Made available in DSpace on 2017-07-10T14:38:25Z (GMT). No. of bitstreams: 1 taiomara.pdf: 1065464 bytes, checksum: af0fbef1e995478eaa14a58b94c0cbd0 (MD5) Previous issue date: 2015-02-27 / Extracellular β-fructofuranosidase production from Aspergillus versicolor was significantly increased after optimization of culture conditions by Central Composite Rotational Design (CCRD). The variables studied were apple pomace, initial pH and cultivation time, and in response to enzymatic activity of β-fructofuranosidase. The maximum yield of β-fructofuranosidase was obtained when the fungus was grown for 12 days in medium supplemented with 3% (w/v) apple pomace as carbon source and the initial medium pH 7.0. In the purification procedure, the extracellular crude extract of A. versicolor was precipitated with ammonium sulfate 75%, and applied to the ion exchange chromatography DEAE-Sephadex, which resulted in two peaks of enzyme activities, named β-fructofuranosidase-I and II. The β-frutofuranosidase- I exhibited optimum temperature and pH of 55°C and 6.0, respectively. The enzyme was stable at temperatures from 40 to 60°C and the pH in a range from 3.0 to 6.0. The activity of β-fructofuranosidase-I was increased in the presence of the Fe2+ ion, and inhibited by Hg2+. The kinetic parameters Vmax and Km with sucrose as substrate were 26.71 mm and 56.980 μmol.min-1, respectively. These biochemical characteristics of the β-fructofuranosidase demonstrate the potential application of this enzyme in the ethanol production industry, which requires high temperature stability and acidic pH. / A produção de β-frutofuranosidase extracelular de Aspergillus versicolor foi significativamente elevada após otimização das condições de cultivo através do Delineamento Composto Central Rotacional (DCCR). As variáveis estudadas foram: bagaço de maçã, pH inicial do meio mineral e tempo de cultivo, tendo como resposta a atividade enzimática da β-frutofuranosidase. A produção máxima de β-frutofuranosidase foi obtida quando o fungo foi cultivado por 12 dias, em meio suplementado com 3% (p/v) de bagaço de maçã como fonte de carbono e pH inicial 7,0 do meio mineral. No procedimento de purificação, o extrato bruto extracelular do A. versicolor foi precipitado com sulfato de amônio 75%, em seguida submetido à cromatografia de troca iônica DEAE-Sephadex, que resultou em dois picos de atividade enzimática, denominadas de β-frutofuranosidase I e II. A β-frutofuranosidase I exibiu temperatura e pH ótimos de 55ºC e 6,0, respectivamente. A enzima mostrou estabilidade nas temperaturas de 40 a 60ºC e foi estável em pH na faixa de 3,0 a 6,0. A atividade da β-frutofuranosidase I foi aumentada na presença do íon Fe2+, mas inibida pelo íon Hg2+. Os parâmetros cinéticos Km e Vmáx utilizando sacarose como substrato, foram 26,71 mM, e 56,980 μmol.min-1, respectivamente. Estas características bioquímicas da β-frutofuranosidase evidenciam o potencial de aplicação desta enzima na indústria de produção de etanol, que exige estabilidade a temperatura alta e pH ácido.

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T10:40:28Z (GMT). No. of bitstreams: 1 Geromel_Clara_D.pdf: 2807679 bytes, checksum: 87559525feb767ee465302f9b3380513 (MD5) Previous issue date: 2006 / Resumo: Sabendo-se que a produtividade da cultura de café está diretamente ligada a três fatores básicos de produção, climáticos, genéticos e fisiológicos, nesse estudo foram abordados alguns aspectos do metabolismo de carboidratos envolvidos no processo de enchimento dos frutos de café ao longo do seu desenvolvimento. A composição de carboidratos, principalmente de polissacarídeos do grão (endosperma) de café é conhecida, assim como a importância dos açúcares sobre a qualidade da bebida, porém a importação de sacarose a partir de folhas e seu desdobramento nos frutos ainda não são completamente esclarecidos. Os açúcares utilizados no metabolismo das sementes são de extrema importância, tendo como exemplo a regulação da relação fonte-dreno, além de controlar a expressão de genes que codificam algumas enzimas envolvidas no metabolismo de açúcares. O trabalho teve como principal objetivo estudar o metabolismo de sacarose nos frutos de café, ao longo do desenvolvimento. Análises histológicas e fornecimento de compostos marcados mostraram que não existem conexões vasculares entre os tecidos, pericarpo,perisperma e endosperma, mas vasos condutores que percorrem o funículo chegam até o perisperma, permitindo um descarregamento direto de fotoassimilados produzidos nas folhas e dele a transferência para o endosperma, além de receber fotoassimilados do pericarpo, pelo transporte de célula a célula. A fotossíntese no pericarpo diminui ao longo do desenvolvimento do fruto. Grãos de amido foram observados nos estados jovens do perisperma e à medida que surge o endosperma, esses grãos vão desaparecendo em regiões próximas ao tecido que está sendo formado. As enzimas e os teores de açúcares endógenos avaliados em tecidos separados ao longo da maturação do fruto apresentaram diferenças entre diferentes tratamentos da lavoura (¿pleno sol¿: condições normais de cultivo, sombreamento e carga reduzida). A sacarose sintase (SUS) apresentou valores de atividades bem maiores que das invertases ácidas (IAV). A sacarose fosfato sintase (SPS) acompanhou o acúmulo de sacarose no estádio inicial no pericarpo de frutos de café. Foi mostrada a existência de duas isoformas de SUS, codificadas por dos genes chamados CaSUS1 e CaSUS2 em C. arabica, que possivelmente desempenham diferentes funções metabólicas nos diferentes tecidos do fruto de café. Esses genes apresentam uma expressão diferencial, com CaSUS1 expressando-se nas fases jovens do desenvolvimento do perisperma e do endosperma; porém a expressão de CaSUS2 sobrepõe os picos de atividade SUS detectados e o acúmulo de sacarose nos estados finais de desenvolvimento do pericarpo e do endosperma. Isso sugere que a isoforma CaSUS1 está relacionada à degradação de sacarose, quando parece claro que a isoforma CaSUS2 está relacionada com a síntese desse açúcar. Foi mostrado que esses dois genes se expressam também in C. racemosa, pois as sondas CaSUS1 e CaSUS2 de C. arabica reconheceram transcritos (mRNA) no endosperma dessa espécie. Portanto, os genes CrSUS1 e CrSUS2 parecem codificar para isoformas de SUS que desempenham funções diferentes daquelas observadas em C. arábica; SUS1 parece estar relacionado com a síntese da sacarose. O sombreamento influenciou na duração do ciclo de desenvolvimento dos frutos, tornando-o mais longo que no pleno sol (respectivamente 260 e 231 dias após o florescimento) e alterando o acúmulo de sacarose nos frutos. Com base nesses resultados fica clara a complexidade do metabolismo de sacarose em frutos de café, visto que nem sempre as atividades enzimáticas seguem o mesmo padrão de acúmulo de açúcares e enzimas de degradação e ressíntese de sacarose atuam simultaneamente, assim como a transferência de açúcares entre os tecidos / Abstract: Since coffee culture productivity is straightly connected to three basic production factors: climate, genetics and physiology, herein some aspects of the carbohydrates metabolism involved in the process of coffee grains filling through its development were analyzed. It is known the carbohydrates composition, mainly the polysaccharides composition of the coffee grain (endosperm) as well as the importance of sugars in the beverage quality, nevertheless the sucrose import from the leaves and its sharing in the fruits are still to be completely clarified. The sugars utilized in the seeds metabolism are extremely important, such as in the regulation of the drain-source relation and in the expression control of genes that codify some enzymes involved in sugars metabolism. The main goal of this work was to study the sucrose metabolism in coffee fruits through their development. Histological analyses and marked compounds supply showed that there are no vascular connections among the tissues of the pericarp, perisperm and endosperm, but conduction vases that run through the funiculus get to the perisperm, enabling an unloading of photoassimilates produced in the leaves. From the perisperm, these assimilates are transferred to the endosperm; The pericarp photosynthesis diminishes through the fruit development. Starch grains were observed in juvenile stages of the perisperm and during the endosperm formation these grains start to disappear in regions close to the forming tissue. The enzymes and the endogenous sugar level evaluated in separated tissues through fruit maturation show some differences among distinct lavoura treatments (¿full sun¿: ordinary culture conditions, shadowing and reduced loading). The sucrose synthase (SUS) showed activity values much higher than the ones presented by the acid invertases (IAV). The sucrose phosphate synthase (SPS) followed the sucrose accumulation in the pericarp initial stage in coffee fruits. It was shown the existence of two isoforms of SUS codificated by genes called CaSUS1 e CaSUS2 in C. Arabica, that possibly play different metabolic roles in different tissues in coffee fruit. These genes show a differential expression, in which CaSUS1 is expressed in the juvenile stage of perisperm and endosperm development; however, CaSUS2 expression overlaps the detected activity peaks of SUS and the sucrose accumulation in the final stages of pericarp and endosperm development. This fact suggests that the CaSUS1 isoform is related to sucrose degradation while it seems clear that the CaSUS2 isoform is related to sucrose synthesis. Both genes were shown to be also expressed in C.racemosa since CaSUS1 and CaSUS2 C. arabica probes recognized transcripts (mRNA) in this species endosperm. Therefore, CrSUS1 and CrSUS2 genes seem to codify SUS isoforms that play different functions from the ones observed in C. arabica; SUS1 seems to be related to sucrose synthesis. The shadowing has influenced the fruits development cycle duration, turning it longer than in pleno sol (respectively, 260 and 231 days after flowering) and altering the sucrose accumulation in fruits. According to these results, it is clear how complex is the sucrose metabolism in coffee fruits, since it is not always that the same pattern of sugar accumulation is followed by the enzymatic activities and enzyme degradation and sucrose (re)synthesis act simultaneously as well as sugar transference among tissues / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

Invertasen sind Schlüsselenzyme in der Kohlenhydratverteilung und haben möglicherweise auch während einer Pathogeninfektion eine zentrale Bedeutung. In vorliegender Arbeit wurde zunächst die Regulation verschiedener Stoffwechselwege in Arabidopsis thaliana nach Infektionen mit einem virulenten oder avirulenten Stamm von Pseudomonas syringae untersucht. Mit Hilfe der Chlorophyllfluoreszenz-Bildgebung konnten räumliche und zeitliche Veränderungen der Photosynthese verfolgt werden. Verschiedene Parameter waren unterschiedlich reguliert. In beiden Interaktionen waren Effekte nur lokal um die Infektionsstellen erkennbar und qualitativ ähnlich. Unterschiede waren im zeitlichen Eintreten und Verlauf sichtbar. Die Methode schien geeignet für die sensitive, nicht-invasive Pathogenfrüherkennung vor dem Auftreten sichtbarer Symptome. Die Regulation verschiedener Gene innerhalb von Source-Sink-Übergängen und die Aktivität von Invertasen war in den beiden Interaktionen qualitativ unterschiedlich. Die Infektion mit virulenten Bakterien resultierte in einer Repression photosynthetischer Gene. Die Aktivität vakuolärer Invertasen stieg vorübergehend nach Infektion mit virulenten Bakterien an, während sie nach Infektion mit avirulenten Bakterien sank. Die Aktivität extrazellulärer Invertasen war in beiden Interaktionen reprimiert. Die erfolgreiche Generierung verschiedener Bakterienstämme von P. syringae, die das grün fluoreszierende Protein exprimieren, kann bei der weiteren Charakterisierung von Pflanze-Pathogen-Interaktionen helfen. Die Regulation von Invertasen erfolgt auf transkriptioneller und posttranslationaler Ebene. Um die Funktion von Invertasen in Pflanze-Pathogen-Interaktionen zu verstehen, wurde zunächst die Regulation von Invertasen durch endogene proteinogene Invertaseinhibitoren untersucht. In Übereinstimmung mit in silico Expressionsdaten konnte durch Untersuchung von Reportergenlinien und in Northern Blot Analysen eine starke Expression von Invertaseinhibitoren in Blättern von A. thaliana festgestellt werden. Nach Applikation biotischer und abiotischer Stressfaktoren wurde diese Expression nahezu vollständig reprimiert. Die indirekte Bestimmung der Invertaseinhibitoraktivität durch Messung der Invertaseaktivität in Mischextrakten zeigte, dass diese nach einer Pathogeninfektion vollständig reprimiert war. In funktionellen Ansätzen wurden transgene Pflanzen generiert, die Invertaseinhibitoren unter Kontrolle induzierbarer Promotoren exprimieren. Die Induktion der Invertaseinhibitorexpression änderte die Sensitivität gegenüber verschiedenen Pathogenen nicht signifikant. In einem pharmakologischen Ansatz wurde der chemische Inhibitor Acarbose zur Hemmung der Invertaseaktivität in A. thaliana verwendet. Eine Behandlung von Blättern bei gleichzeitiger Infektion mit Bakterien verursachte eine erhöhte Sensitivität der Pflanzen gegenüber der Infektion, eine stärkere Repression verschiedener Chlorophyllfluoreszenzparameter sowie ein erhöhtes Bakterienwachstum im Vergleich zu einer Infektion mit den Bakterien allein. Keine Effekte wurden auf transkriptioneller Ebene bei der Untersuchung von Genen verschiedener Stoffwechselwege gefunden. Die Invertaseaktivität nach zusätzlicher Behandlung mit Acarbose war tendenziell niedriger als die Aktivität nach einer Pathogeninfektion alleine. Acarbose erhöhte die Spiegel an Salicylsäure unabhängig von einer Pathogeninfektion. Da das Bakterienwachstum in Mutanten des Salicylsäure-vermittelten Abwehrweges bei zusätzlicher Behandlung mit Acarbose ebenfalls erhöht war, kann eine Beteiligung dieses Abwehrweges am Acarboseeffekt bisher ausgeschlossen werden. Invertasen sind neben ihrer Beteiligung an der Abwehr für die Regulation von Entwicklungsprozessen wichtig. In einem funktionellen Ansatz mit Pflanzen, die Invertaseinhibitoren induzierbar produzieren, wurde die Funktion von Invertasen getestet. Zur Generierung spezifischer Effekte wurden die Inhibitoren unter Kontrolle synthetischer Promotoren in A. thaliana exprimiert. Unerwarteterweise war das Wachstum putativ transgener Keimlinge jedoch im 4-Blatt-Stadium arretiert. Eine Analyse der Aktivität der ß-Glucuronidase in den entsprechenden Reporterlinien zeigte eine Korrelation zwischen der Wachstumsarretierung und einer hohen Aktivität dieser Promotoren unter verschiedenen in vitro Bedingungen. Dieser negative Effekt der Invertaseinhibition auf das Keimlingswachstum wurde in transgenen Tabakpflanzen bekräftigt, die Invertaseinhibitoren unter Kontrolle eines Tetracyclin-induzierbaren Promotors exprimierten. Eine erfolgreiche Induktion des Promotors resultierte in einer Reduktion des Frischgewichtes der Keimlinge. Mittels in silico Expressionsdaten und Northern Blot Analysen konnte für A. thaliana eine spezifische und starke Expression verschiedener Invertaseisoformen in Keimlingen nachgewiesen werden. Diese komplementären Ergebnisse zeigen die Notwendigkeit der Invertaseaktivität für eine normale Keimlingsentwicklung. / Invertases are key metabolic enzymes in carbohydrate partitioning which may also play an important role during pathogen infection. In this study, the regulation of different metabolic pathways in Arabidopsis thaliana plants after infection by a virulent and an avirulent strain of Pseudomonas syringae was investigated. With help of chlorophyll fluorescence imaging spatio-temporal changes in photosynthesis were monitored. The monitored chlorophyll fluorescence parameters were showing differential regulation. The effects were restricted to the vicinity of the infection site and did not spread to uninfected areas of the leaf. Qualitatively similar changes in photosynthetic parameters were observed in both interactions. Major differences between the responses to both strains were evident in the onset and time course of changes. Changes could be detected by chlorophyll fluorescence imaging before symptoms were visible by eye. In contrast to photosynthesis, the regulation of marker genes for source/sink relations and the activities of invertase isoenzymes showed qualitative differences between both interactions. Inoculation of the virulent but not the avirulent strain resulted in downregulation of photosynthetic genes and upregulation of vacuolar invertases. The activity of vacuolar invertases transiently increased upon infection with the virulent strain but decreased with the avirulent strain while extracellular invertase activity was downregulated in both interactions. As an advanced tool for the characterization of the interaction between A. thaliana and P. syringae bacteria expressing the green fluorescing protein were generated. Invertases are regulated on transcriptional and posttranscriptional level. To understand the function of invertases in plant-pathogen-interactions, the regulation of endogenous proteinaceous invertase inhibitors was investigated. According to expression profiling the analysis of reporter gene lines and Northern Blot analyses revealed a strong expression of invertase inhibitors in source leaves of A. thaliana. After application of biotic and abiotic stress factors this expression was completely repressed. Indirect determination of invertase inhibitor activity by measurement of invertase activity in mixed extracts revealed a complete repression of inhibitor activity after pathogen infection. In functional approaches transgenic plants were generated, expressing invertase inhibitor proteins under control of inducible promoters. However, no effect of the induction of inhibitor expression on the sensitivity to different pathogens could be observed so far. In a pharmacological approach acarbose war used as an invertase inhibitor. Simultaneous treatment of plants with acarbose and bacteria revealed an increased sensitivity of the plant towards the bacterial infection and a stronger repression of chlorophyll fluorescence parameters compared to plants treated with bacteria alone. No effects on photosynthesis, carbohydrate metabolism and defence could be observed on transcriptional level. A tendency of lowered invertase activity could be observed after treatment with acarbose and bacteria compared to bacterial treatment alone. Acarbose treatment enhanced the levels of salicylic acid independent of bacterial infection. The acarbose-mediated increase in sensitivity was also detectable in sid2 and cpr6 mutants indicating that the effect of acarbose is independent of the salicylic acid mediated defence pathway. Invertases are important enzymes in higher plants, which are involved in regulating developmental processes and responses to external factors. In a functional approach the role of invertases was investigated using transgenic plants ectopically expressing inhibitor proteins to decrease invertase activity. For generating specific effects, these inhibitor proteins were expressed in A. thaliana under the control of synthetic promoters consisting of tetramers of pathogeninducible elements, which were reported to yield low constitutive expression. Unexpectedly, seedling growth of putative transgenic plants was arrested at the four-leaf stage. Analysis of ß-glucuronidase activity of corresponding reporter gene lines showed a correlation of the growth arrest with high activity of these promoters in seedlings grown under tissue culture conditions. The negative effect of invertase inhibition on seedling growth was substantiated by transgenic tobacco plants expressing an invertase inhibitor under control of a tetracycline inducible promoter. Ectopic induction of the invertase inhibitor during early seedling development resulted in a reduced fresh weight of seedlings. Expression profiling and Northern Blot analyses further supported the importance of invertase in Arabidopsis thaliana seedling development. Different invertases were specifically and strongly expressed. These complementing results show that invertase activity is required for normal seedling development.

Orientador: Luis Henrique Souza Guimarães / Banca: Saulo Santesso Garrido / Banca: Hamilton Cabral / Resumo: As invertases ou β-D-frutofuranosidases (EC 3.2.1.26) são responsáveis pela produção da mistura equimolar de glicose e frutose, conhecida como açúcar invertido, através da hidrólise da ligação β 2-1 da molécula de sacarose. Essa mistura apresenta sabor mais doce que a sacarose e é destinada à diversos fins na indústria alimentícia (doces, xaropes, leite condensado e bebidas). É importante destacar que o produto obtido por hidrólise enzimática é bastante superior ao obtido por hidrólise ácida. A hidrólise catalisada pela enzima β-D-frutofuranosidase produz um xarope de alta qualidade com ausência de hidroximetilfurfural e sem desenvolvimento de cor. Diante do interesse industrial e biotecnológico, e das diversas aplicações dessa enzima, o objetivo deste trabalho foi investigar a produção de β-D-frutofuranosidase extracelular pelo fungo filamentoso Aspergillus niveus através de Fermentação em Estado Sólido (FES) purificando-a e caracterizando-a bioquimicamente. Entre os substratos testados em FES, a maior produção β-D-frutofuranosidásica foi obtida em casca de mandioca, com granulometria de 10 mesh, umidificada na proporção de 1:1 com água de torneira, a uma temperatura de 30ºC com umidade relativa de 30%, por um período de 9 dias de incubação. A β-D-frutofuranosidase extracelular obtida em FES foi purificada 6,53 vezes com recuperação de 5,27%, obtendo-se em SDS-PAGE 8% uma única banda protéica (37 kDa), e massa molecular nativa de 91,2 kDa estimada por Sepharose CL-6B. A temp... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The invertases or β-D-fructofuranosidases (EC 3.2.1.26) are responsible for the production of an equimolar mixture of glucose and fructose, known as invert sugar, by hydrolysis of β 2-1 bond of the sucrose molecule. This mixture is sweeter than sucrose and is intended for various purposes in the food industry (sweets, syrup, condensed milk and drinks). It is important to note that the product obtained by enzymatic hydrolysis is much higher than that obtained by acid hydrolysis. The hydrolysis catalyzed by β-D-fructofuranosidase enzyme produces a high-quality syrup without hydroxymethylfurfural and color development. Before the industrial and biotechnological interest, and the various applications of this enzyme, the aim of this study was to investigate the production of the extracellular β-D-fructofuranosidase by filamentous fungus Aspergillus niveus under Solid Fermentation State (SSF) purifying it and characterizing it biochemically. Among the substrates tested in the SSF, the highest yield of β-D-fructofuranosidase was obtained in cassava hulls with a particle size of 10 mesh, humidified in the ratio 1:1 with tap water and maintained at 30°C with a relative humidity of 30%, for 9 days of incubation. The extracellular β-Dfructofuranosidase obtained in SSF was purified 6.53-folds with a recovery of 5.27%. A single protein band (37 kDa) was obtained in 8% SDS-PAGE and the native molecular mass was estimated as 91.2 kDa by Sepharose CL-6B. The optimum temperature and pH for ac... (Complete abstract click electronic access below) / Mestre

Carbon flow into developing ovaries has been reported to be important in seed retention and seed size. Invertase, which cleaves sucrose into glucose and fructose has been shown to be important in rapidly expanding tissue, such as early root growth or during tiller expansion. The manipulation of invertase activity with over-the-top applications of agrochemicals may prevent the detrimental effects of abiotic stress by altering the source/sink relationship. These experiments examined economically important tissues in rice production during critical developmental stages under abiotic stress. Field and greenhouse studies were conducted under normal growing conditions using local management practices. Plants were treated with exogenous chemicals that affect the activity of invertase during the early-grain fill stage on field grown plants. Other plants were exposed to elevated nighttime temperature of 30°C for 4 d using a free-air, infrared heating device in the greenhouse. Rice was also treated at mid- to late-grain fill stage of the main crop to identify the impact of the exogenous chemicals on developing ratoon tiller buds. The activity of soluble acid invertase (SAI), concentrations of glucose, fructose, sucrose, and starch were determined in penultimate leaves, panicles, and main-crop stem segments during ratoon tiller bud expansion, using the enzyme-coupled stoichiometric production of NADH measured spectrophotometrically at 340nm. The results suggest SAI, carbohydrates, and agronomic characters are influenced by exogenous chemicals at the applied rates. The thidiazuron treatment caused an unidentified stress event. The stress was confirmed by increased hexose concentration and the proportion of hexose concentration to sucrose concentration. This stress reduced the main-crop grain yield, but not the ratoon yield or total grain yield. An interaction between the ammonium molybdate treatment and high nighttime temperature was seen in the panicle. The ranked difference was reduced by the high nighttime temperature from the ambient nighttime temperature for the SAI activity, TSC content, starch content, and TNC content. The tested chemicals and rates are not appropriate for commercial rice production because the effect of the exogenous chemicals do not appear to consistently aid rice plants to counteract the detrimental effects of abiotic stress.

Carbon flow into developing ovaries has been reported to be important in seed retention and seed size. Invertase, which cleaves sucrose into glucose and fructose has been shown to be important in rapidly expanding tissue, such as early root growth or during tiller expansion. The manipulation of invertase activity with over-the-top applications of agrochemicals may prevent the detrimental effects of abiotic stress by altering the source/sink relationship. These experiments examined economically important tissues in rice production during critical developmental stages under abiotic stress. Field and greenhouse studies were conducted under normal growing conditions using local management practices. Plants were treated with exogenous chemicals that affect the activity of invertase during the early-grain fill stage on field grown plants. Other plants were exposed to elevated nighttime temperature of 30°C for 4 d using a free-air, infrared heating device in the greenhouse. Rice was also treated at mid- to late-grain fill stage of the main crop to identify the impact of the exogenous chemicals on developing ratoon tiller buds. The activity of soluble acid invertase (SAI), concentrations of glucose, fructose, sucrose, and starch were determined in penultimate leaves, panicles, and main-crop stem segments during ratoon tiller bud expansion, using the enzyme-coupled stoichiometric production of NADH measured spectrophotometrically at 340nm. The results suggest SAI, carbohydrates, and agronomic characters are influenced by exogenous chemicals at the applied rates. The thidiazuron treatment caused an unidentified stress event. The stress was confirmed by increased hexose concentration and the proportion of hexose concentration to sucrose concentration. This stress reduced the main-crop grain yield, but not the ratoon yield or total grain yield. An interaction between the ammonium molybdate treatment and high nighttime temperature was seen in the panicle. The ranked difference was reduced by the high nighttime temperature from the ambient nighttime temperature for the SAI activity, TSC content, starch content, and TNC content. The tested chemicals and rates are not appropriate for commercial rice production because the effect of the exogenous chemicals do not appear to consistently aid rice plants to counteract the detrimental effects of abiotic stress.

The relationship between extractable invertase activities and sucrose accumulation in the sugarcane (Saccharum spp. hybrids) culm and in vivo invertase mediated sucrose hydrolysis was investigated to determine the significance of invertases in sucrose utilisation and turnover. In vitro activities were determined by assaying the soluble acid invertase (SAI), cell wall bound acid invertase (CWA) and neutral invertase (NI) from internodes three to ten in mature sugarcane plants of cultivar NCo376. Extractable activities were verified by immunoblotting. In vivo invertase mediated sucrose hydrolysis was investigated in tissue discs prepared from mature culm tissue of the same cultivar. Sugarcane NI had a higher specific activity than SAI (apoplastic and vacuolar) in the sucrose accumulating region of the sugarcane culm. CWA was also present in significant quantities in both immature and mature tissue. Sugarcane NI was partially purified from mature sugarcane culm tissue to remove any potential competing activity. The enzyme is non-glycosylated and exhibits catalytic activity as a monomer, dimer and tetramer. Most of the activity elutes as a monomer of native Mr ca 60 kDa. The enzyme displays typical hyperbolic saturation kinetics for sucrose hydrolysis. It has a Km of 9.8 mM for sucrose and a pH optimum of 7.2. An Arrhenius plot shows the energy of activation of the enzyme for sucrose to be 62.5 kJ.mol-1 below 30°C and -11.6 kJ.mol-1 above 30°C. Sugarcane NI is inhibited by its products, with fructose being a more effective inhibitor than glucose. Sugarcane NI is significantly inhibited by HgCI2, AgNO-3, ZnCI2, CuSO4 and CoCI2 but not by CaCI2, MgCI2 or MnCI2. Sugarcane NI showed no significant hydrolysis of cellobiose or trehalose. When radiolabelled fructose was fed to sugarcane internodal tissue, label appeared in glucose which demonstrates that invertase mediated hydrolysis of sucrose occurs. A combination of continuous feeding and pulse chase experiments was used to investigate the in vivo contribution of the invertases and the compartmentation of sugars. Sucrose is synthesised at a rate greater than the rate of breakdown at all stages of maturity in sugarcane culm tissue. The turnover time of the total cytosolic label pool is longer for internode three than internode six. A higher vacuolar:cytosolic sugar molar ratio than previously assumed is indicated. Developmentally, the greatest change in carbon allocation occurs from internodes three to six. The main competing pools are the insoluble and neutral fractions. As the tissue matures, less carbon is allocated to the insoluble and more to the neutral fraction. The neutral fraction consists mainly of sucrose, glucose and fructose. The compartmented nature of sugarcane storage parenchyma carbohydrate metabolism results in a system that is complex and difficult to investigate. A computer based metabolic flux model was developed to aid in the interpretation of timecourse labelling studies. A significant obstacle was the global optimization of the model, while maintaining physiologically meaningful flux parameters. Once the vacuolar:cytosolic molar ratio was increased, the model was able to describe the internode three and six labelling profiles. The model results were in agreement with experimental observation. An increase in the rate of sucrose accumulation was observed with tissue maturation. Only the internode three glucokinase activity was greater than the experimentally determined limit. The rate was however physiologically feasible and may reflect the underestimation of the in vivo rate. SAI and NI contributed to sucrose hydrolysis in internode three but not in internode six. The rates in internode six were set to fixed low values to enable the model to fit the experimental data. This does not however preclude low levels of in vivo SAI and NI activity, which would prove significant over a longer time period. The flow of label through the individual pools, which comprise the experimentally measured composite pools could be observed. This provides insight into the sucrose moiety label ratio, SPS:SuSy sucrose synthesis ratio, and the rate of 14CO2 release. The model provides a framework for the investigation and interpretation of timecourse labelling studies of sugarcane storage parenchyma. / Thesis (Ph.D.)-University of Natal, Durban, 2000.

Orientador: Carlos Alexandre Costa Crusciol / Banca: João Domingos Rodrigues / Banca: Godofredo Cesar Vitti / Banca: Gaspar Henrique Korndorfer / Banca: Rafaella Rossetto / Resumo: Objetivou-se, mediante o presente estudo, avaliar a eficácia da aplicação foliar de boro (B) associado, ou não, aos maturadores etil-trinexapac, etefon e sulfometuron metil na pré colheita da cana-de-açúcar, as alterações fisiológicas e suas implicações na qualidade e produtividade de colmos. O presente projeto foi composto de três subprojetos, sendo o primeiro abordando o uso de B associado ao etil-trinexapac, o segundo ao etefon e o terceiro ao sulfometuron metil. Para cada subprojeto foram conduzidos 4 experimentos em início e 4 em final de safra (2 locais e 2 anos), totalizando 12 experimentos em início de safra e 12 em final de safra, ou seja, 24 experimentos. Nos experimentos em início de safra, foi utilizada a variedade de cana-de-açúcar RB855453 (maturação precoce) nos dois locais e em final de safra foi utilizada a variedade SP80-3280 (maturação tardia) no Grupo Raízen e a variedade RB867515 (maturação média-tardia) no Grupo Tereos. Os tratamentos foram constituídos da aplicação dos maturadores etil-trinexapac, etefon, sulfometuron metil e do controle (maturação natural), associados à aplicação de B foliar. As doses empregadas foram, respectivamente, 200 g i.a. ha-1 (0,8 L p.c. Moddus ha-1), 480 g i.a. ha-1 (0,66 L p.c. Ethrel ha-1), 0,15 g i.a. ha-1 (0,20 g p.c. Curavial ha-1) e para o B, a dose foi 0,18 kg ha-1 (1060 g ácido bórico ha-1). Foram avaliados os parâmetros bioquímicos, biométricos, tecnológicos e nutricionais. Concluiu-se que a deficiência de B limita o acúmulo de sacarose em início e final de safra; a aplicação de maturadores em início de safra, isoladamente ou em associação ao B, possibilitou antecipação no corte da matéria-prima em cerca de 15 dias e o B aplicado isoladamente foi ainda mais eficiente, antecipando a maturação em cerca de 18 dias; em final de ... / Abstract: The aim, through this study, was to evaluate the effectiveness of foliar application of boron (B), associated or not to the ripeners trinexapac-ethyl, ethephon and sulfometuron methyl in pre harvest of sugarcane, the physiological changes and their implications on quality and stalk yield. This project was composed of three subprojects, the first evaluating the use of B associated with trinexapac-ethyl, the second to ethephon and the third to sulfometuron methyl. For each subproject 4 experiments were carried out at the early and at the late season (2 places and 2 years), so were 12 experiments at early season and 12 at late season, totaling 24 experiments. In the experiments at early season, was used the variety of sugarcane RB855453 (early ripening) in both locations and at late season was used the variety SP80-3280 (late ripening) in Raízen Group and variety RB867515 (middle-late season) in Tereos group. The treatments consisted in the application of the ripeners trinexapac-ethyl, ethephon, sulfometuron methyl and control (natural ripening) associated with the application of B. The doses used were, respectively, 200 g i.e. ha-1 (0,8 L c.p. Moddus ha-1), 480 g i.e. ha-1 (0,66 L c.p. Ethrel ha-1), 0,15 g i.e. ha-1 (0,20 g c.p. Curavial ha-1) and for B the dose was 0,18 kg ha-1 (1060 g boric acid ha-1). Biochemical, biometric, technological and nutritional parameters were evaluated. It was concluded that B deficiency in pre-harvest of sugarcane limits the accumulation of sucrose at early and at late season; at early season the ripeners applied alone or in association with B enabled anticipation in the raw material harvesting in about 15 days and B applied alone was even more efficient, anticipating in about 18 days; at late season the ripeners were effective in maintaining the high sucrose concentration longer than control treatment, as well as B applied alone or in association with ... / Doutor

A enzima β-D- frutofuranosídeo frutohidrolase (E.C. 3.2.1.26), também conhecida como invertase, é uma hidrolase capaz de clivar o dissacarídeo sacarose, gerando mistura equimolar de glicose e frutose (‘açúcar invertido’). A aplicação deste, bem como a dos monossacarídeos de modo isolado é bastante comum na indústria alimentícia, por exemplo na manufatura de recheios de doces, além de outras aplicações, como na indústria farmacêutica. O objetivo deste trabalho foi avaliar diferentes suportes e métodos de imobilização de uma invertase de Saccharomyces cerevisiae. Os experimentos feitos com filmes de celulose, macroesferas de quitosana, e o suporte comercial Immobead não apresentaram resultados conclusivos. A imobilização covalente unipontual da invertase em mistura de nano e agregados de nanopartículas de quitosana possibilitou a obtenção dos seguintes resultados: além desse suporte ser de fácil preparação e ativação, oferecendo grande área superficial para a imobilização, o derivado imobilizado apresentou alta recuperação da atividade, sendo utilizado o protocolo que permitiu obter 74,3 % de rendimento e 61,6 % de eficiência de imobilização. A temperatura (55 ºC) e o pH ótimos de atividade (4,5), estabilidade térmica e ao armazenamento não foram modificados pós-imobilização. A afinidade da invertase pelo substrato decaiu cerca de 3 vezes, devido à reduzida acessibilidade da sacarose ao sítio ativo da enzima. Porém, o parâmetro Vmax manteve-se constante, indicando que não houve perda na máxima conversão da sacarose em seus monossacarídeos. Através da imobilização foi possível obter excelente estabilidade operacional: 59 reusos com 100 % da atividade catalítica da enzima (bateladas de 30 min, sob suave agitação, com solução de sacarose 8 %, a 55 ºC). / The enzyme β-D- fructofuranoside fructohydrolase (E.C. 3.2.1.26), also known as invertase, is one hydrolase able to cleave the sucrose disaccharide, generating an equimolar mixture of glucose and fructose (‘invert sugar’). The application of invert sugar, as well the isolated monosaccharides is very common in the food industry, for example in the manufacture of filling of sweets, besides other applications, as in the pharmaceutical industry. The objective of this work was to evaluate different supports and immobilization methods of an invertase from Saccharomyces cerevisiae. The experiments performed with cellulose films, chitosan macrospheres and the commercial support Immobead did not present conclusive results. The unipoint covalent immobilization of invertase in a mixture of chitosan nano and aggregated nanoparticles made possible to obtain the following results: besides the easy preparation and activation of this support, offering high superficial area for enzyme immobilization, the immobilized derivative presented high activity recovery, which allowed getting 74.3 % of immobilization yield and 61.6 % of immobilization efficiency. The optimal temperature (55 ºC) and pH (4.5) for activity, thermal and storage stabilities were not modified after immobilization. The enzyme affinity for its substrate decreased about 3 folds, mainly due to the reduced accessibility of sucrose to the catalytic site of the enzyme. However the parameter Vmax remained constant, indicating that there was not loss in the maximal conversion of sucrose in its monosaccharides. Through the immobilization was possible to obtain excellent operational stability: 59 reuses with 100 % of the catalytic enzyme activity (batches of 30 min, under genlte stirring, with sucrose solution 8 %, 55 ºC).

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Biotecnologia e Biociências, Florianópolis, 2015. / Made available in DSpace on 2016-05-24T17:26:34Z (GMT). No. of bitstreams: 1 338219.pdf: 1806932 bytes, checksum: cb4e68c860e46d1c5b4be98ef954ceed (MD5) Previous issue date: 2015 / Na levedura S. cerevisiae os carboidratos sacarose, maltose e maltotriose são fermentados por vias metabólicas diferentes: a sacarose é hidrolisada pela invertase extracelular (codificada pelos genes SUC), enquanto maltose e maltotriose são ativamente transportadas para dentro da célula e hidrolisadas pelas a-glicosidases presentes no citoplasma (ambas proteínas codificadas pelos genes MAL). Os genes SUC também permitem a síntese de uma forma intracelular da invertase, uma enzima com nenhuma função óbvia em leveduras. No entanto, a sacarose pode também ser metabolizada por células de levedura através dos transportadores e a-glicosidases codificados pelos genes MAL. Nossos resultados mostram que a maltotriose pode ser também eficientemente fermentada pelas células de S. cerevisiae através de seu transporte ativo mediado pela permease AGT1, um transportador MAL necessário para utilização de maltotriose, e sua hidrólise intracelular mediada pela invertase intracelular. A cepa brasileira industrial utilizada na produção de etanol combustível CAT-1 não pode fermentar maltotriose eficientemente devido ao promotor do AGT1 ser defeituoso. Para aumentar a fermentação de maltotriose por esta levedura, colocamos um promotor constitutivo (PGPD) à frente do gene AGT1 presente na cepa CAT-1, gerando a linhagem GMY05. No entanto, esta linhagem não foi capaz de fermentar eficientemente a maltotriose. Por outro lado, quando esta linhagem foi modificada para sobre-expressar a forma intracelular da invertase, por substituição da sequência sinal do gene SUC2 com o promotor constitutivo PADH1, a cepa iSUC2 obtida (GMY08) fermentou maltotriose eficientemente. Utilizando condições em que os genes MAL não são expressos, foi possível mostrar que a forma intracelular da invertase é capaz de hidrolisar a maltotriose (mas não maltose ou p-nitrofenil-a-glicosídico). Assim, os nossos resultados indicam uma sobreposição inesperada no metabolismo de sacarose e maltotriose por células de levedura, indicando que a invertase intracelular pode hidrolisar da maltotriose, e oferece novas abordagens a ser aplicadas para otimizar várias fermentações industriais que utilizam hidrolisados de amido, incluindo a panificação, produção de bebidas destiladas e cerveja, ou inclusive bioetanol.<br> / Abstract : It is well known that in the yeast S. cerevisiae the sugars sucrose, maltose and maltotriose are metabolized by different pathways: sucrose is hydrolyzed by the extracellular invertase (encoded by SUC genes), while maltose and maltotriose are actively transported into the cell and hydrolyzed by intracellular a-glucosidases (both proteins encoded by MAL genes). Furthermore, the SUC genes also allow the synthesis of an intracellular form of invertase, an enzyme with no obvious function in yeasts. We have already shown that sucrose can be metabolized by yeast cells through MAL-encoded transporters and a glucosidases. Now, our results will show that maltotriose can be efficiently fermented by S. cerevisiae cells through its active transport mediated by the AGT1 permease, a MAL transporter required for maltotriose utilization, and its intracellular hydrolysis mediated by the cytoplasmic invertase. The Brazilian industrial fuel-ethanol strain CAT-1 cannot ferment maltotriose efficiently due to a defective promoter of the AGT1 gene. To increase maltotriose fermentation by this strain, we placed a strong promoter (PGPD) in the AGT1 gene of strain CAT-1, generating strain GMY05. While the AGT1 gene was indeed over-expressed in this strain (measured by real-time PCR), maltotriose was still not fermented efficiently. However, when we over-expressed the intracellular form of invertase, by replacing the signal sequence of the SUC2 gene with the strong PPGK promoter, the resulting iSUC2 strain GMY08 fermented maltotriose efficiently. Using conditions were the MAL-encoded a glucosidases could not be expressed, we showed that the intracellular form of invertase could hydrolyze maltotriose (but not maltose or p-nitrophenyl-a-glucoside). Thus, our results indicate an unexpected overlap in sucrose-maltotriose metabolism by yeast cells, showing that the intracellular invertase allows efficient maltotriose hydrolysis, and offers new approaches that can be applied to optimize several industrial fermentation processes that use starch hydrolysates, including production of bread, distilled beverages and beer, or even bioethanol.