation, source of carbon and nitrogen and their concentration, pH and inoculum. The use of molecular markers allowed us to identify the six lipolytic fungi until species, except for strain Fusarium sp. CBMAI 1227. The methodology of screening of fungi for the production of lipase in neutral medium allowed the selection of the isolated Trichodemra harzianum CBMAI 1229 as best producer. The evaluation of improved cultivation conditions was assayed by lipase activity, determined through hydrolysis releasing íon ρ-nitrofenol (pNP) of ρ-nitrophenyl-palmitate (pNPP) as substrate synthetic at 37 °C. The best condition for lipase production (231,58 ± 35,59 U mL-1) was reached after 120 hours of cultivation, 25 °C and 150 rpm in medium mineral Olson & Johnson (1948), suplemented with sunflower oil 3,0% (v/v), peptone 2,0% (w/v), yeast extract 0,2%...

Orientador: João Martins Pizauro Junior / Coorientador: Marta Verardino de Stéfani / Banca: Pietro Ciancaglini / Banca: Cyntia Peralta de Almeida Prado / Resumo: Todos os seres vivos necessitam de um suprimento energético, que deve ser obtido de fontes externas. As substâncias simples da dieta dos animas, como a água, os sais minerais e as vitaminas (exceto a vitamina B12), podem ser absorvidas ao longo do trato digestório sem sofrer transformações físicas ou químicas. Entretanto, macromoléculas tais como carboidratos, proteínas e lipídeos, têm de ser convertidas em moléculas pequenas e menos complexas para serem absorvidas. A digestão química dos nutrientes é realizada por hidrólise enzimática, que consiste na cisão de uma ligação química e inserção de uma molécula de água, sendo este processo catalisado pela ação das enzimas digestivas. O objetivo deste estudo foi o de se analisar a variação nas atividades de cinco hidrolases do sistema digestório de rã-touro (Lithobates catesbeianus) durante o desenvolvimento larval, para fornecer subsídios a nutrição animal. Os girinos foram mantidos em aquários, à 27ºC e separados por estádios de desenvolvimento. Amostras de estômago, pâncreas e intestino foram retiradas de 100 animais de cada estádio, congeladas em nitrogênio liquido e armazenadas a -70ºC para posterior homogeneização e determinação da atividade enzimática. A atividade das enzimas tripsina, -amilase e lipase pancreáticas (44,30 U.mg-1, 3,17 U.mg-1, 2,99 U.mg-1 respectivamente), e da maltase intestinal (26,99 U.mg-1), foram detectadas desde o início da fase de alimentação (estádio 26) enquanto que a atividade de catepsina-E gástrica (13,82 U.mg-1), foi estudada a partir do estádio 28, devido a dificuldade de se obter de estômagos antes deste estádio. Durante a pré-metamorfose a catepsina-E não apresentou grande variação em sua atividade, sendo observado aumento ao final do período da prómetamorfose. A atividade das demais... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: All living beings require energy supply, wich must be obtained from external sources. Simple substances in the diet of the animals, such as water, minerals and vitamins (except vitamin B12) can be absorbed along the digestive tract without undergoing chemical or physical changes. However, macromolecules such as carbohydrates, proteins and lipids, must be converted into smaller and less complex molecules to be absorbed. The chemical digestion of nutrients is accomplished through enzymatic hydrolysis, which means to break a chemical bond and inserti a water molecule. This process is catalysed by the action of digestive enzymes. The aim of this study was to analyze the activity variation of five hydrolases of the bullfrog's (Lithobates catesbeianus) digestive system during larval development, to provide subsidies to animal nutrition. The tadpoles were kept in aquaria, at 27 °C and separated by stages of development. Samples of stomach, pancreas and intestine were taken from 100 animals of each stage, frozen in liquid nitrogen and stored at -70ºC for later homogenization and enzyme activity determination. The activity of trypsin, -amylase and pancreatic lipase (44.30 U.mg-1, 3.17 U.mg-1, 2.99 U.mg-1 respectively), and intestinal maltase (26.99 U.mg-1) were detected since the start of feeding (stage 26) while the gastric cathepsin E (13.82 U.mg-1) activity was studied since stage 28, due to difficulty in obtaining stomachs before this stage. During pre-metamorphosis cathepsin-E did not show great variation in its activity, but an increase was observed at the end of the period of prometamorphosis. The other enzymes activity increased significantly in periods that precede metamorphosis, with the highest activities in stage 38 (261.97 U.mg-1 for trypsin, 28.19 U.mg-1 for -amylase and... (Complete abstract click electronic access below) / Mestre

Orientador: Lara Durães Sette / Coorientador: Eleonora Cano Carmona / Banca: Rubens Monti / Banca: Luis Henrique Souza Guimarães / Resumo: Lipases são hidrolases (triacilglicerol acil hidrolases, EC 3.1.1.3) capazes de hidrolisar ésteres carboxílicos de cadeia longa liberando monoacilgliceróis, diacilgliceróis, ácidos graxos e glicerol. Em adição, as lipases são eficientes em reações de síntese como esterificação, glicerólise, aminólise e transesterificação, essa capacidade relacionada ao deslocamento do equilíbrio da reação no sentido de hidrólise ou síntese dependendo da concentração de água presente no meio, característica que as tornam biocatalizadores muito versáteis, com importantes aplicações biotecnológicas, como em indústrias de alimentos, detergentes, farmacêutica, couro, têxtil, cosméticos e papel. O presente trabalho objetivou identificar as seis espécies de fungos lipolíticos, otimizar a produção dessa enzima pela linhagem selecionada, bem como purificar e caracterizar parcialmente a lipase produzida em condição otimizada. A atividade lipase foi determinada após cultivos submersos, objetivando o aumento da produção da enzima. Os fatores avaliados no processo de otimização da produção de lipase foram: meios minerais, tempo de cultivo, fontes de carbono e nitrogênio e suas concentrações, pH e inóculo. A avaliação das melhores condições de cultivo foi realizada pela atividade lipase, determinada pelo íon ρ-nitrofenol (pNP) liberado na hidrólise do substrato sintético ρ-nitrofenil palmitato a 37 °C. O uso de marcadores moleculares permitiu identificar os seis fungos lipolíticos até espécie, exceto para a linhagem Fusarium sp. CBMAI 1227. A metodologia de triagem dos fungos para produção de lipase em meio neutro permitiu a seleção do isolado Trichodemra harzianum CBMAI 1229 como melhor produtor. A melhor condição para produção de lipase (231,58 ± 35,59 U mL-1) foi alcançada após 120 horas de cultivo a 25 °C e 150 rpm de agitação utilizando meio mineral Olson e Johnson (1948), suplementado com óleo... / Abstract: Lipases are hydrolases (triacylglycerol acylhydrolases EC 3.1.1.3) capable of hydrolyzing carboxyl esters of long-chain acylglycerol release mono- or diacyglycerol, free glycerol and fatty acid. In addition, the lipases are effective for synthesis reactions such as esterification, glycerolysis, transesterification and aminolysis, this capability related to the displacement of the equilibrium of the reaction towards synthesis or hydrolysis inherent in the concentration of water present in the medium, the characteristic that makes biocatalysts very versatile, with important biotechnological applications. Such as food, detergents, pharmaceutical, textile, leather, paper and cosmetics industries. This work aimed to identify six species of fungi that produce lipase, optimize the production of this enzyme by lineage selected and purify and characterize partially the lipase produced in optimal condition. Enzyme activity was determined after submerged cultures, aiming to increase enzyme production. The factors measured in this optimization process of lipase production were: mineral components, time of cultivation, source of carbon and nitrogen and their concentration, pH and inoculum. The use of molecular markers allowed us to identify the six lipolytic fungi until species, except for strain Fusarium sp. CBMAI 1227. The methodology of screening of fungi for the production of lipase in neutral medium allowed the selection of the isolated Trichodemra harzianum CBMAI 1229 as best producer. The evaluation of improved cultivation conditions was assayed by lipase activity, determined through hydrolysis releasing íon ρ-nitrofenol (pNP) of ρ-nitrophenyl-palmitate (pNPP) as substrate synthetic at 37 °C. The best condition for lipase production (231,58 ± 35,59 U mL-1) was reached after 120 hours of cultivation, 25 °C and 150 rpm in medium mineral Olson & Johnson (1948), suplemented with sunflower oil 3,0% (v/v), peptone 2,0% (w/v), yeast extract 0,2%... / Mestre

Orientador: Jonas Contiero / Banca: Rubens Monti / Banca: Heloiza Ferreira Alves do Prado / Banca: Sandra Helena da Cruz / Banca: Eliana Setsuko Kamimura / Resumo: O presente estudo foi desenvolvido sobre aplicações tecnológicas da dextrana produzida pela bactéria Leuconostoc mesenteroides FT045B. Para que fosse possível trabalhar com a enzima, desenvolveu-se metolodogia para purificação com polietilenoglicol (PEG), seguida de filtração em gel cromatográfico. Após análise por SDS-PAGE, foi constatado que a enzima dextranasacarase FT045B apresenta massa molar de aproximadamente 136 kDa. Resultados obtidos pela análise de variância (ANOVA) mostraram que o experimento com 10% de PEG 1500 e 20% de PEG 4000 apresentou melhor purificação da enzima dextranasacarase. Foi confirmado que a enzima dextranasacarase purificada é uma glicosiltransferase e não uma frutosiltransferase, pela análise de atividade em gel de acrilamida sob condições não denaturantes. O ácido ascórbico α-glicosídeo foi sintetizado, pela reação de transglicosilação catalisada pela dextranasacarase previamente purificada, e estruturalmente caracterizado após purificação, por espectrofotômetro de massas. Também com a dextranasacarase purificada, foi realizado um delineamento composto central para otimizar as condições de temperatura, concentração de sacarose e atividade enzimática para a produção de dextrana de massa molar controlada, sendo que o ponto ótimo obtido foi estabelecido em 4,27% de Tween 80 e 1,83g de agitação. O composto, Bromo-dextrana, foi sintetizado pela reação de acetobromação de dextrana, seguido de purificação com etanol, secagem e identificação / Abstract: Development on technological applications of dextran produced by the bacterium Leuconostoc mesenteroides FT045B. Enzyme dextransucrase was purified with polyethyleneglycol (PEG), followed by gel filtration chromatography. After analysis by SDS-PAGE revealed that the enzyme presents dextransucrase FT045B molar mass of approximately 136 kDa. Results obtained by analysis of variance (ANOVA) showed that the experiment with 10% PEG 1500 and 20% PEG 4000 showed better dextransucarase enzyme purification. It was confirmed that the purified enzyme is a glycosyltransferase, but not a fructosyltransferase by activity on acrylamide gel under non denaturants conditions. The α-glycoside ascorbic acid was synthesized by transglycosylation reaction catalyzed by dextransucarase previously purified, and structurally characterized after purification by mass spectrometer ESI-MS. A central compositio design was also purified with dextransucarase performed to optimize the conditions of temperature, concentration and enzyme activity of sucrose to produce dextran of molecular weight controlled, and the optimal point obtained was set at 4.27% Tween 80 and 1,83g of agitation. The compound Bromo-dextran was synthesized by the reaction of acetylation followed by bromation of dextran, and purification with ethanol, drying and identification / Doutor