Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are the small-intestinal glucosidases responsible for catalyzing the last glucose-releasing step in starch digestion. MGAM and SI are each composed of duplicated catalytic domains, N- and C-terminal, which display complementary substrate specificities for the mixture of short linear and branch oligosaccharide substrates that typically make up terminal starch digestion products. As MGAM and SI are involved in post-prandial glucose production, regulating their activities with α-glucosidase inhibitors is an attractive approach to controlling blood glucose levels for the prevention and treatment of Type 2 diabetes. To better understand the complementary activities and mechanism of inhibition of these intestinal glucosidases, this thesis aims to characterize the individual N- and C-terminal MGAM and SI domains using a combination of X-ray crystallographic structural studies, enzyme kinetics, and inhibitor studies. First, the structure of the N-terminal domain of MGAM (ntMGAM) was determined in its apo form and in complex with the inhibitor acarbose. In addition to sequence alignments and kinetics studies, the structures provide insight into the preference of the N-terminal MGAM domain for short linear substrates and the C-terminal domain for longer substrates. Second, the structure of ntMGAM was determined in complex with various α-glucosidase inhibitors, including those currently on the market (acarbose and miglitol), a new class of inhibitors from natural extracts of Salacia reticulata (salacinol, kotalanol and de-O-sulfonated kotalanol) and chemically synthesized derivatives of salacinol. These studies reveal the features of the Salacia reticulata inhibitors that are essential for inhibitory activity and highlight their potential as future drug candidates. Third, the crystal structure of the N-terminal domain of SI (ntSI) was determined in apo-form and in complex with kotalanol. Structural comparison of ntSI and ntMGAM reveal key differences in active site architectures, which are proposed to confer differential substrate specificity.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Atualmente, a preocupaÃÃo com a qualidade de vida tem aumentado a demanda por alimentos que melhorem a saÃde e reduzam os riscos de doenÃas, dentre os quais destacam-se os oligossacarÃdeos prÃ-biÃticos. Nos Ãltimos anos, hà um crescente interesse no desenvolvimento de novos produtos alimentÃcios contendo esses compostos, incluindo-se os sucos de frutas. Desta forma, este trabalho visou à produÃÃo de oligossacarÃdeos prÃ-biÃticos em sucos de frutas e a secagem desses produtos pela tÃcnica de spray-driyng. Para a sÃntese enzimÃtica de oligossacarÃdeos utilizou-se a enzima dextrana-sacarase produzida pelo microorganismo L. mesenteroides B512F e realizou-se um planejamento experimental composto central, variando-se as concentraÃÃes de sacarose e aÃÃcares redutores nos sucos, com os ensaios conduzidos durante 24h a 30ÂC. Os resultados obtidos demonstraram que os sucos de abacaxi, melÃo e laranja apresentaram-se como uma excelente alternativa para a sÃntese de oligossacarÃdeos prÃ-biÃticos com elevados graus de polimerizaÃÃo. Os sucos prÃ-biÃticos obtidos das diferentes frutas submetidos ao processo de Spray Drying, no qual variaram-se os agentes encapsulantes utilizados, maltodextrina e goma arÃbica, assim como, a temperatura do ar de entrada (160 e 180 ÂC). As melhores condiÃÃes de secagem dos sucos de abacaxi, melÃo e laranja prÃ-biÃticos foram verificadas, ao utilizar-se 20% de maltodextrina e temperatura de 180 ÂC. Foram realizadas secagens dos sucos prÃbiÃticos, nas melhores condiÃÃes de secagem previamente determinadas, e observou-se que os pÃs obtidos apresentaram baixos valores para atividade de Ãgua, umidade, higroscopicidade e tempo de reidrataÃÃo, conferindo uma maior estabilidade fisico-quÃmica e microbiolÃgica ao produto, bem como, rÃpido preparo. A atividade antioxidante dos sucos tambÃm foi avaliada e verificou-se uma reduÃÃo neste parÃmetro. Conforme os resultados obtidos, constatou-se que os sucos de abacaxi, melÃo e laranja sÃo excelentes substratos para a obtenÃÃo de uma bebida prÃ-biÃtica inovadora, a apresentar-se na forma lÃquida para consumo imediato ou na forma desidratada, para preparo instantÃneo. / Nowadays, people have been concerned with their quality of life and wellness,increasing the consumption of foods, with prebiotic oligosaccharides, which may improve the health and decrease the disease risks. In recent years, a lot of new foods have been developed with these compounds (prebiotic oligosaccharides)including the fruit juices. The aim of this work was the prebiotic oligosaccharides production in orange, pineapple and melon juices, and their spray drying.The dextransucrase, produced by L. mesenteroides NRRL B-512F, was used for enzymatic synthesis following a central composite experimental design, with sucrose and reducing sugars variation, at 30 ÂC/ 24h. The prebiotic juices obtained were dried by spray drying, varying the maltodextrin and arabic gum concentration (10 and 20%) and the inlet temperatures (160 and 180 ÂC). Pineapple, orange and melon juices showed better results when maltodextrin(20%) and the inlet temperature 180 ÂC were utilized. The juice powders obtained showed low water activity, moisture,higroscopicity and rehydration time. These results give better microbiology and physicochemical stability, as well as, fast preparation. There was a decrease of the antioxidant activity at all products. The fruit juices used demonstrated an excellent way to produce, by enzymatic synthesis, prebiotic oligosaccharides with high polymerization degrees.

Der erste Teil der Dissertation behandelt die Substanz Phenylacetaldehyd, welche im Honig ausgehend von der Aminosäure Phenylalanin als natürlicher Stoff, aber auch als Rückstand nach Einsatz als Bienenvertreibungsmittel vorliegen kann. Die in dieser Arbeit durchgeführten Untersuchungen zeigen, dass der Gehalt an Phenylalanin sowie äußere Bedingungen, denen Honige ausgesetzt sind, für die Konzentration an Phenylacetaldehyd maßgebend sind. Diese Parameter müssen mindestens bekannt sein, um entscheiden zu können, ob Phenylacetaldehyd als Rückstand im Honig vorliegt. Der zweite Teil befasst sich mit der Filtration von Honig, welche in manchen Ländern durchgeführt wird, um eine Kristallisation zu herauszuzögern. Es wurde eine Methode entwickelt, um illegale Beimischungen gefilterter Honige zu ungefilterten Honigen nachzuweisen. Dazu wird das Enzym Saccharase gelchromatographisch isoliert und diese Fraktion elektrophoretisch untersucht. Die Veränderung des Proteinspektrums lässt sich mit Hilfe einer densitometrischen Auswertung quantifizieren und zeigt gefilterten Honig auch in Mischungen bis zu einem Anteil von mindestens 15 % an.

Amadori-Produkte werden spontan während der ersten Phase der Maillard-Reaktion aus reduzierenden Zuckern und Aminen wie Lysin gebildet. Sie entstehen während der Erhitzung von Lebensmitteln und in vivo. Der enzymatische Abbau solcher spontan gebildeten Produkte ist Thema dieser Arbeit. Ein Teil untersuchte die Rolle von Oligosaccharid-Amadori-Produkten während der Verdauung von Kohlenhydraten im Dünndarm. Aufgrund ihrer strukturellen Ähnlichkeit mit bekannten Glycosidase-Inhibitoren wurde eine hemmende Wirkung der Amadori-Produkte auf die Kohlenhydratverdauung vermutet. Der andere Teil beschäftigte sich mit Fructosamin-3-kinase (FN3K) und dessen verwandtem Enzym Fructosamin-3-kinase-related Protein (FN3K-RP) aus humanen Erythrocyten. Diese Ketosaminkinasen werden als Proteinreparaturenzyme betrachtet, sogar als enzymatische Verteidigung gegen Glykierung in vivo diskutiert. Durch ihre Reaktion entstehen jedoch auch hoch-reaktive 1,2-Dicarbonylverbindungen, die weitere Proteinschäden bewirken können. Noch ist nicht klar, ob die Ketosaminkinasen die pathophysiologischen Folgen der Glykierung verhindern oder fördern. In dieser Arbeit wurde die Substratspezifität von Ketosaminkinasen mit einer Reihe von Amadori-Produkten untersucht. Damit könnten Inhibitoren zur weiteren Enzymcharakterisierung oder sogar für pharmazeutische Anwendungen identifiziert werden. Außerdem wurde die Variabilität der Enzymaktivitäten von Mensch zu Mensch in einer Kohorte von 100 Probanden untersucht. Als Modell für die menschliche Kohlenhydratverdauung im Dünndarm wurden Caco-2-Zellen als Monolayer etabliert. Deren Sucrase-Isomaltase kann die alpha-glycosidische Bindung in Amadori-Produkten von Maltose und Maltotriose mit Lysin und auch in Maltulose hydrolysieren. Trotz der Aminogruppe hemmen diese Amadori-Produkte die Maltosehydrolyse nur schwach als konkurrierende Substrate. Lactulosyllysin konnte nicht durch die Lactase der Caco-2-Zellen hydrolysiert werden. Tagatosyllysin und die Heyns-Produkte Glucosyllysin und Mannosyllysin hemmten die Lactosehydrolyse schwach. Alle beobachteten Hemmeffekte sind wahrscheinlich zu schwach, um während der Verdauung in vivo bedeutsam zu sein. Für FN3K konnte Desoxypiperidinofructose als kompetitiver Inhibitor identifiziert werden (Kic 0,006 mM). FN3K zeigte nur geringe Selektivität gegenüber Amadori-Produkten verschiedener Amine, ausgenommen aromatischer Amine. FN3K-RP war in Erythrocyten wesentlich aktiver als FN3K, auch wenn die Aktivität nicht selektiv inhibiert werden konnte. Beide Enzymaktivitäten unterscheiden sich unter den 100 Probanden, mit einer Spannweite von 3 bis 12 mU/g Hämoglobin für FN3K und 60 bis 135 mU/g Hb für FN3K und FN3K-RP zusammen. Es scheint eine Verbindung zwischen der Ketosaminkinase-Aktivität in Erythrocyten und Nierenerkrankungen, familiär auftretendem Diabetes mellitus, sowie familiär aufgetretenen Herzinfarkten oder Schlaganfällen zu bestehen, wie orientierende Auswertungen zeigten. Deshalb ist eine genauere Untersuchung der physiologischen Bedeutung der Ketosaminkinasen nötig. / Amadori products are formed spontaneously from reducing sugars and amines, e.g. lysine, during the first phase of the Maillard reaction. They occur in heated food and in vivo. The thesis focuses on the enzymatic degradation of such spontaneously formed compounds. One part of this work investigated the faith and impact of oligosaccharide derived Amadori products during small intestinal carbohydrate digestion. Due to their structural similarity with known glycosidase inhibitors, an inhibitory action of Amadori products towards carbohydrate digestions was assumed. The other part dealt with fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) from human erythrocytes. Such ketosamine kinases are regarded as protein repair enzymes, maybe even an enzymatic defence against glycation in vivo. While deglycating protein bound Amadori products, however, they produce highly reactive 1,2-dicarbonyl compounds, which can lead to further protein damage. It is unclear, whether the ketosamine kinase action prevents or supports the pathophysiological effects of glycation. This work studied the substrate specifity of ketosamine kinases with a variety of Amadori products, which could result in inhibitors for further enzyme characterisation or even pharmaceutical uses. Further, the variability of both enzyme activities in a cohort of 100 subjects was examined. As a model for human small intestinal carbohydrate digestion, a Caco-2 cell monolayer was employed. Their sucrase-isomaltase is able to hydrolyse the alpha-glucosidic linkage in Amadori products of maltose and maltotriose with lysine, as well as in maltulose. Despite their amino group, those amadori products inhibited maltose hydrolysis merely weakly as competing substrates. Lactulosyl lysine on the other hand could not be hydrolysed by Caco-2 lactase. Tagatosyl lysine and the Heyns products glucosyl lysine and mannosyl lysine showed weak inhibition of lactose hydrolysis. All observed inhibitory effects are probably too weak to be of importance during carbohydrate digestion in vivo. Deoxypiperidinofructose was identified as a competitive inhibitor of FN3K (Kic 0,006 mM). FN3K acted rather non-specific towards Amadori products of different amines, except aromatic amines. FN3K-RP showed much higher activity in erythrocytes than FN3K, although its activity could not be inhibited selectively. Both enzyme activities vary among 100 subjects, with a range of 3 to 12 mU/g hemoglobin for FN3K and 60 to 135 mU/g hb for FN3K and FN3K-RP together. Relations of ketosamine kinase activity in erythrocytes with renal diseases, familial diabetes mellitus and familial cardiovascular events seem to exist. Thus, investigating the physiological impact of ketosamine kinases is necessary.

FONTES, Claudia Patrícia Mourão Lima. Produção de oligossacarídeos pre-bióticos em suco de frutas. 2013. 119 f. : Tese (doutorado) - Universidade Federal do Ceará, Programa de Pós-Graduação em Biotecnologia – RENORBIO, Fortaleza-CE, 2013. / Submitted by demia Maia (demiamlm@gmail.com) on 2016-05-20T15:01:32Z No. of bitstreams: 1 2013_tese_cpmlfontes.pdf: 1453775 bytes, checksum: 41a991c356e68b4bdf906f873dd5281e (MD5) / Approved for entry into archive by demia Maia (demiamlm@gmail.com) on 2016-05-20T15:02:03Z (GMT) No. of bitstreams: 1 2013_tese_cpmlfontes.pdf: 1453775 bytes, checksum: 41a991c356e68b4bdf906f873dd5281e (MD5) / Made available in DSpace on 2016-05-20T15:02:03Z (GMT). No. of bitstreams: 1 2013_tese_cpmlfontes.pdf: 1453775 bytes, checksum: 41a991c356e68b4bdf906f873dd5281e (MD5) Previous issue date: 2013 / Nowadays, people have been concerned with their quality of life and wellness,increasing the consumption of foods, with prebiotic oligosaccharides, which may improve the health and decrease the disease risks. In recent years, a lot of new foods have been developed with these compounds (prebiotic oligosaccharides)including the fruit juices. The aim of this work was the prebiotic oligosaccharides production in orange, pineapple and melon juices, and their spray drying.The dextransucrase, produced by L. mesenteroides NRRL B-512F, was used for enzymatic synthesis following a central composite experimental design, with sucrose and reducing sugars variation, at 30 °C/ 24h. The prebiotic juices obtained were dried by spray drying, varying the maltodextrin and arabic gum concentration (10 and 20%) and the inlet temperatures (160 and 180 °C). Pineapple, orange and melon juices showed better results when maltodextrin(20%) and the inlet temperature 180 °C were utilized. The juice powders obtained showed low water activity, moisture,higroscopicity and rehydration time. These results give better microbiology and physicochemical stability, as well as, fast preparation. There was a decrease of the antioxidant activity at all products. The fruit juices used demonstrated an excellent way to produce, by enzymatic synthesis, prebiotic oligosaccharides with high polymerization degrees. / Atualmente, a preocupação com a qualidade de vida tem aumentado a demanda por alimentos que melhorem a saúde e reduzam os riscos de doenças, dentre os quais destacam-se os oligossacarídeos pré-bióticos. Nos últimos anos, há um crescente interesse no desenvolvimento de novos produtos alimentícios contendo esses compostos, incluindo-se os sucos de frutas. Desta forma, este trabalho visou à produção de oligossacarídeos pré-bióticos em sucos de frutas e a secagem desses produtos pela técnica de spray-driyng. Para a síntese enzimática de oligossacarídeos utilizou-se a enzima dextrana-sacarase produzida pelo microorganismo L. mesenteroides B512F e realizou-se um planejamento experimental composto central, variando-se as concentrações de sacarose e açúcares redutores nos sucos, com os ensaios conduzidos durante 24h a 30°C. Os resultados obtidos demonstraram que os sucos de abacaxi, melão e laranja apresentaram-se como uma excelente alternativa para a síntese de oligossacarídeos pré-bióticos com elevados graus de polimerização. Os sucos pré-bióticos obtidos das diferentes frutas submetidos ao processo de Spray Drying, no qual variaram-se os agentes encapsulantes utilizados, maltodextrina e goma arábica, assim como, a temperatura do ar de entrada (160 e 180 °C). As melhores condições de secagem dos sucos de abacaxi, melão e laranja pré-bióticos foram verificadas, ao utilizar-se 20% de maltodextrina e temperatura de 180 °C. Foram realizadas secagens dos sucos prébióticos, nas melhores condições de secagem previamente determinadas, e observou-se que os pós obtidos apresentaram baixos valores para atividade de água, umidade, higroscopicidade e tempo de reidratação, conferindo uma maior estabilidade fisico-química e microbiológica ao produto, bem como, rápido preparo. A atividade antioxidante dos sucos também foi avaliada e verificou-se uma redução neste parâmetro. Conforme os resultados obtidos, constatou-se que os sucos de abacaxi, melão e laranja são excelentes substratos para a obtenção de uma bebida pré-biótica inovadora, a apresentar-se na forma líquida para consumo imediato ou na forma desidratada, para preparo instantâneo.