Made available in DSpace on 2014-07-29T15:16:29Z (GMT). No. of bitstreams: 1 Dissertacao_Eduardo_Fernandes_Barbosa.pdf: 661327 bytes, checksum: 816b815568b664e6a1393b1196e634ea (MD5) Previous issue date: 2010-02-19 / This work describes the immobilization of invertase on chemically synthesized polyaniline and activated with glutaraldehyde (PANIG) for production of invert syrup from sugarcane juice. Free invertase activity present in crude extract (E.B.) obtained from cells of Saccharomyces cerevisiae, was characterized for an evaluation of interferents present in the extract on enzyme activity (optimum conditions: temperature 50 ° C, pH of 4.5 in sodium acetate buffer 0.1 mol L-1 and reaction time of 10 minutes, with an activity of 11.31 ± 0.36 EU mL-1). We tested some parameters optimization of enzyme immobilization, such as amount of enzyme, immobilization time, pH and temperature of immobilization. The optimal immobilization was obtained in buffer sodium acetate 0.1 mol L-1 pH 4.5, immobilization time of 1 hour at 50°C and 169.55 EU mg-1 PANIG. The efficiency of immobilization was 0.86. The stability of the system PANIG-Invertase was tested against the storage time and thermostability, and after 75 days storage in buffer sodium acetate 0.1 mol L-1 pH 4.5 was obtained for 94% of initial activity with only 17% retained for the free enzyme. The immobilized invertase didn t change the optimal conditions compared to the free, but the immobilized was more stable in adverse conditions such as pH below and above optimum conditions showed an increase in thermostability. Some features of the hydrolysis product were evaluated (water activity, viscosity and color), compared to the sugarcane juice in nature, showing that the reactors allowed changes in sugarcane juice that expand the possibilities for using syrup obtained in the production of sweets, ice cream and syrups rich in fructose. The high stability of the system tested, along with its high retention of activity strongly suggests the use of the system in reactors. / Este trabalho descreve a imobilização da enzima invertase em polianilina sintetizada quimicamente e ativada com glutaraldeído (PANIG) para produção de xarope de açúcar invertido a partir de caldo de cana. Atividade da invertase livre, presente no extrato bruto (E.B.), obtido a partir de células de Saccharomyces cerevisiae, foi caracterizada e estabelecida em temperatura de 50°C; pH de 4,5 em tampão acetato de sódio 0,1 mol L-1 e tempo de reação de 10 minutos, com uma atividade de 11,31 ± 0,36 UE mL-1. A partir da imobilização por ligação covalente, testou-se alguns parâmetros de otimização da imobilização enzimática, como quantidade de enzima, tempo de imobilização, pH e temperatura de imobilização. A imobilização ótima foi obtida em tampão acetato de sódio 0,1 mol L-1 pH 4,5, tempo de imobilização de 1 hora, temperatura de 50°C e 169,55 UE mg-1 de PANIG. A eficiência de imobilização foi de 0,86. A estabilidade do sistema PANIG-Invertase foi testada frente ao tempo de armazenamento e termoestabilidade, sendo que após 75 dias de armazenamento em tampão acetato de sódio 0,1 mol L-1 pH 4,5 obteve-se 94% da atividade inicial, com apenas 17% retidos para a enzima livre. A invertase imobilizada não apresentou variação nas condições ótimas em comparação à livre, porém a imobilizada apresentou-se mais estável em condições adversas, como pH abaixo e acima das condições ótimas e apresentou aumento da termoestabilidade. Algumas características do produto hidrolisado foram avaliadas (Atividade de água, viscosidade e coloração), em comparação ao caldo de cana in natura, mostrando que o sistema testado possibilita modificações no caldo de cana que ampliam as possibilidades de utilização do xarope obtido na produção de doces, sorvetes e xaropes ricos em frutose. A elevada estabilidade do sistema testado, junto à sua retenção elevada de atividade, sugerem a aplicação do sistema em reatores.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-12-20T16:28:52Z No. of bitstreams: 2 Dissertação - Juliana Macêdo Silvestre - 2015.pdf: 815128 bytes, checksum: 3bea2ad6d4ab6f0e783b901523c0d838 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-12-26T14:12:10Z (GMT) No. of bitstreams: 2 Dissertação - Juliana Macêdo Silvestre - 2015.pdf: 815128 bytes, checksum: 3bea2ad6d4ab6f0e783b901523c0d838 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-12-26T14:12:10Z (GMT). No. of bitstreams: 2 Dissertação - Juliana Macêdo Silvestre - 2015.pdf: 815128 bytes, checksum: 3bea2ad6d4ab6f0e783b901523c0d838 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-05-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Cerrado is a biome that occupies about 25% of Brazilian territory. The seasonality, hot-humid and cold-dry periods, is one of the most striking features of the Cerrado. Reduced rainfall reduces the availability of water in the surface layers of the soil and increases the evaporative demand of air leading to the formation of litter cooperating in cycling soil nutrients. Another important factor in the Cerrado is the occurrence of fires that influence the dispersion, bloom, sprouting and diversity of certain species of plants. The relationship between seasonality, low fertility of soils and naturally occurring fires has shaped vegetative and reproductive strategies of plants from the Cerrado biome. Ichthyothere terminalis (Spreng.) Blake Asteraceae species occurring in the Cerrado, accumulates large amounts of fructans in the underground organs as reserve carbohydrates. Fructans are fructose polymers which are synthesized by the enzymes 1-SST (sucrose: sucrose fructosyltransferase) and FFT (fructan: fructan fructosyltransferase) and depolymerized by 1-FEH (exohidrolase fructan) and invertase. This study investigated the effect of freezing in the production of the enzymes 1-SST, 1-FEH and Invertase in I. terminalis. The partial characterization of these enzymes was performed. The samples were frozen immediately after collection (EB2) or after 6 hours of transport to the laboratory (EB1). The highest amount of protein was found in EB1 (97.81 μg.mL-1) as compared to EB2 (11,90 μg.mL-1). The results showed that the extraction method was efficient, since there was a significant reduction in reducing sugars and the activity of enzymes 1-SST, 1-FEH and Invertase were detected, mainly in the EB1. Enzymes 1-SST and Invertase showed a pH optimum between pH 6 and 7, while the 1-FEH enzyme had a greater spectrum ranging from pH 5 to 7. The three enzymes had an optimum temperature in the range 40 to 50 (°C). This work represents an important step towards understanding the role of these enzymes in the fructan metabolism in I. terminalis. / O Cerrado é um bioma que ocupa cerca de 25% do território brasileiro. A sazonalidade climática, períodos quente-úmido e frio-seco, é uma das características mais marcantes do Cerrado. A redução pluviométrica diminui a disponibilidade de água nas camadas superficiais do solo e eleva a demanda evaporativa do ar levando a formação de serrapilheira que coopera na ciclagem de nutrientes do solo. Outro fator importante no Cerrado é a ocorrência de queimadas que influenciam na dispersão, floração, rebrota e diversidade de certas espécies de plantas. A relação entre sazonalidade climática, baixa fertilidade dos solos e ocorrência natural de queimadas tem moldado estratégias vegetativas e reprodutivas das plantas do bioma Cerrado. Ichthyothere terminalis(Spreng.) Blake, espécie da família Asteraceae ocorrente no Cerrado, acumula grandes quantidades de frutanos em seus órgãos subterrâneos como carboidratos de reserva. Os frutanos são polímeros de frutose sintetizados pelas enzimas 1-SST (sacarose: sacarose frutosiltransferase) e FFT (frutano:frutano frutosiltransferase) e despolimerizados pela 1-FEH (frutano exohidrolase) e invertase. O presente trabalho objetivou estudar o efeito do tempo de congelamento na produção das enzimas 1-SST, 1-FEH e Invertase em I. terminalis. Além disso, a caracterização parcial destas enzimas foi realizada. As amostras coletadas foram congeladas imediatamente após a coleta (EB2) ou após 6 horas de transporte até o laboratório (EB1). A análise da quantidade de proteína extraída mostrou que EB1 apresentou maior quantidade de proteínas (97,81 μg.mL-1) quando comparada com EB2 (11,90 μg.mL-1). O método de extração foi eficiente, pois houve uma redução significativa em açúcares redutores e a atividade das enzimas 1-SST, 1-FEH e Invertase foram detectadas, principalmente no EB1. As enzimas 1-SST e Invertase apresentaram um pH ótimo entre pH 6 e 7, ao passo que a enzima 1-FEH teve um espectro maior variando de pH 5 a 7. As três enzimas apresentaram uma temperatura ótima na faixa de 40 a 50 (°C). Este trabalho representa um passo importante para o entendimento do papel destas enzimas no metabolismo de frutanos em I. terminalis.

A banana tem sido comumente indicada como uma boa fonte de frutooligossacarídeos (FOS), que são considerados componentes funcionais de alimentos. Contudo, diferenças significantes em suas quantidades têm sido referidas na literatura. Portanto, uma parte do trabalho foi destinada à identificação e quantificação de FOS durante o amadurecimento de cultivares de bananas pertencentes aos grupos genômicos mais comumente cultivados no Brasil. Considerando as diferenças de cultivar, estágio do amadurecimento e metodologia usada para análise de FOS, os conteúdos dos açúcares foram analisados por cromatografia líquida de alta performance (HPAEC-PAD) e cromatografia a gás (CG-MS). Uma pesquisa inicial entre oito cultivares no estágio maduro, mostrou acúmulo de 1-cestose, primeiro membro da série de FOS, em todas elas (quantidades entre 297 e 1600 µg/g M.S). A nistose, o segundo membro, foi detectado somente na cultivar Prata. Com bases nestes dados, foram escolhidas cinco cultivares, para que fossem analisadas durante todo o amadurecimento. Os resultados mostraram uma forte correlação entre a chegada a um nível específico de sacarose (~200 mg/g M.S) e a síntese de 1-cestose. Em uma segunda fase, os níveis de sacarose e FOS total foram quantificados em diferentes fases de amadurecimento de banana Prata, armazenada em temperatura ambiente e em baixa temperatura. As supostas enzimas envolvidas em sua síntese também foram avaliadas. Para explorar a possibilidade da invertase ser responsável pela atividade de frutosiltransferase em banana, foi medido o efeito do inibidor Piridoxal HCl, os níveis de concentração do substrato e as atividades de hidrólise e transglicosilação, e o efeito do tempo no estudo cinético da enzima. A baixa temperatura atrasou todos os eventos analisados por 15 dias e os níveis de sacarose tiveram um pequeno aumento, porém constante, enquanto a banana estava armazenada ao frio, e uma rápida elevação no final do amadurecimento. Foi detectado FOS total desde o primeiro dia pós-colheita, enquanto que a 1-cestose permaneceu indetectável até os níveis de sacarose atingirem aproximadamente 200 mg/g M.S., em ambos os grupos. Os níveis de sacarose e FOS total foram ligeiramente maiores em bananas armazenadas em baixas temperaturas do que em frutos controle. Em ambas as amostras os níveis de FOS total foram maiores que de 1-cestose. Os perfis de carboidratos por HPLC e TLC sugeriram a presença de neocestose, 6-cestose e bifurcose. A enzima supostamente responsável pela atividade de transglicosilação em banana parece ser a invertase. Contudo, os altos níveis de sacarose encontrados em banana armazenadas em baixa temperatura, poderiam ser resultado de várias mudanças de enzimas degradativas e biossíntéticas, como sacarose-sintase (SuSy), sacarose-fosfato-sintase (SPS), invertase e outras, uma vez que a sacarose possui um papel central, direta ou indiretamente, em diversas vias do metabolismo de carboidrato em banana. Assim, na última parte do trabalho foram analisados o acúmulo de sacarose e a síntese e atividade de enzimas sintéticas, hidrolíticas e fosforolíticas, importantes no metabolismo de amido-sacarose, durante o amadurecimento de banana Prata nos dois tratamentos. A baixa temperatura não danificou os frutos, aumentando a vida de prateleira deles. As amostras do frio apresentaram pequeno aumento no nível de degradação de amido e um acréscimo de 20 % na sacarose acumulada durante o amadurecimento. Foi verificado o atraso na produção de etileno, CO2, e no início de degradação de amido durante o acondicionamento ao frio, concomitante ao atraso no pico de atividade de α-amilase. O atraso no climatério também manteve alta a atividade e síntese protéica de SuSy durante o armazenamento a frio, que declinaram após a retirada do frio, como no controle. As enzimas β-amilase, fosforilase (forma citosólica e plastidial) e SPS reagiram positivamente, sofrendo uma indução positiva na síntese e atividade enzimática durante o armazenamento ao frio, que poderia ser parte do mecanismo necessário para os maiores níveis de açúcares e para o processo de tolerância do fruto à baixa temperatura. / Banana has been currently indicated as a good source of fructooligosaccharides (FOS), which are considered to be functional components of foods. However, significant differences in their amounts in bananas have been observed in the literature. So, a part of this work aims to identify and quantify FOS during ripening in different banana cultivars belonging to the most common genomic groups cultivated in Brazil. Considering that these differences can be due to cultivar, stage of ripening, and the methodologies used for FOS analyses, sugar contents were analyzed by high performance anion exchange chromatography pulsed amperiometric detection (HPAEC-PAD) and gas chromatography- mass spectrometry (GC-MS). An initial screening of eight cultivars in a full-ripe stage showed that 1-Kestose, the first member of the FOS series (amounts between 297 and 1600 µg/g of D.M), was accumulated in all of them. Nystose, the second member, was detected only in Prata cultivar. Five of the cultivars were analyzed during ripening, and a strong correlation could be established with a specific sucrose level (~200 mg/g of D.M.), which seems to trigger the synthesis of 1-Kestose. In a second part of this work, the levels of sucrose and total-FOS were quantified in different phases of banana Prata ripening stored at ambient and low temperature. The supposed enzymes involved in their synthesis were also evaluated. To explore the possibility that invertase could be responsible for the fructosyltransferase activity in banana, we measured the effect of the inhibitor Pyridoxal HCl, the level of substrate concentration on both hydrolyze and transglycosylase activity in the same protein extract and the effect of time on kinetic study of the enzyme. The cold temperature delayed all the analyzed events for 15 days and sucrose levels increased low, but constantly, while banana were stored at low temperature and had a burst when it increased. Total-FOS were detected in the first days after harvest, while 1-kestose remained undetectable until the sucrose levels were around 200 mg.g (dry weight), in both groups. Total-FOS and sucrose levels were higher in banana stored at low temperature than in control. In both samples total-FOS levels were higher than 1-kestose. The carbohydrate profiles by HPLC and TLC suggest the presence of neokestose, 6-kestose and bifurcose. The enzyme supposed to be responsible for the transglycosilation activity in banana, seems to be an invertase. However, the higher sucrose levels found in banana stored at low temperature could be result of several changes in biosynthetic and degradative enzymes, such sucrose-synthase, sucrose-phosphate-synthase, invertase and others, once that sucrose plays a central role in a lot of direct and indirect carbohydrate pathways in banana fruits. So, in the last part of this work, we analyzed the sucrose accumulation and synthesis and activity of synthetic, hydrolytic and phosphorolytic enzymes that are important in the starch-sucrose metabolism during ripening of banana Prata stored at ambient and low temperature. The levels of starch degradation and sucrose accumulation (around 20% over) showed high levels in cold fruits as compared with control, during the ripening. The cold temperature delayed the ethylene and CO2 production, and the beginning of the starch degradation, concomitantly with a delay in the profile of α-amylase synthesis and activity. The late climateric also maintained the high synthesis and activity of SuSy during the cold storage that decreased just after ending the cold exposure. The β-amylase, phosphorylase (plastidial and citossolic forms) and the SPS enzymes showed a positive induction in the both activity and synthesis of protein during the cold storage. It could be important to the higher sugars levels showed at low temperature and that could contribute to the process of cold resistance in banana fruit.

Invertasen spielen eine zentrale Rolle im Metabolismus der Pflanzen und werden über eine Vielzahl von Faktoren in ihrer Regulation beeinflusst. Invertasen mit pH-Optimum im sauren Bereich liegen dabei als vakuoläre und zellwandgebundene Isoformen einer Genfamilie in den Pflanzen vor. Im Rahmen dieser Arbeit wurden zum ersten Mal 9 putative Invertasen aus der Nutzpflanze Brassica napus, sowie 4 weitere putative Mitglieder der bereits bekannten Tabakinvertasenfamilie isoliert und Expressionsprofile für die neu klonierten Invertasen erstellt. Symplastisch isolierte Zellen, wie z. B. Pollen und Pollenschläuche, sind auf die Expression zellwandgebundener Invertasen besonders angewiesen, da sie Kohlen-hydrate primär in Form von Fructose und Glucose aufnehmen, die über die Invertasen-vermittelte Spaltung aus Saccharose gebildet werden. An Tabak hatten Goetz et al. (2001) gezeigt, dass eine Inhibierung dieser pollen¬spezi¬fischen Invertaseaktivität zu männlich sterilen Pflanzen führt. Im Rahmen dieser Arbeit wurde nachgewiesen, dass durch Inhibierung der Zellwandinvertase¬aktivität mittels Antisense-Technik oder Expression des proteinogenen Invertase-Inhbitors AtC/VIF2 auch männlich sterile Arabidopsis-Pflanzen generiert werden können. Ein Aktivitätsvergleich der Invertase-promotoren von Nin88 aus Tabak und AtcwINV2 aus Arabidopsis im jeweiligen homo- und heterologen System zeigte jedoch, dass der Einsatz dieser pollenspezifischen Promotoren zur Erzeugung männlich steriler Pflanzen über Familiengrenzen hinweg nicht möglich ist. Neben ihrer Funktion als Energieträger stellen Kohlenhydrate auch Signalmoleküle dar, die in die Regulation zentraler Prozesse der Pflanzen eingreifen. Anhand von Keimungsanalysen mit Arabidopsis-Pollen wurde festgestellt, dass Hexokinase-unabhängige Signalingwege involviert sein müssen, um ein Auskeimen der Pollen zu ermöglichen. Dabei kann die Hexokinase-vermittelte Inhibition der Pollenkeimung vermutlich durch die Beteiligung anderer Signaling-Wege aufgehoben werden. Es wurde außerdem die zuckerabhängige Ausbildung blasenartiger Strukturen an Arabidopsis-Pollen nachgewiesen, die vermutlich durch einen Zucker-spezifischen Abbruch des Pollenschlauchwachstums gebildet werden. / Invertases with acid pH-optimum play a central role in plant metabolism and are regulated by numerous biotic and abiotic factors. They consist of vacuolar and extracellular isoforms of a gene family in plants. For the first time 9 putative members of the invertase family of Brassica napus, as well as 4 additional putative tobacco acid invertases were cloned in this work and expression profiles were analysed. Symplastically isolated cells, like pollen and pollen tubes, in particular depend on the expression of extracellular invertases, since they prefer the uptake of glucose and fructose. These hexoses are generated by the invertase-mediated cleavage of sucrose. Goetz et al. (2001) have shown on tobacco that inhibition of the pollen specific invertase activity lead to male sterile plants. In this work it was shown that inhibition of cell wall invertase activity via antisense technology or expression of the proteinaceous invertase inhbitor AtC/VIF2 also causes male sterility in Arabidopsis thaliana. Comparison of the promoter activities of the invertases Nin88 (tobacco) and AtcwINV2 (Arabidopsis) in the corresponding homo- and heterologous systems revealed that the use of these pollen specific promoters for generating male sterile plants is not possible in diverse plant families. Besides their function as energy source, carbohydrates are signaling molecules that influcence regulation of central processes in the plants. On the basis of germination assays with Arabidopsis pollen, it was shown that hexokinase-independent signaling pathways must be involved to permit normal pollen germination. In this context, hexokinase-mediated inhibition of pollen germination might be overrode by the involvement of other signaling pathways. In addition, the sugar-dependent generation of bubble-like structures at Arabidopsis pollen was demonstrated, which might be caused by the sugar-specific interruption of pollen tube growth.

ABSTRACT BIOSENSOR BASED ON INTERPENETRATED POLYMER NETWORK OF ALGINIC ACID AND POLY (1-VINYLIMIDAZOLE) Kartal, M&uuml / jgan M.S., Department of Chemistry Supervisor : Prof. Dr. Levent Toppare January 2008, 63 pages A new proton conductor polymer was prepared using alginic acid (AA) and poly (1-vinylimidazole) (PVI). The polymer network was obtained by mixing AA and PVI at various stoichiometric ratios, x (molar ratio of the monomer repeat units). The AA/PVI network was characterized by elemental analysis (EA) and FT-IR spectroscopy. Potential use of this network in enzyme immobilization was studied. Enzyme entrapped polymer networks (EEPN) were produced by immobilizing invertase and tyrosinase (PPO) in the AA/PVI network. Additionally, the maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were investigated for the immobilized invertase and enzymes. Also, temperature and pH optimization, operational stability and shelf life of the polymer network were examined.

The aim of this study was to investigate the viability of patterning by immobilization, photocatalytic removal, and re-immobilization steps of the enzyme on photocatalytically active thin films for biosensor fabrication purposes. For this aim, TiO2-SiO2-Au sol-gel colloids were synthesized and deposited on glass substrates as thin films by dip coating. Cysteamine linker was assembled on gold nanoparticles to functionalize thin films with amine groups for immobilization of model enzyme invertase. Effect of immobilization temperature, enzyme concentration of the immobilization solution and immobilization period on invertase immobilization were investigated. The immobilized invertase activity was found independent from the immobilization temperature in the range tested (4oC-room temperature). The optimum enzyme concentration and period for immobilization was determined as 10&micro / g/ml and 12 hours respectively. The resulting invertase immobilized thin films showed high storage stability retaining more that 50% of their initial activity after 9 weeks of storage. Photocatalytic enzyme removal and re-immobilization studies were carried out by irradiating the invertase immobilized thin films with blacklight. Upon 30 minutes of irradiation, immobilized invertase was completely and irreversibly inactivated. Initial immobilized invertase activity (before the irradiation) was attained when invertase was re-immobilized on thin films that were irradiated for 5 hours. Thus it was inferred that with sufficient exposure, enzymes can be completely removed from the surfaces which makes the re-immobilization possible. The possibility of enzyme removal with photocatalytic activity and re-immobilization can pave the way to new patterning techniques to produce multi-enzyme electrode arrays.

Synthesis and characterization of conducting copolymers were achieved by using thiophene-3-yl acetic acid cholesteryl ester (CM) and poly (3-methylthienyl methacrylate) (PMTM). A new polythiophene containing a cholesteryl side chain in the &amp / #946 / -position was chemically polymerized in nitromethane/carbon tetrachloride using FeCl3 as the oxidizing agent. Polymerization was also achieved by constant current electrolysis in dichloromethane. Subsequently, conducting copolymers of thiophene-3-yl acetic acid cholesteryl ester (CM), PCM1 (obtained from chemical polymerization method), PCM4 (obtained from constant current electrolysis) with pyrrole were synthesized. Thiophene functionalized methacrylate monomer (MTM) was synthesized via esterification of the 3-thiophene methanol with methacryloyl chloride. The methacrylate monomer was polymerized by free radical polymerization in the presence of azobis (isobutyronitrile) (AIBN) as the initiator. Graft copolymers of poly (3-methylthienyl methacrylate)/polypyrrole, (PMTM2/PPy) and poly (3-methylthienyl methacrylate)/polythiophene, (PMTM2/PTh) were synthesized by constant potential electrolyses. PMTM2 coated Pt electrodes were utilized as the anode in the polymerization of pyrrole and thiophene. Moreover, oxidative polymerization of PMTM1 was studied by galvanostatic and chemical techniques. Characterizations of the samples were performed by CV, FTIR, NMR, DSC, TGA and SEM analyses. Electrical conductivities were measured by the four-probe technique. Immobilization of invertase in conducting copolymer matrices, poly (3-methylthienyl methacrylate) with pyrrole and thiophene was achieved by constant potential electrolysis using the sodium dodecyl sulfate as the supporting electrolyte. Polythiophene was also used for immobilization matrices. Cholesterol oxidase has been immobilized in conducting copolymer of thiophene-3-yl acetic acid cholesteryl ester with polypyrrole (CM/PPy) and polypyrrole (PPy) by the electropolymerization method. p-Toluene sulfonic acid was used as a supporting electrolyte. Kinetic parameters (Kinetic parameters / Vmax and Michaelis-Menten constant / Km) and operational stability of enzyme electrodes were investigated. Surface morphology of the films was also examined.

A new thiophene derivative containing menthyl group (MM) was synthesized and polymerized via chemical and electrochemical methods. Polymers obtained and MM itself were used to synthesize copolymers with pyrrole under conditions of constant potential electrolysis. Cyclic Voltammetry, thermal analysis and scanning electron microscopy analyses were performed for the characterization of samples. Immobilization of invertase and polyphenol oxidase enzymes was performed in the matrices obtained via copolymerization of MM with pyrrole. Immobilization was carried out via entrapment of enzyme in matrices during the polymerization of pyrrole. Temperature optimization, operational stability and shelf-life of the enzyme electrodes were investigated. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined. It is known that wine includes phenolic groups that give astringency in high concentrations. Polyphenol oxidase (PPO) converts mono and diphenols to quinone. By analyzing the product, one can find out the amount of phenolic groups. By using obtained enzyme electrodes via immobilization of PPO, amount of phenolics in different wines were analyzed.

Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: The goals of this project were firstly to develop the tissue preparation and in situ hybridisation protocols for sugarcane culm tissue, and secondly to use the developed techniques to examine the expression patterns of three invertase isoforms in sugarcane internodes of various developmental stages. Sugarcane invertases have been the focus of intense research for many years, yet almost nothing is known of their tissue-specific distribution. It was thought that by characterising their expression patterns using in situ hybridisation, more knowledge of their functions and involvement in sucrose accumulation would be gained. Although in situ hybridisation is now regularly used to study gene expression in plants, there is to date only a single publication describing its use on immature sugarcane tissue. Therefore this technique needed further development, and this was achieved by comparing different tissue preparation methods, as well as by systematically testing the various parameters pertaining to each method. The in situ hybridization technique was also developed by testing and comparing a number of key parameters. It was found that fixing whole mount tissue for 48 h preserved sugarcane tissue adequately. High hybridization temperatures and probe concentrations provided the best signal, and including pre-treatment with HCl and Pronase was essential in sensitizing the tissue to the probe. A less viscous detection buffer reduced both osmotic effects and time required for signal detection. In the second part of this study, the developed method was used to examine the expression patterns of the three invertase isoforms in young, maturing and mature internodes of sugarcane, and the results were complemented with Northern blot analysis. Transcript of all three isoforms was found to be present in the storage parenchyma and in the phloem tissue. Transcript levels of all three isoforms declined in maturing tissue, with soluble acid invertase declining sharply and dropping below detection in maturing and mature tissue. Transcript levels of cell wall invertase and neutral invertase declined only gradually, and appreciable levels of both were still present in mature tissue. Acid invertase is suggested to be mainly involved in internode elongation, while cell wall invertase would appear to play important roles in phloem unloading and turgor control. Neutral invertase is suggested to be involved in either sucrose cycling or maintenance of hexose pools, however the function of this enzyme remains unclear. This study has demonstrated the value of in situ hybridization, yet at the same time has shown its limitations, especially when more traditional biochemical techniques are not employed to complement the results. Although the precise functions of the invertase isoforms in sugarcane remain inconclusive, this study has opened up the way for tissuespecific promoter design and future in situ studies of sugarcane invertases / AFRIKAANSE OPSOMMING: Die doel van hierdie projek was tweeledig: eerstens om weefselvoorbereiding en in situhibridisasie- protokolle vir die stingelweefsel van suikerriet te ontwikkel; en tweedens om die ontwikkelde tegnieke te gebruik om die uitdrukkingspatrone van drie invertaseisovorme in die suikerriet-internodes van verskeie ontwikkelingstadia te ondersoek. Suikerriet-invertases is al vir jare lank die fokus van intense navorsing, maar baie min is bekend oor hulle weefselspesifieke verspreiding. Die idee was om meer kennis oor suikerriet-invertases se funksies en betrokkenheid by sukrose-akkumulasie te verkry deur in situ-hibridisasie te gebruik om hulle uitdrukkingspatrone te karakteriseer. Alhoewel in situ-hibridisasie deesdae gereeld gebruik word om geenuitdrukking in plante te bestudeer, is daar tot op datum slegs een publikasie wat die gebruik daarvan in onvolwasse suikerrietweefsel beskryf. Hierdie tegniek moes dus verder ontwikkel word, en dit is gedoen deur verskillende weefselvoorbereidingsmetodes te vergelyk en sistematies die verskillende parameters wat op elke metode van toepassing is te toets. Die in situ-hibridisasie-tegniek is ook ontwikkel deur die toetsing en vergelyking van 'n aantal sleutelparameters. Daar is gevind dat suikerrietweefsel voldoende gepreserveer word deur die intakte gemonteerde weefsel vir 48 uur te fikseer. Hoë hibridisasietemperature en hoë peilerkonsentrasies het die beste sein gegee; die insluiting van voorbehandeling met HCl en Pronase was noodsaaklik om die weefsel meer gevoelig vir die peiler te maak. Osmotiese invloede en die tyd nodig vir seindeteksie is verminder deur die viskositeit van die buffer te verminder. In die tweede deel van die studie is die ontwikkelde metode gebruik om die uitdrukkingspatrone van die drie invertase-isovorme in jong, ontwikkelende en volwasse internodes te ondersoek en die resultate is deur 'n noordelike oordraganalise gekomplementeer. Transkripte van al drie isovorme is in die stoorparenchiem en floëemweefsel gevind. Transkripvlakke van al drie isovorme het afgeneem in ontwikkelende weefsel, met oplosbare suurinvertase wat skerp afgeneem en tot onder die deteksie-limiet gedaal het in ontwikkelende en volwasse weefsel. Transkripvlakke van selwandinvertase en neutrale invertase het slegs geleidelik afgeneem en merkbare vlakke van albei was teenwoording in ontwikkelende en volwasse weefsel. Daar word voorgestel dat suurinvertase hoofsaaklik betrokke is by internodeverlenging, terwyl selwandinvertase skynbaar 'n belangrike rol in floëem-ontlading en turgor-beheer speel. Daar word voorgestel dat neutrale invertase betrokke is óf by die sukrose-sirkulering óf by die onderhoud van heksose-poele; die funksie van hierdie ensiem is egter steeds nie duidelik nie. Hierdie studie het die waarde van in situ-hibridisasie gedemonstreer maar terselfdetyd ook die beperkinge daarvan uitgewys, veral as meer tradisionele biochemiese tegnieke nie gebruik word om die resultate aan te vul nie. Alhoewel daar onsekerheid is oor die presiese funksies van die invertase-isovorme in suikerriet, het die studie die weg gebaan vir weefselspesifieke promotorontwerp en toekomstige in situ-studies van suikerrietinvertases.

Thesis (MSc (Genetics. Institute for Plant Biotechnology))--University of Stellenbosch, 2006. / The main goal of this project was to elucidate the apparent role of neutral invertase (NI) in sucrose accumulation in sugarcane. In the first part of the study putative transgenic cell lines (transformed with antisense NI constructs) were characterised to confirm the stable integration and expression of the transgene. Batch suspension cultures were used to initiate replicate cultures of several of these transgenic lines as well as a control, and the metabolism of the cultures during a 14 day growth cycle was examined. The transgenic lines had substantially reduced levels of NI activity. While the activities of the other invertases remained unchanged, the activity of sucrose synthase (SuSy) was significantly higher in the transgenic suspension cultures relative to the control. Throughout the growth cycle, sucrose concentrations in the transgenic lines were consistently higher, and glucose and fructose concentrations lower, than the control. The transgenic cultures also exhibited a decreased growth rate in comparison to the control. Labelling studies confirmed a decrease in the in vivo rate of invertase-mediated sucrose hydrolysis in the transgenic lines, as well as indicating a decline in the partitioning of carbon to respiratory pathways in these cultures. In the second part of the study, which focussed on greenhouse-grown transgenic plants, similar results were reported. NI activity was significantly decreased, and SuSy activity increased in all of the tissues sampled. The sucrose concentration and purity were also higher in the transgenic tissues, while the in vivo sucrose hydrolysis rate was lower. Allocation of carbon to respiration was lower in the transgenic plants, suggesting that a decrease in sucrose breakdown reduces the availability of hexoses for growth and respiration. Overall, the results suggest that NI plays a key role in the control of sucrose metabolism, and that changes in the activity of this enzyme have far-reaching effects on cellular metabolism. The fact that the trends reported in the whole-plant studies parallel those of the suspension cultures confirms that suspension cultures can be used as a model system in metabolic engineering research in sugarcane. Thus the possibility now exists to analyse large numbers of transgenic lines in a quicker time frame and at a reduced cost in comparison to conventional methods.