Dissertations / Theses on the topic 'Bowman-Birk inhibitor'

MARTINS, Thiago Fernandes. Purificação e caracterização bioquímica de um inibidor tipo Bowman-Birk de sementes Luetzelburgia auriculata (Allemão) Ducke. 2015. 102 f. Dissertação (mestrado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2015.
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Plant protease inhibitors are proteins of low molecular weight, usually present in high concentrations in storage tissues, particularly in seeds of species belong to the Fabaceae family. In this study, a Bowman-Birk protease inhibitor (LzaBBI) was purified from the saline extract of the Luetzelburgia auriculata seeds After boiling of this saline extract, the inhibitor was purified by affinity chromatography on Sepharose® 4B-anhydrotrypsin, followed by ion exchange chromatography on DEAE Sepharose and reverse phase chromatography. Under reducing conditions or not, LzaBBI showed an apparent molecular mass of 17.3 kDa and a single polypeptide chain. The NH2-terminal sequencing of the LzaBBI showed high similarity with other Bowman-Birk inhibitors of legumes. LzaBBI remained stable after boiling at 98 °C for 120 min and also remained stable with trypsin inhibitory activity after incubation in pH buffers with pHs varying from 2 to 11. In addition to its relevant structural stability, of importance in future biotechnological applications, LzaBBI showed negative impact on the Staphylococcus aureus development when at low doses.
Inibidores de proteases vegetais são proteínas de baixa massa molecular, geralmente presentes em altas concentrações nos tecidos de armazenamento, particularmente em sementes de plantas pertencentes à família Fabaceae. Neste estudo, um inibidor de proteases pertencente à família Bowman-Birk (LzaBBI) foi purificado a partir do extrato salino de sementes de Luetzelburgia auriculata. Após fervura desse extrato salino, o inibidor foi purificado por cromatografia de afinidade em matriz de anidrotripsina-Sepharose® 4B, seguido de cromatografia em matriz de troca iônica (DEAE Sepharose) e cromatografia em fase reversa. Em condições redutoras, ou não, o LzaBBI apresentou massa molecular aparente de 17,3 kDa, e uma única cadeia polipeptídica. Sua sequência NH2-terminal possui alta similaridade com inibidores do tipo Bowman-Birk de leguminosas. O LzaBBI permaneceu estável após fervura a 98 °C por 120 min, bem como após incubado na faixa de pHs entre 2 a 11. Além de possuir relevante estabilidade estrutural, de importância para futuras aplicações biotecnológicas, apresentou efeito negativo no crescimento da bactéria Staphylococcus aureus, quando em baixas concentrações.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Inibidores de proteases vegetais sÃo proteÃnas de baixa massa molecular, geralmente presentes em altas concentraÃÃes nos tecidos de armazenamento, particularmente em sementes de plantas pertencentes à famÃlia Fabaceae. Neste estudo, um inibidor de proteases pertencente à famÃlia Bowman-Birk (LzaBBI) foi purificado a partir do extrato salino de sementes de Luetzelburgia auriculata. ApÃs fervura desse extrato salino, o inibidor foi purificado por cromatografia de afinidade em matriz de anidrotripsina-Sepharose 4B, seguido de cromatografia em matriz de troca iÃnica (DEAE Sepharose) e cromatografia em fase reversa. Em condiÃÃes redutoras, ou nÃo, o LzaBBI apresentou massa molecular aparente de 17,3 kDa, e uma Ãnica cadeia polipeptÃdica. Sua sequÃncia NH2-terminal possui alta similaridade com inibidores do tipo Bowman-Birk de leguminosas. O LzaBBI permaneceu estÃvel apÃs fervura a 98 ÂC por 120 min, bem como apÃs incubado na faixa de pHs entre 2 a 11. AlÃm de possuir relevante estabilidade estrutural, de importÃncia para futuras aplicaÃÃes biotecnolÃgicas, apresentou efeito negativo no crescimento da bactÃria Staphylococcus aureus, quando em baixas concentraÃÃes.
Plant protease inhibitors are proteins of low molecular weight, usually present in high concentrations in storage tissues, particularly in seeds of species belong to the Fabaceae family. In this study, a Bowman-Birk protease inhibitor (LzaBBI) was purified from the saline extract of the Luetzelburgia auriculata seeds After boiling of this saline extract, the inhibitor was purified by affinity chromatography on Sepharose 4B-anhydrotrypsin, followed by ion exchange chromatography on DEAE Sepharose and reverse phase chromatography. Under reducing conditions or not, LzaBBI showed an apparent molecular mass of 17.3 kDa and a single polypeptide chain. The NH2-terminal sequencing of the LzaBBI showed high similarity with other Bowman-Birk inhibitors of legumes. LzaBBI remained stable after boiling at 98 ÂC for 120 min and also remained stable with trypsin inhibitory activity after incubation in pH buffers with pHs varying from 2 to 11. In addition to its relevant structural stability, of importance in future biotechnological applications, LzaBBI showed negative impact on the Staphylococcus aureus development when at low doses.

Os inibidores inibidores de serino proteinases do tipo Bowman-Birk (BBI) possuem dois sítios ativos e são encontrados em plantas das famílias Fabaceae e Poaceae. Neste trabalho foi apresentada a estrutura primária e o padrão de expressão de 14 seqüências expressas (EST, expressed sequence tags) de BBI putativos encontradas no banco de dados do "Projeto Transcriptoma da Cana-de-açúcar" (SUCEST). Estas quatorze seqüências foram utilizadas em conjunto com outras 87 seqüências de BBI previamente descritas e depositadas no banco de dados "GenBank" para a construção de árvores filogenéticas da família BBI. A análise filogenética mostrou que os BBI de monocotiledôneas e dicotiledôneas podem ser claramente separados em diferentes grupos e a topologia das árvores filogenéticas sugere um padrão evolutivo diferente das famílias de BBI em plantas. Os inibidores de dicotiledôneas são bem conservados e acumularam diferenças sutis durante a evolução. Em contrapartida, os inibidores de monocotiledôneas são altamente variáveis, indicando a ocorrência de um processo evolutivo interessante, baseado em eventos de duplicação intragênica e mutação. Dois inibidores de serino proteinases, um de tripsina e outro de quimotripsina, derivados do gene que codifica o inibidor Bowman-Birk de soja, foram expressos na superfície do fago filamentoso M13 e utilizados para a construção de bibliotecas de variantes. Para tal foram feitas mutações em quatro aminoácidos do sítio ativo destes inibidores e duas bibliotecas de expressão na superfície de fagos filamentosos foram construídas. Posteriormente, estas bibliotecas foram utilizadas para a seleção de variantes que melhor interagiam com a tripsina bovina e de Diatraea saccharalis e com as enzimas digestivas presentes no extrato intestinal desta praga. Os variantes selecionados foram seqüenciados, analisados e caracterizados. Os resultados mostraram que a técnica de expressão na superfície de fagos filamentosos foi eficiente para selecionar novos inibidores. Além disto, com as mutações realizadas, foi possível transformar a alça de inibição de quimotripsina em uma alça de inibição de tripsina.
The Bowman-Birk inhibitors (BBIs) are double headed inhibitors of serine proteinase found in plants from Fabaceae and Poaceae families. We describe the primary structure and the gene expression profile of 14 putative BBIs from the sugarcane expressed sequence tag (SUCEST) database and show how we used these newly discovered sequences together with 87 previously described BBI sequences from the "GenBank" database to construct phylogenetic trees for the BBI family. Phylogenetic analysis revealed that BBI-type inhibitors from monocotyledonous and dicotyledonous plants could be clearly separated into different groups, while the overall topology of the BBI tree suggests a different pattern of evolution for BBI families in flowering plants. We also found that BBI proteinase inhibitors from dicotyledonous plants were well conserved, accumulating only slight differences during their evolution. In addition, we found that BBIs from monocotyledonous plants were highly variable, indicating an interesting process of evolution based on internal gene duplications and mutation events. Two serine-type proteinase inhibitors, a trypsin and a chymotrypsin, both derived from the soybean Bowman-Birk inhibitor, were expressed on the surface of a filamentous phage. Site mutations were made in four positions of the reactive sites of these inhibitors and two phage-display libraries were constructed. Later, these libraries were used to select better ligands to the bovine and Diatraea saccharalis trypsin and to the midgut enzymes of this insect pest. The selected variants were sequenced, analyzed and characterized. The results showed that the phage-display technique is efficient to select new proteinase inhibitors. Furthermore, it was possible to modify the chymotrypsin loop into a trypsin loop using the library constructed by the insertion of a degenerated primer.

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.

Proteins that recognise the sugar surface structures on cells have an enormous potential to be used as tools in the characterisation of these structures. A group of proteins, called lectins, have been identified that can bind to carbohydrate complexes on the receptors of cells. The crude extract from Grevillea robusta seeds was found to contain lectin-like proteins that were different from most other lectins, as they would specifically target the receptors of white blood cells and not those found on red blood cells. Therefore, the lectin isolated from G.robusta could be used as a tool to identify the specific surface structures on white blood cells. The lectin was isolated using affinity chromatography where a complex (oligosaccharide) matrix was used. Agglutination, binding and sugar inhibition assays confirmed the isolated protein was a lectin. The lectin was found in low amounts (up to 5% of the total protein content) within the seeds of G.robusta. As a result of this low yield, the identification of the lectin by PAGE was difficult because the levels of protein were beyond the detection limit of the commercial staining reagents. The lectin was called the GR2 protein and was characterised as a monocot mannose binding lectin based on its sugar specificity for only mannose. A serine protease inhibitor was isolated from the seeds of G.robusta using two different chromatography methods, reverse phase HPLC (GR1.HPLC) and gel filtration chromatography (GR1.GF). Ion exchange chromatography was used to initially separate the proteins in the crude extract and the fraction containing the GR1 protein was further purified using reverse phase HPLC (GR1.HPLC). N-terminal sequencing results of the GR1.HPLC protein, showed evidence of proteolytic cleavage during the extraction process, which lead to the second purification method being established. Protease inhibitors were added to the buffers prior to being purified by gel filtration chromatography, which resulted in the GR1 protein being isolated from the crude extract without the presence of the contaminating protein. Mass spectroscopy identified the molecular weight of the GR1 protein to be 6669Da and the full amino acid sequence was derived by cDNA techniques. Sequence alignment studies of the GR1 protein showed significant similarities with the Bowman-Birk inhibitor. The positioning of the cysteine residues were conserved throughout the Bowman-Birk superfamily, however these residues were not conserved within the GR1 protein. Competitive inhibition assays on the GR1 protein revealed the protein could inhibit both trypsin and chymotrypsin at similar levels to that seen for the Bowman-Birk inhibitor. Therefore, the GR1 protein was characterised as a member of the Bowman-Birk superfamily of serine protease inhibitors. The three-dimensional structure of the GR1 protein was determined using two-dimensional NMR spectroscopy. Computer programs such as XEASY, DYANA and SYBYL® were used to tabulate the information taken from the 2D experiments, generate structures and minimise these structures respectively. The solution structure of the GR1 protein was found to contain a region of antiparallel β-sheet structure that corresponded to the trypsin binding site and the remainder of the protein consisted of loops and turns that were held together by disulfide bridges (the chymotrypsin-binding region). Structural similarities between the GR1 protein and the Bowman-Birk inhibitor existed only in the trypsin-binding site of the Bowman-Birk inhibitor. The GR1 protein is the first member of the Proteaceae family to be characterised as a Bowman-Birk inhibitor. This thesis outlines the isolation and biochemical characterisation of the two proteins found within Grevillea robusta and also describes the steps involved and results obtained in determining the three-dimensional structure of the GR1 protein.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Science
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Die vorliegende Arbeit beschäftigt sich mit der Herstellung von funktionellen Magnetit-Nanokompositen durch Sprühtrocknung für die Anwendung in der Bioseparation. Dabei liegen die Schwerpunkte auf der Anwendung von Polyelektrolyten als Ionenaustauscher sowie auf der Nachhaltigkeit des Herstellungsprozesses. Basierend auf einem existierenden Herstellungsprozess wurde die Aufgabenstellung konkretisiert. Es wurden Möglichkeiten zur nachhaltigen Prozessgestaltung der Synthese von kationischen bzw. anionischen magnetischen Funktionspartikeln zur Proteinabtrennung vorgestellt. Als magnetische Komponente wurde Magnetit verwendet. Aufgrund seines pseudo-amphiphilen Charakters und seiner besonderen Eigenschaften in Bezug auf die Stabilisierung von Magnetit-Kolloiden wurde Polyvinylbutyral (Mowital B 30T) als Matrixpolymer bei der Sprühtrocknung benutzt. Für die nachhaltige Prozessgestaltung wurden Isopropanol und Tetrahydrofuran als Dichlormethan-Ersatz bei der Sprühtrocknung verwendet. Bei der Synthese kationischer Magnetic Beads wurden verzweigtes Polyethylenimin und lineares Poly(Allyamin) als Anionenaustauscher verwendet. Beide Polykationen sind schwache Polyelektrolyte mit Aminogruppen. Für die Verarbeitung der Polykationen als funktionelle Liganden in magnetischen Funktionspartikeln wurde zwei Herstellungsmethoden vorgestellt: eine Synthese ohne Oberflächenmodifizierung, wobei die mechanische und chemische Stabilität der Funktionspartikeln einzig von der chemischen Struktur der eingesetzten Materialien bzw. vom Matrixpolymer abhängt, und eine Synthese mit Oberflächenmodifizierung. Die Synthese mit Oberflächenmodifizierung ist gekennzeichnet durch die kovalente Bindung von Polyethylenimin bzw. Poly(Allyamin) an der Oberfläche der Funktionspartikeln (Polyvinylbutyral). Dafür wurde 1,1’-Carbonyldiimidazol als „zero length“-Crosslinker benutzt. Die nach beiden Methoden hergestellten Funktionspartikeln wurden charakterisiert, um ihre technische Eignung beurteilen zu können. Für die Charakterisierung der sorptiven Eigenschaften wurde unter anderem der Bowman-Birk Inhibitor (BBI) verwendet. Das Protein ist ein Sojaprodukt und für seine krebsvorbeugende Wirkung bekannt. Um die Selektivität der Abtrennung zu untersuchen, wurden BBI-Produkte mit unterschiedlichen Reinheitsgraden benutzt. Durch die zwei vorgestellten Methoden konnten kationische magnetische Funktionspartikeln erfolgreich hergestellt werden. Alle Funktionspartikeln sind superparamagnetisch, und der Medianwert ihrer Partikelgrößenverteilung liegt im einstelligen Mikrometerbereich. Aufgrund eines höheren Polykationanteils ist die Bindungskapazität der Funktionspartikeln ohne Oberflächenmodifizierung um den Faktor 2,4 größer als die BBI-Bindungskapazität der Funktionspartikeln mit Oberflächenmodifizierung (Qmax=322 mg/g). Das Fehlen eine feste Anbindung des funktionellen Liganden an den Funktionspartikeln ohne Oberflächenmodifizierung verleiht jedoch diesen eine sehr schlechte chemische Stabilität in Lösungen. Es wurde auch gezeigt, dass oberflächenmodifizierte Funktionspartikeln mit ähnlichen Eigenschaften durch den Einsatz von Dichlormethan bzw. Tetrahydrofuran als Lösungsmittelersatz während der Sprühtrocknung hergestellt werden können. Durch den Einsatz von mit Poly(allylamin) oberflächenmodifizierten Funktionspartikeln konnte BBI von technischen Sojamolken unterschiedlicher Reinheitsgrade erfolgreich abgetrennt werden. Anionische Magnetic Beads wurden mit Kationenaustauscherharz als funktionellem Ligand hergestellt. Dabei wurde Isopropanol als organisches Lösungsmittel während der Sprühtrocknung verwendet. Die Synthese wurde analog zur Synthese der kationischen Magnetic Beads ohne Oberflächenmodifizierung durchgeführt. Es wurde auch hier gezeigt, dass anionische magnetische Funktionspartikeln mit guten sorptiven Eigenschaften durch den Einsatz von Isopropanol als organisches Lösungsmittel hergestellt werden können. Die anionischen Funktionspartikeln besitzen im Vergleich zu Literaturwerten höhere Bindungskapazitäten (bis 280 mg/g; ermittelt mit Lysozym). Im letzten Kapitel wird der kritische Prozessschritt des Lösungsmittelaustausches behandelt. Nach dem Lösungsmittelaustausch sollten die Magnetitnanopartikeln in der organischen Phase stabil sein. Dies ermöglicht sowohl eine homogene Verteilung der Nanopartikeln in der Matrix als auch deren bessere Verkapselung während der Sprühtrocknung. Es wurde festgestellt, dass sich eine vollständige Abtrennung von Dichlormethan durch die angewendete Destillationsmethode nicht erreichen lässt. Anhand von zwei Modellsystemen — Rizinolsäure- und Ölsäure-beschichteten Magnetitnanopartikeln — und Lösungsmittelgemischen wurde die Stabilität von sterisch stabilisierten Magnetitpartikeln in binären Lösungsmittelgemischen untersucht. Der Fokus bei dieser Untersuchung lag auf der Untersuchung der Stabilität der beschichteten Magnetitnanopartikeln in einer möglichst Dichlormethan- bzw. Isooktan-freien organischen Phase. Als zweites Lösungsmittel (als Lösungsmittelersatz betrachtet) wurden neben Tetrahydrofuran und Isopropanol technisch verbreitete Lösungsmittel wie Isooktan und 1-Butanol eingesetzt. Die Untersuchungsergebnisse zeigen, dass die Anwendung der technischen Rizinolsäure bzw. Ölsäure einen zusätzlichen Einfluss auf die Stabilität der Magnetitpartikeln hat, da diese aus anderen Fettsäuren mit unterschiedlichen chemischen Strukturen bestehen. Die Diskrepanz zwischen der berechneten HANSEN-Distanzen und der Stabilität der Magnetitnanopartikeln mit reinen Fettsäuren lässt vermutet, dass die Zusammensetzung der Lösungsmittelgemische an der fest/flüssig-Grenzfläche anders ist als im freien Volumen. Ein Modell zur Beschreibung der Stabilität der Nanopartikeln, das auf einer Anreicherung des Ausgangslösungsmittels an der Grenzfläche basiert, wurde postuliert. Solange die Diffusion des zweiten Lösungsmittels in die Adsorptionsschicht nicht ausreichend genug ist, um die Löslichkeit der Fettsäureketten entscheidend zu reduzieren und somit einen Abfall der Abstoßungskräfte zwischen der Partikeln hervorzurufen, bleiben alle beschichteten Magnetitnanopartikeln stabil im Lösungsmittelgemisch. Dies ist der Fall für die mit der reinen Rizinolsäure beschichteten Magnetitnanopartikeln in allen verwendeten Lösungsmittelgemischen mit 0,5 Vol. % DCM in der kontinuierlichen Phase. Durch die vorgestellten Herstellungsmethoden wurde gezeigt, dass magnetische Funktionspartikeln sowohl mit festen partikelförmigen Ionenaustauschern als auch mit flüssigen schwachen Polyelektrolyten erfolgreich synthetisiert werden können. Eine nachhaltige Prozessgestaltung durch die Reduzierung der Dichlormethanmenge im Sprühtrocknungsprozess ist auch möglich. Für eine erfolgreiche industrielle Anwendung der Funktionspartikeln müssen aber ihre chemischen sowie mechanischen Eigenschaften deutlich verbessert werden. Dies könnte z.B. durch die Verwendung eines anderen Matrixpolymers oder durch die Entfernung von nicht gebundenen Bestandteilen durch gezielte Waschung der Funktionspartikeln erfolgen. Die Bindungskapazität sowie die Selektivität der oberflächenmodifizierten Funktionspartikeln sollte ebenfalls verbessert werden. Dafür wurde einen Ansatz durch die Quaternisierung der Aminogruppen präsentiert. Schließlich würde die Untersuchung der Stabilität der beschichteten Magnetitnanopartikeln in einphasigen reinen Lösungsmitteln nähere Erkenntnisse über das postulierte Modell der Anreicherung von Dichlormethan in der Adsorptionsschicht erbringen. Dabei könnte die Dichlormethanmenge durch mehrstufige Destillation bzw. Rektifikation beim Lösungsmittelaustausch entfernt werden. Eine vollständige Untersuchung dieses Effekts würde zusätzlich eine Antwort auf zahlreiche Fragestellungen der Kolloidchemie in Bezug auf das Stabilitätsverhalten von Pigmentdispersionen (Lacke) oder von beschichteten Nanopartikeln in Polymerlösungen erbringen.

La biodiversità naturale è un’importante risorsa per l’uomo sin da tempi antichi. Tuttavia, sta sperimentando un critico declino e molte specie sono a rischio estinzione. Il cambiamento climatico e l’attività umana sono i maggiori drivers di una conservazione e gestione del territorio non sostenibili. L’opportunità di ammortizzare gli effetti del cambiamento climatico è fornita da specie indigene, domesticate in contesti locali ma poco investigate. In questo contesto, questa tesi mira a sviluppare strategie per valorizzare la biodiversità naturale, considerando diversi e integrati aspetti scientifici. Il primo obiettivo è quello di riscoprire un legume tradizionale africano, Vigna unguiculata (L.) Walp., e verificare la sua adattabilità a condizioni di stress tipiche del cambiamento climatico o pratiche agricole poco esigenti. La ricerca di composti bioattivi è un valore aggiunto al fine di dare coscienza della potenzialità della specie. Per questa ragione, i successivi obiettivi della tesi sono l’esplorazione della diversità genetica di composti bioattivi dei legumi, gli inibitori delle proteasi Bowman-Birk (BBIs), e la valutazione delle proprietà nutraceutiche. Un design sperimentale multidisciplinare è stato applicato integrando diversi approcci per creare un flusso di lavoro coerente. Un esperimento di campo e analisi di produzione e metaboliti è stato organizzato per dimostrare l’adeguatezza di Vigna unguiculata come coltivazione per il cambiamento climatico. La diversità genetica di questa specie è stata esplorato con tecniche di biologia molecolare e analisi computazionali e filogenetiche. Le proprietà nutraceutiche sono state determinate grazie a procedure biochimiche e test su modelli, cellulari e in vivo, di cancro ed invecchiamento. Dal punto di vista della coltivazione, Vigna unguiculata può essere considerata una specie poco esigente in termini di richiesta d’acqua e pratiche agronomiche. Ciò rende questo legume adatto a pratiche di agricoltura conservativa in paesi in via di sviluppo o in quei paesi colpiti fortemente dal cambiamento climatico. Questo legume si dimostra importante come risorsa di macronutrienti essenziali, e, per promuovere la sua diffusione a livello globale, abbiamo indagato anche la presenza di molecole con azione dirette per la salute umana. L’esplorazione genetica ha considerato quasi 200 accessioni, tovando13 diverse isoforme di BBI tra accessioni selvatiche e domesticate distribuite sul continente Africa e in altre parti del mondo. In aggiunta, abbiamo sviluppato una metodica estrattiva e purificative che ha permesso l’isolamento e caratterizzazione delle singole isoforme di BBI. La dimostrazione di attività correlate a BBI nei diversi modelli, rende questa famiglia di proteine un valore aggiunto per la salute umana. L’azione verso linee cellulari tumorali suggerisce possibili applicazioni terapeutiche anche in sinergia con farmaci d’elezione. Ciò apre opportunità per la futura ricerca in specie e generi affini e la valutazione di isoforme maggiormente efficaci anche in sistemi in vivo. Concludendo, questo progetto dimostra che i) la bioprospezione per la ricerca di molecole bioattive in specie regionali è un importante passo per la loro salvaguardia, ii) conoscere evoluzione e diversificazione di piante di interesse è uno strumento per migliorare azioni bioprospettive e identificare migliori varianti di composti bioattivi, iii) analisi di efficacia funzionale in vitro e in vivo è un passaggio fondamentale per dedicare ricerca scientifica al miglioramento della biodiversità in contesti operativi. Quest’ultima è una fase importante per stimolare investitori, sia privati che pubblici, al fine di portare valore economico e sociale alla conservazione della biodiversità.
Natural biodiversity is an important source for humans since ancient times. However, biodiversity is experiencing a dramatic decline and many species are at extinction risk. Climate change and human activity are the main drivers of non-sustainable landscape conservation and management. The opportunity to dampen climate change effects is provided by indigenous species, domesticated in local contexts but are little investigated. In this framework, this PhD thesis aims at developing strategies to valorise natural biodiversity, considering different integrative scientific aspects. The first objective of this thesis is the rediscovery of a traditional African legume, Vigna unguiculata (L.) Walp., and assess its adaptability to stressful conditions typically caused by climate change or undemanding agricultural practices. Moreover, the research for bioactive compounds is an added value to give consciousness of the species potential. For this purpose, exploration of genetic diversity of known legume bioactive compounds, the Bowman-Birk protease inhibitors (BBIs) and appraisal of their nutraceutical properties are the second objectives of the project. A multidisciplinary experimental overview has been applied by integrating different approaches to create a coherent workflow. The demonstration of Vigna unguiculata L. as suitable species for climate change was carried out with a field experiment and subsequent laboratory analyses to evaluate production parameters and metabolic features. The genetic diversity of this species was explored through molecular biology techniques and in silico computational and phylogenetic analyses. The nutraceutical features were established by biochemical procedures and cellular biology by testing compounds on different ageing and cancer models. From the point of view of cultivation needs, it is possible to consider V. unguiculata (L.) Walp. as undemanding in terms of water demand and agronomic practices. This makes this legume suitable for conservation agriculture practices in developing countries and where climate change is having a dramatic impact on indigenous crop. This legume is also an important resource of essential macronutrients and to enhance this species and promote its cultivation globally, we also wanted to focus on the presence of bioactive molecules with direct action on humans. The genetic exploration considered almost 200 accessions and found 13 isoforms of BBI were identified in different wild and cultivated accessions, distributed in the African continent and in other areas of the world. Furthermore, we managed to develop an extraction and purification procedure to isolate single isoforms and characterise them. Our data suggest that V. unguiculata BBIs possess a great natural genetic and biochemical diversity. Moreover, the demonstration of BBI-related bioactivities on different models makes them very promising as a high-value natural compound for human wellbeing. The direct action on different tumour cell lines suggests a possible therapeutic application also in synergy with some drugs (i.e. Cetuximab). This opens opportunities for future research on similar related species and genera, and on the analyses to evaluate the most effective isoforms also in in vivo systems. In conclusion, this PhD project demonstrates that i) bioprospection of local species directed to the search for bioactive molecules represents an important lever for safeguarding; ii) the knowledge of evolution and diversification of the plants of interest is a tool to improve bioprospecting actions and identify molecular variants of bioactive compounds; iii) analyses of functional efficacy of bioactive compounds in in vitro and in vivo systems is a fundamental step to dedicate scientific research to the enhancement of biodiversity in an operational context. This is an essential phase to stimulate private investors and businesses to bring economic and social value and biodiversity conservation.

Orientador: Maria Lígia Rodrigues Macedo
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Os inibidores de proteinases extraídos de plantas têm se mostrado promissores como um método alternativo no combate aos insetos-pragas. Neste estudo, um inibidor de peptidase foi isolado de sementes de Clitoria fairchildiana (Papilionoideae), denominado CFPI, caracterizado funcional e estruturalmente e sua atividade inseticida foi avaliada. CFPI foi purificado por exclusão molecular, seguido por coluna de interação hidrofóbica e apresentou um pico majoritário com atividade inibitória após ter sido submetido à filtração com alta resolução. Estudos cinéticos realizados com CFPI purificado mostraram uma atividade inibitória do tipo competitiva contra tripsina e quimotripsina bovinas, com uma estequiometria de inibição de 1:1 para ambas as enzimas. A constante de inibição de CFPI contra tripsina e quimotripsina bovinas foram 3,3 x 10-10 e 1,5 x 10-10 M, respectivamente, revelando uma forte capacidade de ligação. Eletroforese em SDS-Page mostrou que CFPI possui uma única cadeia polipeptídica, com uma massa molecular aparente de 15 kDa, sob condições não redutoras. Entretanto, o inibidor apresentou uma massa acurada de 7973 Daltons determinada por MALDI-TOF, sugerindo que CFPI forme dímeros em solução. Essa característica, aliada à estequiometria de inibição para tripsina e quimotripsina, à constante de inibição (Ki) para ambas as enzimas e ao sequenciamento e alinhamento N-terminal, permitiram classificar CFPI como membro da família Bowman-Birk de inibidores. O inibidor manteve-se estável ao aquecimento progressivo por 30 min a cada temperatura, variando de 37 até 100 ?C e a análise de dicroísmo não mostrou mudanças no espectro a 207 nm após aquecimento à 90 ?C e subsequente resfriamento. Além disso, CFPI mostrou atividade sobre uma ampla faixa de pH (2-10). Em contraste, a redução de CFPI com DTT resultou em perda de atividade inibitória contra tripsina e quimotripsina. CFPI exibiu atividade inibitória considerável contra enzimas tripsinas de Anagasta kuehniella (76%), Diatraea saccharalis (59%) e Heliothis virescens (49%). Suas propriedades inseticidas foram confirmadas a partir do impacto negativo causado no crescimento de A. kuehniella e C. cephalonica. O inibidor exerceu efeito antinutricional sobre A. kuehniella tanto na geração F0 como em F1
Abstract: Proteinase inhibitors isolated from plants have shown a promising alternative method against insect pests. In this study, a proteinase inhibitor was isolated from Clitoria fairchildiana seeds (CFPI). CFPI was functional and structurally characterized and its insecticidal activity was evaluated. CFPI was purified by molecular exclusion, following by hydrophobic interaction column and showed a majoritarian peak with inhibitory activity after high resolution filtration gel column. Kinetic studies of the purified inhibitor showed a competitive¿type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3 ×10?10 and 1.5 × 10?10 M, respectively, displaying a tight binding property. SDS¿PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15 kDa under non¿reducing conditions. However, MALDI¿TOF analysis demonstrated a molecular mass of 7.973 Da, suggesting that CFPI forms dimers in solution. This feature, combined with the stoichiometry of inhibition for trypsin and chymotrypsin, the inhibition constant (Ki) for both enzymes and the N-terminal sequencing, allowed classifying CFPI as a member of Bowman-Birk family inhibitors. CFPI remained stable to progressive heating for 30 min to each temperature range of 37 up to 100 °C and CD analysis exhibited no changes in spectra at 207 nm after heating at 90 °C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2¿10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited remarkable inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on growth of A. kuehniella and C. cephalonica. The inhibitor exhibited antinutritional effect on A. kuehniella in the F0 and F1 generations
Doutorado
Bioquimica
Doutora em Biologia Funcional e Molecular

碩士
國立成功大學
化學系碩博士班
93
A 16 kDa protease inhibitor had been purified from rice coleoptiles grown in hypoxia condition. This protease inhibitor showed a competitive inhibitor toward trypsin. In order to study the optimum inhibitory ability of this protease, we had measured its activity against trypsin under various pHs and in the presence of Mg2+. It was found that under weak basic condition, it showed better activity to inhibit trypsin. To further study the effect of the presence of Mg2+, various concentration of Mg2+ were added. It was found that at pH7.8 and with the presence of 0 M, 0.5 M, 1.0 M, 1.5 M, and 2.0 M of Mg2+, the respective Ki values were 4.84 x 10-10 M, 4.56 x 10-10 M, 4.39 x 10-10 M, 3.07 x 10-10 M, and 2.45 x 10-10 M, showing that the inhibitory activity in the presence of 2.0 M Mg2+ was about twofold of that without the presence of Mg2+. By contrast, same measurements for soybean protease inhibition activity, it was found that with the increase of the concentration of Mg2+ the inhibition activity decreased with the respective Ki values were 4.21 x 10-11 M, 6.24 x 10-11 M, 8.46 x 10-11 M, 9.34 x 10-11 M, and 1.18 x 10-10 M. It was found that the secondary structures were change with the presence of 2 M of Mg2+, whereas the tertiary structure was unchanged. The fluorescence study showed that the tryptophan fluorescence intensity increased indicating that there was a micro-environmental change around the tryptophan residues. ANS fluorescence also showed there was an increase for hydrophobic area in the presence of Mg2+. The MIANS fluorescence study showed that rice protease inhibitor resulted in the increase of intensity, however, with the presence of Mg2+, the intensity decreased.

碩士
臺南師範學院
自然科學教育學系碩士班
92
After joining the WTO, the agriculture transformation in Taiwan is necessity. The government promotes "Few amount & diverse exquisite agriculture" at the present time. In all crops that expanded, the black soybeans and vegetable soybeans were expected as the most potential crops for development. Many reports indicated the black soybean has the better nutrition composition than soybean, in addition Chinese medicine also belief the effect of health care of black soybean is more important than soybean. The vegetable soybean is important export crops in our country; it occupies almost 88% in whole export freeze vegetables. After earning more income, the consumers mainly concern in purchasing the agricultural product are good quality and diversity. To compete with the cheaper imported soybean, the most important things are improving the varieties and studying its function of composition of the domestic soybean. On the medical science research, soybean constituents, including proteases inhibitors, lignans, saponins and isoflavone, have been shown to have anticarcino- genic ability and other therapeutic function. The Bowman-Birk inhibitors in the soybean are capable of preventing carcinogenesis in a wide variety of in vivo and in vitro model system. In addition, data from epidemiological reports ifoflavones have multi-biological effects, including estrogenic and antiestrogenic effects, antiangio- genesis, antioxidant effects, and suppress cancer cells. The black soybean and vegetable soybean that expand currently in Taiwan were used as materials for this study. Quantification and investigation into the difference and variation of BBI and isoflavones content were compared in different soybean varieties. Result showed that the BBI content of soybean were affected by varieties and crop years. The BBI content of the vegetable soybean variety of KS5 that grow in different locations also showed significant differences (p <0.05). In addition, interactions between the crop year and crop location were remarkable. However, the BBI content of the black soybean is higher than that in soybean, but on exception, the vegetable soybean variety of TN1. The isoflavone content of the domestic soybean also under the influence of variety, crop years and location, but the degree is different. In this research, soybeans were separated into their anatomical parts, such as hull, hypocotyls, and cotyledon, in order to analysis for their isoflavone content. The results indicated discrepancy in isoflavone content and composition in different parts of soybean. The concentration of aglycone isoflavones on a weight basis was the lowest in the hull and was the highest in the hypocotyl of the seed. Different crop years affected isoflavone content to a greater extent than the crop location. But they were no obvious difference in the ratio of isoflavone content that affected by crop years or location. It suggests that the ratio of isoflavone composition in soybean controled by heredity. In all domestic soybean varieties that measured, the hereditable variation of BBI and isoflavones were maximum in the vegetable soybean variety of KS6. And a negative association was observed between the amount of isoflavones and BBI in all soybean varieties’ seeds. Finally, this research found that the quality of commercial soybean is uneven. In a field survey, organic black bean named soybean species, was not a reel a soybean species. The data told that even consumers pay higher price to get better quality product but sometimes can’t.

碩士
國立清華大學
生物資訊與結構生物研究所
95
The 15-kDa trypsin inhibitors from rice bran (RBTI) are members of the Bowman-Birk protease inhibitor (BBI) family. The crystal structure of a 1:1 complex between RBTI and bovine pancreatic trypsin (BPT) was determined by combination of molecular replacement and electron density modification methods. This complex model has been refined to a crystallographic R-factor of 26.2% and free R-factor of 31.6% at 3.0 Å resolution. The RBTI structure consists of seven beta-strands and loops without alpha-helices structure and folds into two compact domains (N- and C-domain) which are similar to each domain from barley BBI. However, orientation between two domains is quite different to barley BBI, this makes the distance between two P1 residues (17Lys and 83Lys) in RBTI is only 23 Å apart rather than distance of 40 Å in barley BBI. The closer distance provides evidence to support result from activity assay that two domains show different abilities to inhibit trypsin. RBTI C-domain with protruding 84Met at P1’ position, moreover, mainly leads into breakdown of the classically canonical conformation of reactive site loop. Major interaction with BPT is achieved by RBTI N-domain but C-domain plays an auxiliary role to block trypsin molecule.

碩士
國立臺灣大學
生物化學暨分子生物學研究所
92
Abstract Protease inhibitors are a class of well-established cancer chemopreventing agents. Among several types of protease inhibitors, the Bowman-Birk inhibitor (BBI), a soybean-derived protein with the well-characterized ability to inhibit trypsin and chymotrypsin activities, has been shown to be an effective suppressor of carcinogenesis and treated in phaseⅡa clinical trial in human. However, the precise mechanisms by which BBI suppresses carcinogenesis are unknown. In this study, we demonstrated that BBI specifically and potently inhibits the proteasomal chymotrypsin-like activity in vitro and in vivo in MCF7 breast cancer cells. Inhibition of the proteasome by BBI is associated with accumulation of ubiquitinated proteins and the known proteasome substrates, p21Cip1/WAF1 and p27Kip1, accompanied with down-regulation of cyclin D1 and cyclin E which led to arrest cell cycle at G1/S phase. Furthermore, BBI suppressed MCF7 cell growth without showing cytotoxicity and had a novel effect on a loss of phoshporylated extracellular signal-related kinases (ERK1/2) when comparison with well-characterized chemopreventive agents. BBI was unable to inactivate ERK1/2 in the presence of a phosphatase inhibitor, sodium orthovanadate, or a transcriptional inhibitor, actinomycin D, suggesting the involvement of a specific phosphatase. We found an induction of dual specific MAP kinase phosphatase-1 (MKP-1) in dose- and time-dependent fashion which was correlated with dephosphorylation of ERK1/2 in BBI-treated MCF7 cells. In addition, BBI exhibited no inhibitory effects on EGF-induced activation of ERK1/2 and Akt. Taken together, we demonstrated that BBI indeed suppressed ERK1/2 activity via up-regulation of MKP-1 mediated by blockade of proteasome function. Our results supported the notion that the inhibition of proteasome activity by BBI is a novel mechanism that might contribute to cancer preventative effects of BBI. This study further provides the evidence that soybean products intake indeed have the potential to advance as chemopreventive agents. Inhibitors of proteasome are currently emerging as novel cancer preventing and therapeutic agents. To determine whether the tea polyphenols, including theaflavin, theaflavin-3-gallate, theaflavin-3’-gallate, theaflavin-3,3’-digallate (TF3), and (-)-epigallocatechin-3-gallate (EGCG) were potential proteasome inhibitors, we treated purified 20S or 26S proteasome and 26S proteasome in leukemia and cancer cell lines with these compounds. TF3 displayed a potent inhibitory effect on the growth of U937 cells with an estimated IC50 value of 6μM. Furthermore, TF3 had more efficient inhibition on the proteasomal chymotrypsin-like activity of purified proteasome derived from different sources and 26S proteasome in four kinds of cancer cell extracts as compared to those of EGCG. In addition, the theaflavins, especially TF3, inhibited the proteasomal chymotrypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities of 26S proteasome in a concentration-dependent manner in human breast cancer MCF7 cells which have been described to be resistant to apoptosis induced by proteasome inhibitors. These results illustrated that proteasome inhibition by theaflavins lead to anti-proliferation of MCF7 cells. Furthermore, comparing well-characterized polyphenols on proteasome inhibition, the ester bond-bearing compounds, with some exceptions, exhibited more potently inhibitory effects on the proteasomal chymotrypsin-like activity than those without ester bond(s). Interestingly, the gallic acid and n-propyl gallate also had slightly inhibition on proteasome activities. Therefore, we suggest that the galloyl moiety might be the other important structure of polyphenols that contributes to the proteasome inhibition.