Orientador: Helia Harumi Sato / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-05T16:51:34Z (GMT). No. of bitstreams: 1 Guimaraes_DanielaPaiva_M.pdf: 17454686 bytes, checksum: 95c3e2eb551f853fabd105f2d0d03911 (MD5) Previous issue date: 2006 / Mestrado / Mestre em Ciência de Alimentos

A utilização descontrolada de pesticidas tem provocado no decorrer dos anos a intoxicação de milhares de pessoas no mundo, uma vez que, seus resíduos têm sido depositados em alimentos, em solos e em ambientes aquáticos. Assim, a construção de duas novas plataformas sensoras para a detecção de pesticidas é o objetivo desse trabalho. Na primeira plataforma foi utilizado o polieletrólito catiônico polietilenoimina (PEI) em conjunto com o polissacarídeo extracelular algal (PSE) produzido pela microalga criptofícea Cryptomonas tetrapirenoidosa preparados através da técnica de deposição \"spin-coating\". E a segunda plataforma foi produzida por eletrodeposição pulsada, entre um potencial de redução e um de oxidação, utilizando nanoestruturas de óxido de zinco (ZnO). Para caracterizar as plataformas, foram utilizadas as técnicas de microscopia eletrônica de varredura com fonte de emissão de campo (FEG-SEM), difração de raios X (XRD), espectroscopia de absorção ultravioleta-visível (UV-Vis), microscopia de força atômica (AFM) e espectroscopia de reflexão-absorção no Infravermelho com modulação da polarização (PM-IRRAS). Através da imobilização da enzima polifenol oxidase na forma de extrato bruto em sua fonte natural (fruto abacate), as plataformas de PEI/PSE e ZnO, foram avaliadas como biossensores de catecol e do inseticida carbaril. De modo comparativo, as plataformas de PEI/PSE sem a presença imobilizada da enzima também foram estudadas para a detecção do catecol e do carbaril. A simplicidade na formação e na construção dessas plataformas vem qualificá-las como viáveis a serem produzidas em escala industrial e com baixo custo de processamento. E diante dos resultados obtidos no desenvolvimento desses biossensores destaca-se a eficiência e a rapidez de detecção, o que os tornam economicamente promissores e competitivos em termos de aplicações ambientais. / The uncontrolled use of pesticides has resulted over the years the intoxication of thousands of people in the world, since their waste has been deposited in food, in soil and aquatic environments. Thus, the construction of two new sensors platforms for pesticide detection is the objective of this work. At first platform was used cationic polyelectrolyte polyethyleneimine (PEI) along with the extracellular algal polysaccharide (EPS) produced by microalgae criptofícea Cryptomonas tetrapirenoidosa prepared by deposition technique \"spin-coating\". The second platform was produced by pulsed electrodeposition between a reduction and an oxidation potential using nanostructures zinc oxide (ZnO). To characterize the platforms, we used the techniques of field emission gun scanning electron microscopy (FEG-SEM), X-ray diffraction (XRD), ultraviolet visible absorption spectroscopy (UV-Vis), atomic force microscopy (AFM), and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). By immobilization of the polyphenol oxidase enzyme as a crude extract in their natural source (avocado fruit), platforms PEI/PSE and ZnO, they were evaluated as catechol and carbaryl insecticide biosensors. In a comparative way, the platforms PEI/PSE without the presence of immobilized enzyme were also studied for detection of catechol and carbaryl. The simplicity in the formation and construction of these platforms comes qualify them as viable to be produced on an industrial scale and low cost processing. And on the results obtained in the development of such biosensors stand out the efficiency and speed of detection, which make them economically promising and competitive in terms of environmental applications.

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-09-02T15:02:36Z No. of bitstreams: 2 Dissertação - Jhessica Cavalcante de Souza Golveia - 2016.pdf: 2178992 bytes, checksum: bbc320d2f858d086790c3448278f38c2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-05T13:03:33Z (GMT) No. of bitstreams: 2 Dissertação - Jhessica Cavalcante de Souza Golveia - 2016.pdf: 2178992 bytes, checksum: bbc320d2f858d086790c3448278f38c2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-05T13:03:33Z (GMT). No. of bitstreams: 2 Dissertação - Jhessica Cavalcante de Souza Golveia - 2016.pdf: 2178992 bytes, checksum: bbc320d2f858d086790c3448278f38c2 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Laccase (EC 1.10.3.2, benzenediol: oxygen oxidoreductase) belongs to multicobre oxidases family, produced mainly by fungi. Due to its ability to catalyze oxidation of a wide variety of substrates, laccase has been used in various industrial applications such as delignification and bleaching of pulp, discoloration of dyes in textile effluents and detoxification of environmental pollutants. To meet the high demand of laccase at low cost is necessary to increase their production. One strategy is use of inducers such as metal ions and aromatic compounds. However, these compounds may have high toxicity and high cost. The use of agro-industrial and agricultural wastes containing laccase inducers is a sustainable and promising alternative strategy. The main of this study was to evaluate "in vitro" the capacity of ten agro-industrial wastes to induce the production of laccase from Pycnoporus sanguineus. We evaluated two different culture conditions (1% Malto extract and potato dextrose broth 0.5%) and the cultivation under stirring and static condition. The plant materials were added at a concentration of 1% (w / v) in 250 ml Erlenmeyer flasks containing 60 ml of culture medium. The best growing conditions have been established and the results of maximum laccase activity were with the medium CBD 0.5%, stirring 150 rpm at 28 ° C. Positive controls were performed (with induction xylidine and copper) and negative controls (no addition of inducer). All tests were performed in triplicate. The wastes that induced highest amount of enzyme were the cupuassu (1642U.mL-1) and cocoa (1589U.mL-1), guariroba´s straw (1258 U.mL-1) and açaí (886 U.mL-1), being greater than the induced positive control with 2,5-xylidine and copper (511 U.mL-1). Cupuassu and cocoa was selected for characterization. The enzymatic extract induced with cupuassu proved to be more stable at high temperatures and at different pHs, in addition to being able to oxidize different substrates. The extract was choice for the remediation of ethinylestradiol. Obtained 86% removal after 4 hours reaction and after 8 hours the concentration of hormone was below the method detection limit. Analysis of MS-ESI-TOF showed that hormone molecule was biodegraded during the enzymatic process. / A enzima lacase (EC 1.10.3.2, benzenodiol: oxigênio-oxidorredutase) pertence à família das multicobre oxidases, produzida principalmente por fungos. Devido à sua habilidade de catalisar a oxidação de uma ampla variedade de substratos, a lacase tem sido utilizada em várias aplicações, dentre elas a detoxificação de poluentes ambientais. Para atender a alta demanda de lacase a baixo custo é necessário aumentar a sua produção. Para isso utilizam-se indutores, como íons metálicos e compostos aromáticos, que possuem elevada toxicidade e alto custo. A utilização de resíduos agroindustriais contendo indutores de lacase é uma estratégia alternativa sustentável e promissora. O objetivo deste trabalho foi avaliar “in vitro” a capacidade de dez resíduos agroindustriais em induzir a produção de lacase de Pycnoporus sanguineus e aplicar o extrato com melhor atividade enzimática na biorremediação do 17-α-Etinilestradiol. Os resíduos foram adicionados na concentração de 1% (p/v) em erlenmeyers de 250 mL, contendo 60 mL de meio de cultura. Foram realizados controles positivos (com indução por xilidina e cobre) e controles negativos (sem adição de indutores). Para a biorremediação, um volume referente a 200 U de atividade enzimática foi adicionado a frascos erlenmeyer de 150 mL, contendo 10 mL de tampão acetato 50 mM pH 5,0 e 10 mL de solução de Etinilestradiol 10 mg.mL-1. Os frascos foram colocados sob agitação de 150 rpm a 25ºC. A porcentagem de remoção foi avaliada por Cromatografia Líquida de Alta Eficiência-CLAE após 1,2,4,8,10,12 e 24 horas de reação. Dos resíduos testados, os que levaram à maior produção de lacase foram os de cupuaçu (1642 U.mL-1), cacau (1589 U.mL-1), palha de guariroba (1258 U.mL-1) e açaí (886 U.mL-1) no sexto dia de cultivo, sendo superiores ao controle positivo induzido com 2,5-xilidina e cobre (511 U.mL-1). O extrato enzimático induzido com resíduo de cupuaçu se mostrou ser estável a elevadas temperaturas, bem como a diferentes pHs, além de ter sido capaz de oxidar diferentes substratos, sendo o extrato de escolha para a remediação do 17-α-Etinilestradiol. Obteve-se 86% de remoção após 4 horas de reação e após 8 horas a concentração do hormônio foi abaixo do Limite de Detecção do método. Análises de MS-ESI-TOF mostraram que a molécula de hormônio pode ter sofrido biodegradação durante o processo de biorremediação.

Nesta dissertação, a polifenol oxidase (PFO) como extrato bruto de abacate (Persea americana) foi imobilizada em filmes de polipirrol (PPI) sintetizados eletroquimicamente utilizando o glutaraldeído (GA) como um agente de ligação entrecruzada. Os filmes PPI e PPI/PFO-GA foram caracterizados por eletroquímica, principalmente voltametria cíclica, sendo avaliadas a eletroatividade e a reversibilidade eletroquímica. A detecção de compostos fenólicos em soluções padrão foi feita por cronoamperometria, tendo um controle sobre a concentração dos compostos. O processo de transferência de massa foi monitorado com uma microbalança de cristal de quartzo eletroquímica. Os resultados indicaram uma boa reprodutibilidade das medidas na detecção dos compostos fenólicos. A estabilidade do biossensor em uma solução tampão manteve-se durante 27 dias, um resultado aceitável já que é encontrado na literatura um tempo de vida estável para sistemas semelhantes em tomo de 30 dias / In this dissertation, polyphenol oxidase (PPO) as crude extract of avocado (Persea americana) was immobilized on electrochemical1y synthesized polypyrrole (PPY) films using glutaraldehyde (GA) as a crosslinking agent. PPY and PPY/PPO-GA films were electrochemical1y characterized, mainly by cyclic voltametry, where electroactivity and electrochemical reversibility were evaluated. The detection of phenolic compounds in standard samples was made by chronoamperometry with a control over the compound concentration. The process of mass transfer was monitored with an electrochemical quartz crystal microbalance (EQCM). Our results indicated a good repeatability of the measurements for the detection of phenolic compounds. The stability of biosensor in a buffer solution has remained for 27 days, a result acceptable since it is found in the literature a time of life stable for similar systems around 30 days

Neste trabalho, foram fabricados biossensores amperométricos a partir do uso da polifenol oxidase (PFO), obtida do abacate como fonte enzimática, imobilizada em filmes de polipirrol (PPI) e que foram eletrossintetizados em meio de três diferentes eletrólitos de suporte (NaCl e NaClO4 e NaDDS). O método de imobilização enzimática foi o da adsorção, sendo a solução de enzima adicionada à solução com o pirrol e o eletrólito durante o processo de eletropolimerização. Os filmes de PPI/PFO foram caracterizados por técnicas eletroquímicas, principalmente por voltametria cíclica. A detecção de compostos fenólicos (catecol e pirogalol) foi realizada pela técnica de cronoamperometria após se variar a concentração do analito. A morfologia dos filmes foi estudada por microscopia de força atômica (AFM), sendo observado que a presença da enzima no filme polimérico assim como o uso de diferentes eletrólitos de suporte levou a diferenças na superfície dos filmes. Além disto, verificou-se que o biossensor construído a partir do uso do NaCl, apresentava uma resposta mais eficiente, ou seja, ele foi capaz de detectar catecol e pirogalol em um menor limite de detecção. / In this study amperometric biosensors were manufactured from the use of polyphenol oxidase (PPO) obtained from avocado as a source of enzyme immobilized in polypyrrole (PPY) films that were electrosynthesized with three different support electrolytes (NaCl, NaClO4 and NaDDS). The method of enzyme immobilization was the adsorption. The PPO was added in the solution containing pyrrole and electrolyte during electropolymerization. The PPY/PPO films were characterized by electrochemical techniques mainly by cyclic voltammetry. Detection of phenolic compounds (catechol and pyrogallol) was performed by the technique of chronoamperometry after varying the concentration of the analyte. The morphology of the films was studied by the atomic force microscopy (AFM) and observed that the presence of the enzyme in the polymer film and the use of different electrolytes support led to differences in the surface of films. However it was found that the biosensor constructed from the use of NaCl showed more efficient response and it was able to detect catechol and pyrogallol in a lower limit of detection.

No description available.

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting copolymer poly(N-(4-(3-thienyl methylene)-oxycarbonylphenyl)maleimide)-co-pyrrole(P(MBThi-co-Py)). A copolymer was electrochemically synthesized by using sodium dodecyl sulfate (SDS) as supporting electrolyte and characterized by FTIR, scanning electron microscopy (SEM) and conductivity measurements. Immobilization of glucose oxidase (GOD) and polyphenol oxidase (PPO) enzymes were performed in conducting PPy and P(MBThi-co-Py) matrices by electropolymerization. Kinetic parameters, maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes by help of Lineweaver-Burk plot. Effect of temperature and pH on GOD and PPO activity was examined. Operational stability and long term stability of the enzyme electrodes were investigated. The immobilized GOD and PPO electrodes were used for determination of glucose amount in Turkish orange juices and analyzing the concentration of phenolic compounds in Turkish red wines respectively.

Electrochemical polymerization of pyrrole functionalized polystyrene (PStPy) with pyrrole was carried out in water-sodium dodecyl sulfate solvent-electrolyte couple. Characterization of the resulting copolymer was performed via Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and four probe conductivity measurements. Glucose oxidase and polyphenol oxidase enzymes were immobilized in polypyrrole (PPy) and conducting copolymer of pyrrole functionalized polystyrene with pyrrole (P(PStPy-co-Py). Resulting enzyme electrodes were characterized by kinetic parameters / Vmax and Km. Behavior of enzyme electrodes upon temperature and pH changes were investigated. Glucose oxidase electrode was used for the determination of glucose in orange juice and polyphenol oxidase electrode was used for the determination of polyphenolic compounds in red wine.

In this study, two symbiotic fungi of Southern Pine Beetle (SPB), Entomocorticium peryii and Entomocorticium sp.A were evaluated in terms of polyphenol oxidase (PPO) production. The effect of different inhibitors, inducers and assay parameters such as temperature and pH on enzyme activity were investigated and maximum PPO activity was observed at 30&deg / C, pH 8.0 and when tannic acid was used as an inducer. Copper-chelator salicyl hydroxamic acid (SHAM) and pcoumaric acid, both indicated as inhibitors of tyrosinase and catechol oxidase significantly reduced the activity. For biochemical characterization studies, the enzyme was concentrated by ultrafiltration. To determine type of the enzyme, activity staining after Native-PAGE was carried out. Type of polyphenol oxidase produced by E. peryii and E. sp.A was determined as catechol oxidase by activity staining. However higher activity was observed on hydroquinone (p-diphenol) rather than catechol (o-diphenol). The enzyme obeys Michealis-Menten kinetics with Km and Vmaxvalues being 10.72 mM hydroquinone and 59.44 U/ml for E. peryii and 8.55 mM hydroquinone and 73.72 U/ml for E. sp.A respectively..

ABSTRACT IMMOBILIZATION OF INVERTASE, POLYPHENOL OXIDASE AND GLUCOSE OXIDASE IN CONDUCTING COPOLYMERS OF THIOPHENE-CAPPED POLYTETRAHYDROFURAN AND PYRROLE B&ouml / y&uuml / kbayram, AySe Elif Ph.D., Department of Chemistry Supervisor: Prof. Dr. Levent Toppare January 2005, 123 pages Immobilization of invertase, polyphenol oxidase (PPO) and glucose oxidase (GOD) enzymes were performed in electrochemically synthesized two types of conducting copolymers. One end and two end thiophene-capped polytetrahydrofuran (TPTHF-1 and TPTHF-2) were copolymerized with pyrrole under conditions of constant potential electrolysis. The copolymers were characterized by thermal, spectroscopic and scanning electron microscopy analyses. Immobilization was carried out via entrapment of enzymes in two types of matrices during the copolymerization of pyrrole with the insulating polymers in the presence of sodium dodecyl sulphate (SDS). Kinetic parameters: Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for the enzyme electrodes. Temperature optimization, pH optimization, operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase and glucose oxidase were used to determine the amount of their substrates in samples. Polyphenol oxidase converts mono and diphenols to quinone. Amount of phenolic compounds in two kinds of wines were determined by analyzing the quinone amount. Glucose oxidase converts &amp / #61538 / -D-glucose to D-glucono-1,5-lactone. Glucose amount was determined in two kind of factory-produced orange juices by analyzing D-glucono-1,5-lactone.