A captação de maltose em bactérias é feita por um sistema transportador do tipo ABC composto por uma proteína ligadora de substrato (MalE), duas proteínas transmembrana e uma ATPase. No presente trabalho descrevemos a clonagem, expressão e análise bioquímica e estrutural da proteína MalE da bactéria fitopatogênica Xanthomonas axonopodis pv citri (Xac) O gene malE de Xac foi clonado em vetor de expressão pET28a, a proteína recombinante foi expressa em Escherichia coli e purificada por cromatografia de afinidade ao níquel. Amostras da proteína solúvel foram analisadas quanto à estrutura secundária, interação com possíveis ligantes, estabilidade frente a diferentes condições físico-químicas. Ensaios de cristalização possibilitaram a obtenção de cristais em diferentes condições, um deles apresentou grupo espacial P6122, mas não foi possível resolver a estrutura. Com base nas estruturas conhecidas de ortólogos de MalE, geramos um modelo estrutural para a proteína de Xac e foram feitas análises quanto à interação com trealose e maltose. Modelos estruturais dos componentes transmembrana (LacF e LacG) e ATPase (UgpC) do sistema transportador de maltose de Xac também foram gerados. Os resultados representam uma contribuição importante para o conhecimento sobre a fisiologia e sistemas de transporte de Xac. / Maltose uptake in bacteria is mediated by an ABC transporter comprising a substrate binding protein (MalE), two transmembrane proteins, and one ATPase. In the present study, we describe the cloning, expression and biochemical as well as structural analyses of the MalE protein of the phytopagen Xanthomonas axonopodis pv citri (Xac). The malE gene of Xac was cloned in the pET28a expression vector, the recombinant protein was expressed in Escherichia coli and, subsequently, purified by nickel affinity chromatography. Samples of soluble protein were analyzed regarding secondary structure, interaction with putative ligants and stability under different physico-chemical conditions. Crystallization trials were carried out under different conditions, one particular condition yielded crystal with a P6122space group, but the structure was not solved. Based on known ortholog structures, a structural model for Xac MalE was obtained allowing interaction with modeled threhalose and maltose. Structural models the transmembrane (LacF and LacG) and ATPase (UgpC) components were also obtained. The present results represent an important contribution to the knowledge of the physiology and transporter systems found in Xac.

Das multiple Myelom ist eine seltene maligne Knochenerkrankung bei insbesondere älteren Menschen. Dabei vermehren sich im Knochenmark in hohem Maße unkontrolliert entartete Plasmazellen. Diese Myelomzellen unterdrücken einerseits die Bildung von normalen Plasmazellen, andererseits wird das Gleichgewicht zwischen Knochenaufbau und –abbau empfindlich gestört, woraus eine erhöhte Knochenresorption resultiert. Neben den bisher angewandten Chemo- und Strahlentherapien gewinnen innovative Medikamente, wie Proteasominhibitoren und Bisphosphonate, in der Therapie an Bedeutung. Diese Medikamente reduzieren das Myelomzellwachstum und wirken hemmend auf den Knochenabbau. Durch das Auffüllen von durch Resorptionsprozesse geschädigten Knochendefekten mit wirkstoffbeladenen Calciumphosphatzementen (CPC) wird nicht nur der Knochen stabilisiert, sondern im Vergleich zur herkömmlichen oralen oder intravenösen Medikamentenverabreichung eine gezielte Freisetzung des Wirkstoffes direkt am Wirkort in wesentlich reduzierten Dosen ermöglicht. Durch die Kombination des Knochenzementes mit anderen effizienten Drug-Delivery-Systemen (DDS), wie z. B. Polymeren, kann eine optimale Anpassung der Wirkstofffreisetzung ermöglicht werden. Insbesondere haben sich bereits dendritische Polymere aufgrund ihrer globularen Struktur und Vielzahl an peripheren Funktionalitäten als besonders geeignete Wirkstoffträgersysteme herausgestellt. Bei der Anwendung im physiologischen System spielt insbesondere die Biokompatibilität dieser polymeren DDS eine entscheidende Rolle. Durch Modifizierung der peripheren Gruppen mit biokompatiblen Einheiten, wie Oligosacchariden oder Aminosäuren, kann die physiologische Verträglichkeit signifikant erhöht werden. Für die Behandlung des multiplen Myeloms am Knochen sollte in dieser Arbeit ein geeignetes dendritisches DDS auf Basis von hochverzweigtem Polyethylenimin (PEI) synthetisiert und charakterisiert werden. Das DDS sollte dabei verschiedene Anforderungen, wie eine hohe Wasserlöslichkeit und Biokompatibilität, erfüllen. Weiterhin sollten die mechanischen Eigenschaften des CPC nicht negativ beeinflusst werden und der Wirkstoff sollte effektiv vom DDS aufgenommen und kontrolliert aus dem generierten Komposit (Wirkstoff/DDS/CPC) freigesetzt werden. In der sogenannten N-Carboxyanhydrid (NCA)-Polymerisation wurden am PEI(5) (5 ≙ Mw 5 kDa) benzylgeschützte Polyglutaminsäure bzw. Polyasparaginsäureketten aufgepfropft. Durch hydrolytische Abspaltung der Schutzgruppen an den PBLG-Ketten von PEI(5)-PBLG-346 und PEI(5)-PBLA-346 erfolgte die Generierung der wasserlöslichen DDS PEI(5)-PGlu-346 und PEI(5)-PAsp-346. Die Charakterisierung der synthetisierten Kern-Schale-Architekturen PEI(5)-PBLG-346, PEI(5)-PBLA-346, PEI(5)-PGlu-346 und PEI(5)-PAsp-346 zeigte, dass nur wenige lange Polyaminosäureketten an wenigen primären und sekundären Aminogruppen des PEI(5) aufgebaut wurden. Aufgrund der noch freien primären und sekundären Aminogruppen am PEI(5) und den peripheren Aminogruppen an den Polyaminosäureketten wurden durch die Anbindung von Maltose- bzw. Laktoseeinheiten Kern-Schale-Architekturen mit einer binären Doppelschalenstrukturen erzeugt. Im Gegensatz zu reiner Polyglutaminsäure zeigten die mit Glutaminsäure modifizierten Polymerstrukturen PEI(5)-PGlu-346 und PEI(5)-PGlu-346-Mal interessante strukturelle Eigenschaften in wässriger Umgebung. Aufgrund des pH-abhängigen Ladungszustandes resultiert bei reinen Polyglutaminsäureketten normalerweise der typische Helix-Coil-Übergang. Dabei findet eine Konformationsumwandlung der α-helikalen Struktur zur ungeordneten Sekundärstruktur statt. Im Falle der PEI(5)-PGlu-346- und PEI(5) PGlu-346-Mal-Copolymere wurde jedoch keine α-helikale Konformation bei niedrigem pH-Wert nachgewiesen. Die PGlu-Ketten der wasserlöslichen Kern-Schale-Architekturen bildeten sowohl im sauren, als auch im basischen pH-Wertbereich eine ungeordnete Sekundärstruktur aus. Zusätzlich konnte nachgewiesen werden, dass die Kern-Schale-Architekturen in Abhängigkeit vom pH-Wert als isolierte Makromoleküle bzw. Aggregate mit unterschiedlich lang gestreckten Peptidketten vorliegen. Die Ursache dafür sind nicht-kovalente, intra- und intermolekular wirkende Kräfte. Zur Beurteilung der Kern-Schale-Architekturen als geeignete DDS wurde die Komplexierung des Proteasominhibitors Bortezomib (BZM) in die reinen Copolymere PEI(5)-PGlu-346, PEI(5)-PGlu-346-Mal und PEI(25)-Mal B (25 ≙ Mw 25 kDa, ohne Polyglutaminsäureketten) sowie deren Polyelektrolytkomplexe untersucht. Dabei wurden Copolymer/BZM- bzw. PEK/BZM-Komplexe in verschiedenen Verhältnissen hergestellt und die Komplexierungskapazität durch zeitabhängige Ultrafiltration UV/Vis-spektroskopisch ermittelt. Im Vergleich zu den glutaminsäuremodifizierten Copolymeren wurde durch PEI(25)-Mal B etwa doppelt so viel Wirkstoff in verschiedenen wässrigen Systemen aufgenommen. Der Grund dafür ist der größere PEI-Kern und die dementsprechend höhere Anzahl an peripheren Aminogruppen mit gebundenen Maltoseeinheiten. Die PEK zeigten im Vergleich zu den Copolymeren keine Verbesserung der Komplexierungskapazität. Um eine effektive Wirkstofffreisetzung für eine dosierte Langzeittherapie aus dem Kompositmaterial zu erhalten, ist eine stark verzögerte Freisetzung des Copolymers bzw. PEK selbst aus dem CPC notwendig. In Abhängigkeit von der Konzentration wurde für PEI(25)-Mal B eine geringere Freisetzung aus dem Copolymer/CPC- und PEK/CPC ermittelt. Aufgrund der nanoskaligen Dimension der polymeren Strukturen wird die Diffusion durch das offene CPC-Porensystem erschwert. Für die PEI(5)-PGlu-346, PEI(5)-PGlu-346-Mal und die zugehörigen PEK wurde hingegen keine messbare Freisetzung aus dem CPC nachgewiesen. Die Glutaminsäureeinheiten können Calciumionen komplexieren und beeinflussen dadurch die Keimbildung und das Wachstum der CaP-Phase. Die Copolymerstrukturen werden somit in den CPC integriert und können nur durch Abbau des schwerlöslichen Zementes freigesetzt werden. Bei den Untersuchungen der BZM-Freisetzung aus den BZM/Copolymer/CPC- und BZM/PEK/CPC-Kompositen kristallisierte sich BZM/PEI(5)-PGlu-346-Mal/CPC als effektivstes DDS heraus. Im Vergleich zum reinen BZM in CPC wurde nach 24 h nur etwa die Hälfte des Wirkstoffes aus dem Komposit freigesetzt. Weiterhin steigerte sich die Freisetzungsrate über den gesamten Zeitraum von 14 Tagen auf nur etwa 60 %. Aus dem BZM/CPC-Komposit wurden nach 14 Tagen mehr als 75 % BZM freigesetzt. In Kooperation mit der Arbeitsgruppe von Prof. Michael Gelinsky vom Zentrum für Translationale Knochen-, Gelenk- und Weichgewebeforschung (TU Dresden) wurde keine signifikante Änderung der Druckfestigkeit des CPC durch die Integration der glutaminsäuremodifizierten Copolymere festgestellt. Weiterhin wurde in in vitro-Untersuchungen mit osteogen stimulierten humanen mesenchymalen Stammzellen (hMSC) kein entscheidender Einfluss der in dieser Arbeit hergestellten PEI(5)-PGlu-346- und PEI(5)-PGlu-346-Mal-Copolymere auf die Proliferation der Zellen beobachtet. Zudem war bei beiden Copolymeren eine osteogene Differenzierung der hMSC zu knochenbildenden Osteoblasten nachweisbar, wobei PEI(5)-PGlu-346-Mal die Entwicklung der Stammzellen zu knochenbildenden Zellen sogar zu fördern scheint. Durch die Kombination von hochverzweigtem PEI mit Polyglutaminsäure und Maltose wurde in dieser Arbeit ein innovatives DDS für die kontrollierte und effektiv verzögerte Freisetzung von BZM aus CPC erzeugt, welches die einleitend erwähnten Anforderungen erfüllt. Das Copolymersystem weist eine hohe Biokompatibilität auf, ohne die mechanischen Eigenschaften des CPC zu verändern. Diese Arbeit hat daher einen entscheidenden Beitrag im Bereich der Wirkstofffreisetzung aus festen Materialien geliefert und bildet die Grundlage für zukünftige polymere DDS in CPC.

<p>Sarcoptes scabiei is a disease-causing parasitic mite of humans and animals that is prevalent worldwide. The parasite lives in burrows in the epidermis of its host. These burrows are formed by a combination of mechanical destruction by the mite and secretion of various factors.</p><p>The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. In this project, the enzyme was expressed in transformed Escherichia coli cells. The SOD cDNA from S. scabiei was ligated into two different expression vectors: pPU16 and pET-14b. The S. scabiei SOD open reading frame reported here is 696 nucleotides long and yields a protein with a molecular weight of  69.5 kDa. Only one of the constructs was successfully created, using pPU16. The construct was designated pPU110 and has a sequence coding for a hexahistidine tag downstream of the SOD cDNA and has a sequence coding for the maltose binding protein (MBP) upstream.</p><p>The expression plasmid pPU110 was verified by DNA-sequencing and the tested in different expression experiments. Analysis using SDS-PAGE showed that recombinant fusion SOD could be readily expressed in E.coli.</p>

No description available.

Sarcoptes scabiei is a disease-causing parasitic mite of humans and animals that is prevalent worldwide. The parasite lives in burrows in the epidermis of its host. These burrows are formed by a combination of mechanical destruction by the mite and secretion of various factors. The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. As such, it is an important antioxidant defense in nearly all cells exposed to oxygen. In this project, the enzyme was expressed in transformed Escherichia coli cells. The SOD cDNA from S. scabiei was ligated into two different expression vectors: pPU16 and pET-14b. The S. scabiei SOD open reading frame reported here is 696 nucleotides long and yields a protein with a molecular weight of  69.5 kDa. Only one of the constructs was successfully created, using pPU16. The construct was designated pPU110 and has a sequence coding for a hexahistidine tag downstream of the SOD cDNA and has a sequence coding for the maltose binding protein (MBP) upstream. The expression plasmid pPU110 was verified by DNA-sequencing and the tested in different expression experiments. Analysis using SDS-PAGE showed that recombinant fusion SOD could be readily expressed in E.coli.

No description available.

New catalytic synthetic methods in organic chemistry that satisfy increasingly stringent environmental constraints are in great demand by the pharmaceutical and chemical industries. Studies over last 15 years have revealed that activity of enzymes can be increased in organic solvents rather than their natural aqueous environment. Because of their ease of use, high selectivity and environment friendliness, enzymes are enjoying increasing popularity in today's synthesis world. Chapter 1 describes chemo-enzymatic synthesis of various glycolipid analogs. A highly regioselective macrolactonization was achieved using lipase from Candida antarctica as a catalyst. It also describes evaluation of lipases from different source and their efficiency in catalyzing the macrolactonization reaction. These analogs were synthesized using commercially available agriculture based disaccharides (maltose, lactose, cellobiose, melibiose). These glycolipid analogues have potential applications in the cosmetic industry, formulation, food production, and pharmaceutical industry.In Chapter 2, ring opening polymerization of epsilon-caprolactone in ionic liquid, [bmim][PF6] was investigated. A comparative study of ROP in different solvents (toluene, Ionic liquid, and bulk condition) was conducted. Effect of time and enzyme concentration on molecular weight and % yield was investigated. It was concluded that enzymatic ring opening polymerization of epsilon-caprolactone in ionic liquid, [bmim][PF6 ] is a very competitive and environmental friendly way of synthesizing high molecular weight polyesters.

Dissertação (mestrado) - Universidade Federal de Santa Catarina. Programa de Pós-Graduação em Biotecnologia. / Made available in DSpace on 2012-10-22T16:56:48Z (GMT). No. of bitstreams: 1 231338.pdf: 2306929 bytes, checksum: d6024b930edc5b3c0a8fcc17e42eedfd (MD5) / In the present report we have analyzed the kinetics of growth, sugar consumption and ethanol production on synthetic or rich medium containing maltose or maltotriose by a S. cerevisiae strain with the AGT1 permease as its unique a-glucoside transporter. Our results show that in order to efficiently consume and ferment these sugars from the medium it is required constitutive expression of the transporter gene, which can be achieved by the presence of constitutive MAL (e.g. MAL63c) regulator. Besides constitutive expression of the permease, growth on rich medium also increased the fermentation performance of the cells. While a good correlation between the activity the AGT1 transporter and the fermentation performance of the cells was observed, the same was not true for the activity of the intracellular a-glucosidase. This AGT1-containing yeast strain ferments maltotriose more efficiently than maltose due to higher expression of the AGT1 permease during growth on maltotriose, leading to higher uptake rates of this sugar into the cells. Our results also indicate that during maltotriose fermentation by yeasts with constitutive expression of the AGT1 permease transport across the plasma membrane may no longer limit sugar utilization by S. cerevisiae cells.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Agrárias. Programa de Pós-Graduação em Ciência dos Alimentos / Made available in DSpace on 2012-10-26T12:32:04Z (GMT). No. of bitstreams: 1 301566.pdf: 5310006 bytes, checksum: 950d02c60470c1ac92a9b79a81c931e3 (MD5) / A cevada ocupa a quarta posição em produção de cereais no mundo. Porém, tem sido pouco utilizada na indústria de alimentos, sendo destinada, em sua maioria, para alimentação animal e produção de malte cervejeiro. No entanto, a cevada possui alto teor de fibra alimentar e alta proporção de fibras solúveis, especialmente ?-glucanas. Estudos têm demonstrado que as ?-glucanas possuem efeitos fisiológicos benéficos à saúde. Além da importância nutricional, as ?-glucanas apresentam importante papel tecnológico em alimentos processados. Portanto, a extração e comercialização das ?-glucanas já é uma realidade no mercado internacional. Outro importante polissacarídeo da cevada é o amido; porém, esta origem tem sido pouco valorizada, sendo desprezado nos atuais processos de extração das ?-glucanas. Considerando as inúmeras possibilidades de aplicação do amido na indústria de alimentos, é possível sugerir que este resíduo possui grande potencial tecnológico. Os amidos podem ser modificados para atender a demanda da indústria, sendo uma das modificações a hidrólise enzimática para produção de xaropes com alto teor de maltose. Assim, os objetivos deste estudo foram extrair as ?-glucanas de cevada e produzir xarope de alto teor de maltose a partir do amido residual da extração. A farinha de cevada do cultivar BRS 195 foi utilizada no estudo. A sua composição química foi determinada, as ?-glucanas e o amido foram extraídos, quantificados e parcialmente caracterizados. A fração amido residual foi hidrolisada enzimaticamente para a produção do xarope de maltose. Os resultados indicaram que a cevada utilizada no estudo é uma boa fonte de obtenção de ?-glucanas e amido, devido ao elevado teor destes componentes, 4,74 % e 58,07 %, respectivamente, e ao reduzido teor de lipídios, 2,44 %. A extração de ?-glucanas da cevada foi influenciada pela temperatura do processo, e a máxima concentração na fração extraída foi de 53%; porém, esta apresentou impurezas como amido e proteína. O comportamento reológico das ?-glucanas extraída foi semelhante ao de ?-glucanas comerciais, tendo a viscosidade diminuída com o aumento da temperatura. A semelhança com as ?-glucanas comerciais também foi observada na espectroscopia no infravermelho, que confirmou a contaminação com amido. A microscopia eletrônica de varredura demonstrou imagens características de um produto esponjoso tanto para ?-glucanas extraída, quanto para a comercial. A fração de amido residual apresentou 77% deste componente. As características de viscosidade (RVA) foram semelhantes ao amido disponibilizado comercialmente. A espectroscopia no infravermelho apresentou picos característicos de amido. Através da microscopia eletrônica de varredura foi possível visualizar grânulos de amido intactos, tipo A e tipo B. A hidrólise enzimática do amido resultou em alta produção de maltose e teve influencia linear da concentração de substrato, o que indica que a taxa de hidrólise para a produção de maltose diminui com o aumento da concentração do substrato. O teor de sólidos solúveis presentes na maltose está de acordo com a concentração inicial de amido utilizada no experimento. Considerando os resultados, pode-se concluir que o amido residual da extração das ?-glucanas de cevada é uma excelente alternativa para o aproveitamento de resíduos, ampliando a utilização deste cereal na indústria de alimentos / Barley is the fourth crop produced worldwide. However, it has been poorly utilized in the food industry, been left mostly for animal feed and malting barley. Nevertheless, barley is rich in dietary fiber and has a high content of soluble fibers, especially ?-glucans. Several studies have demonstrated that the ?-glucans have beneficial physiological effects on health. Beyond the nutritional importance, the ?-glucans show an important technological role in processed foods. So, the extraction and commercialization of ?-glucans is already a reality in the international market. Another important barley polysaccharide is the starch; however, this source has been undervalued, being despised in current processes for ?-glucans extraction. Considering the unlimited possibilities for starch application in the food industry, it is possible to suggest that this residue has a high technological potential. The starches can be modified to meet the industrial expectations, including enzymatic hydrolysis to produce high maltose syrups. The objects of this study were to extract ?-glucans barley and produce maltose syrup from residual starch. Barley flour from cultivar BRS 195 was used. Its chemical composition was determined, the ?-glucans and starch were extracted, quantified and partially characterized. The residual starch fraction was enzymatically hidrolyzed to produce maltose syrup. The results showed that the barley used in the study was a good source of ?-glucans and starch because of the high contents of these components, 4.74% and 58.07% respectively, and to the reduced fat content, 2.44%. The extraction of ?-glucanas was influenced by process temperature; the maximum concentration in the extracted fraction was of 53%, however, this showed impurities like starch and protein. The rheological behavior of the extracted ?-glucans was similar to that of commercial ones, with a decrease in viscosity upon increasing temperatures. The similarity with commercial ?-glucans was also observed with infrared spectroscopy that confirmed starch contamination. Scanning electron microscopy showed images characteristic of a spongy product for the extracted ?-glucans as well as for the commercial ones. The residual starch fraction contained 77% of starch and its viscosity (RVA) was similar to the one of commercially available starch. The infrared spectroscopy, presented characteristic starch peaks. Through scanning electron microscopy it was possible to visualize intact starch granules A type and B type. The enzymatic hydrolysis of the starch resulted in a high maltose production and had linear influence of the substrate concentration, which means that the hydrolysis rate for maltose production decreases with the increase in the substrate´s concentration. The amount of soluble solids present in the maltose is in agreement with the initial starch concentration used in the experiment. Considering the results, it can be concluded that the residual starch from the barley ?-glucans extraction is an excellent alternative to make good use of waste products, widening the utilization of this cereal in the food industry

Comparative studies of taste perception have found that primates may differ markedly in their sensitivity for substances perceived as sweet by humans. These findings raise questions about the reason that may underlie these differences in sweet-taste sensitivity between species. The aim of the present study was to assess the taste responsiveness of chimpanzees (Pan troglodytes verus) to seven substances tasting sweet to humans and to compare the results with those of other primate species. Using a two-bottle preference test (1 min) I found that the taste preference thresholds of the chimpanzees for five food-associated carbohydrates ranged between 20-30 mM for sucrose, 20-50 mM for fructose, 60-80 mM for glucose, 50-80 mM for maltose, and 30-80 mM for lactose. Taste preference thresholds for two steviol glycosides ranged from 0.04-0.05 mM for stevioside, and 0.03-0.05 mM for rebaudioside A. The chimpanzees displayed clear preferences for all sweet-tasting substances presented. In line with data obtained in other primates, the taste preference threshold of the chimpanzees for sucrose was lower compared to the other carbohydrates presented and the taste preference thresholds for stevioside and rebaudioside A were lower compared to sucrose. In general, the taste sensitivity of the chimpanzees fell into the range of data reported in other nonhuman primate species. Interestingly, the taste preference thresholds of the chimpanzees reported here are similar to the taste detection thresholds obtained in humans, despite the fact that the former are only a conservative approximation of an animal’s taste sensitivity. This suggests that chimpanzees may be as sweet-taste sensitive as humans.