<p>Predmet doktorske disertacije je ispitivanje mogućnosti karakterizacija novih biorazgradivih kompozitnih filmova na<br />bazi biljnih proteina. Osnovno istraživanje se bazira na<br />ispitivanju mogućnosti dobijanja kompozitnih filmova na bazi<br />obnovljivih sirovina, vlažnim postupkom (&quot;casting&quot; metoda),<br />karakterizaciji dobijenih filmova i modifikaciji uslova dobijanja<br />radi pobolj&scaron;anja osobina formiranog filma. Istraživanja se<br />zasnivaju na dobijanju filmova na bazi pogače uljane tikve<br />golice (Cucurbita pepo L. c. v. Olinka) (pumpkin oil cake &ndash;<br />PuOC), njenog proteinskog izolata i njihove kombinacije sa<br />drugim filmogenim polimerima (proteinima i polisaharidima).<br />Istraživanje podrazumeva procenu mogućnosti primene PuOC<br />radi delimične zamene op&scaron;te poznatih filmogenih materijala,<br />kao i produkciju filmova od PuOC i od proteinskog izolata<br />PuOC. Za produkciju filmova, od važnosti je ispitivanje<br />procesnih parametara (temperatura, pH, period denaturacije,<br />uslovi su&scaron;enja, itd.) i komponenti koje formiraju film (količina<br />polimera sa sposobno&scaron;ću formiranja filma, količina i vrsta<br />plastifikatora, količina i vrsta agenasa za umrežavanje, itd.). S<br />obzirom na velik broj parametara koji utiču na formiranje filma,<br />kao i na osobine formiranih filmova, ispitano je međusobno<br />delovanja vi&scaron;e faktora na mogućnost produkcije i osobine<br />dobijenog filma. Odabir i optimizacija procesnih parametara i modelovanje produkcije filmova izvedeno je implementiranjem<br />nove kompjuterske i analitičke metodologije. Osobine značajne<br />za dalju primenu dobijenih filmova podrazumevaju mehaničke<br />osobine (zateznu jačinu i izduženje pri kidanju), barijerne<br />(propustljivost gasova) i fizičko-hemijske karakteristike<br />(rastvorljivost, količinu rastvorljivih proteina, biolo&scaron;ku<br />aktivnost u vidu antioksidantne aktivnosti), a ispitane su u cilju<br />deklarisanja potencijalne aplikacije filma. Dodatno, ispitana je<br />mogućnost produkcije kompozitnih filmova proteinski<br />izolat/hitozan, kao i primena enzimskog umrežavanja (crosslinking)<br />enzimom transglutaminaza (TGaza), radi dobijanja<br />filmova sa unapređenim karakteristikama. Istraživanja su<br />vođena i u smeru karakterizacije strukture nastalih filmova,<br />primenom tehnika elektron skenirajuće mikroskopije,<br />deferencijalne skening kalorimetrije, gasne hromatografije i<br />Furije transformi&scaron;uće infracrvene spektrofotometrije.</p> / <p>The subject of the doctoral dissertation is to examine the possibility of<br />production and characterization of new biodegradable composite<br />films based on plant proteins. The research is based on an<br />examination of the possibility of obtaining composite films based on<br />renewable raw materials, using the casting method, on the<br />characterization of the obtained films and the requirement for the<br />modification to improve the properties of the formed film. The<br />research is aimed to evaluate the possibility of the production<br />the new biodegradable films based on hull-less pumpkin<br />(Cucurbita pepo L. c. v. Olinka) oil cake (pumpkin oil cake -<br />PuOC), its protein isolates and their combinations with other<br />filmogenous polymers (proteins and polysaccharides). The<br />study involves partial replacement of commonly well-known<br />filmogenous materials with PuOC, and production of films<br />based on the whole PuOC and the protein isolates from PuOC.<br />For film production, it is important to investigate the process<br />parameters (temperature, pH, denaturation period, drying conditions,<br />etc.), film-forming components (the amount of polymer with filmforming<br />ability, the amount and type of plasticizer, the amount and<br />type of cross-linking agents, etc..). As the large number of parameters<br />influence the film formation, as well as the properties of formed<br />films, the interaction of several factors which affect the possibility of<br />the production and properties of the obtained film, was examined.<br />Selection and optimization of process parameters and modeling of<br />film production will be carried out by implementing a new computer<br />and analytical methodology. Characteristics important for further<br />application of the obtained films include mechanical properties<br />(tensile strength and elongation at break), barrier (gas permeability)<br />and physical-chemical properties (solubility, the amount of soluble<br />proteins, biological activity in the form of antioxidant activity) and<br />were tested for the purpose of declaring potential application of the produced films. Additionaly, the possibility of production of composite films protein isolate/chitosan, and the application of enzymatic networks (cross-linking) by the enzyme<br />transglutaminase, in order to obtain films with improved<br />properties, was examined. Research was conducted in the<br />direction of the characterization of films formed by applying the<br />techniques of scanning electron microscopy, diferential<br />scanning calorimetry, gas chromatography and Fourier<br />transforming infrared spectrophotometry.</p>

The journey of structural studies on lectins, starting with ConA in the 70s, has crossed many milestones. Lectins, multivalent carbohydrate-binding proteins of non-immune origin, specifically bind diverse sugar structures. They have received considerable attention in recent times on account of the realization of the importance of protein-sugar interactions, especially at the cell surface, in biological recognition. They occur in plants, animals, fungi, bacteria and viruses. Plant lectins constitute about 40% of the lectins of known structure. They can be classified into five structural groups, each characterized by a specific fold. Among them, legume lectins constitute the most extensively investigated group. Basic Winged bean lectin (WBAI) is a glycosylated, homodimeric, legume lectin with Mr 58000. The structure of WBAI complexed with methyl-a-galactose, determined earlier in this laboratory, provided information about the oligomeric state and the carbohydrate specificity of the lectin in terms of lectin-monosaccharide interactions. The present work was initiated to understand the carbohydrate specificity of the lectin, especially at the oligosaccharide level, with special reference to its blood group specificity. The hanging drop method was used for crystallizing WBAI and its complexes. Intensity data were collected on Mar Research imaging plates mounted on Rigaku RU-200 or ULTRAX-18 X-ray generators. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program AMoRe was used for structure solution. Structure refinement was carried out using the CNS software package. Model building was done using the molecular graphics program O. INSIGHT II, ALIGN, CONTACT and PROCHECK of CCP4 were used for the analysis and validation of the refined structures. WBAI exhibits differential affinity for different monosaccharide derivatives of galactose. In order to elucidate the structural basis for this differential affinity, the crystal structures of the complexes of basic winged bean lectin with galactose, 2-methoxygalactose, N-acetylgalactosamine and methyl-a-N-acetylgalactosamine have been determined. Lectin-sugar interactions involve four hydrogen bonds and a stacking interaction in all of them. In addition, a N-H O hydrogen bond involving the hydroxyl group substituted at C2 exists in the galactose and 2-methoxygalactose complexes. The additional hydrophobic interaction, involving the methyl group, in the latter leads to the higher affinity of the methyl derivative. In the lectin - N- acetylgalactosamine complex the N-H O hydrogen bond is lost, but a compensatory hydrogen bond involving the oxygen atom of the acetamido group is formed. In addition, the CH3 moiety of the acetamido group is involved in hydrophobic interactions. Consequently, the 2-methyl and the acetamido derivatives of galactose have nearly the same affinity for the lectin. The methyl group, a-linked to the galactose, takes part in additional hydrophobic interactions. Therefore, methyl-a- N-acetylgalactosamine has higher affinity than N-acetylgalactosamine to the lectin. The structures of basic winged bean lectin-sugar complexes provide a framework for examining the relative affinity of galactose and galactosamine for the lectins that bind to them. The complexes also lead to a structural explanation for the blood group specificity of basic winged bean lectin, in terms of its monosaccharide specificity. The Tn-determinant (GalNAc-a-O-Ser/Thr) is a human specific tumor associated carbohydrate antigen. Having epithelial origin, it is expressed in many carcinogenic tumors including breast, prostate, lung and pancreatic cancers. The crystal structure of WBAI in complex with GalNAc-a-O-Ser (Tn-antigen) has been elucidated, in view of its relevance to diagnosis and prognosis of various human cancers. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the formation of the complex of the lectin with Tn-antigen. Modelling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein. WBAI binds A-blood group substance with higher affinity and B-blood group substance with lesser affinity. It does not bind the O substance. The crystal structures of the lectin, complexed with A -reactive and B - reactive di and tri saccharides, have been determined. In addition, the complexes of the lectin with fucosylated A- and B-trisaccharides and with a variant of the A-trisaccharide have been modelled. These structures and models provide valuable insights into the structural basis of blood group specificities. All the four carbohydrate binding loops of the lectin contribute to the primary combining site while the loop of variable length contributes to the secondary binding site. In a significant advance to the current understanding, the interactions at the secondary binding site also contribute substantially, albeit in a subtle manner, to determine the blood group specificity. Compared to the interactions of the B- trisaccharide with the lectin, the third sugar residue of the A -reactive trisaccharide forms an additional hydrogen bond with a lysine residue in the variable loop. In the former, the formation of such a hydrogen bond is prevented by a shift in the orientation of the third sugar resulting from an internal hydrogen bond in it. The formation of this bond is also facilitated by an interaction dependent change in the rotamer conformation of the lysyl residue of the variable loop. Thus, the difference in the interactions at the secondary site is generated by coordinated movements in the ligand as well as the protein. A comparison of the crystal structure and the model of the complex involving the variant of the A-trisaccharide results in the delineation of the relative contributions of the interactions at the primary and the secondary sites in determining blood group specificity. At the disaccharide level, WBAI exhibits higher affinity for á1-3 linked Gal/GalNAc containing oligosaccharides, compared to that of other á linked oligosaccharides. With an objective of understanding the preferential binding of WBAI for á 1-3 linked Gal/GalNAc containing oligosaccharides, crystal structure of the complexes of the lectin with Galá1-4Gal, Galá1-4GalâEt and Galá1-6Gal have been determined. The reducing sugar of the disaccharides with linkages other than á1-3 binds to the lectin through a water bridge whereas the same sugar moiety with á 1-3 linkage makes direct interactions with the loop L4 of the protein. The modelling study on the complex of the lectin with Galá1-2Gal further upholds this observation. Different structures involving WBAI, reported earlier and presented here, were used to investigate the plasticity of the lectin. The front curved â-sheet, which nestles the metal binding region and on which the carbohydrate binding loops are perched, is relatively rigid. On the contrary, the flat back â-sheet, involved in the quaternary association in legume lectins, is flexible. This flexibility is probably necessary to account for the variation in quaternary structure. With the results presented in this thesis, 14 crystal structures of WBAI, in the free form and in complex with different sugars, have been reported, all from this laboratory. It is now, perhaps, appropriate to examine the new information and insights gained from these investigations, on the structure and function of the lectin. Earlier X-ray studies of WBAI contributed substantially in establishing that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to large alterations in quaternary association. Structural studies on WBAI, particularly those reported here, also contributed to the elucidation of the nuances of carbohydrate recognition by lectins. A comparative study of the available structures also revealed the flexible and rigid regions of the protein. The study of the influence of covalently linked sugars on the structure of Erythrina corallodendron lectin (ECorL), a homolog of WBAI, is the content of appendix of the thesis. The three-dimensional structure of the recombinant form of Erythrina Corallodendron lectin(rECorL) complexed with lactose, has been elucidated by X-ray crystallography. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites(Asn17). These changes take place in such a way that hydrogen bonds with the neighbouring protein molecules in rECorL compensate those made by the glycan with the protein in ECorl. contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

MEDEIROS, Jackeline Lima de. Caracterização bioquímica e funcional de isolados proteicos e genótipos de excelência de feijão-caupi [Vigna unguiculata (L.) Walp.]. 95 f. : Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2013. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-25T12:56:25Z No. of bitstreams: 1 2013_dis_jlmedeiros.pdf: 790389 bytes, checksum: 4fadbf512e230d0abc973e12db6c63af (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-07-05T19:55:38Z (GMT) No. of bitstreams: 1 2013_dis_jlmedeiros.pdf: 790389 bytes, checksum: 4fadbf512e230d0abc973e12db6c63af (MD5) / Made available in DSpace on 2016-07-05T19:55:38Z (GMT). No. of bitstreams: 1 2013_dis_jlmedeiros.pdf: 790389 bytes, checksum: 4fadbf512e230d0abc973e12db6c63af (MD5) Previous issue date: 2013 / The cowpea (Vigna unguiculata (L.) Walp) is an important plant due to their nutritional potential, revealed to mainly high protein content. The highlight proteins present in the functional properties (technological) and interest in new sources of protein at affordable costs leads to increasing interest in studying the legume. Thus, the present study aimed to obtain protein isolates from bean cowpea genotypes, as the biochemical and functional characterization of these isolates and their seeds. The results showed the seeds are good sources of protein (23.19 to 24.02%), fiber (17.75 to 19.78%) and have a low lipid content (1.67%). The protein isolated obtained showed high purity (above 90%) and yield (35.11 and 38.12%). Both isolated as seeds not proved toxic but exhibited trypsin inhibitor (24.2 to 28.2 IU), chymotrypsin (26.66 to 32.17 IU) and lectin (80.000-320.000 IU). The digestibility of isolated (63.23 and 63.81%) was higher than the seeds (50.07 to 54.37%). The predominant proteic fraction was globulin, with the highest concentration in the isolates. The seeds and isolated solubilities presents U-shaped, with lower solubility at the isoelectric point. The isolated protein showed greater capacity for absorb water (2.41 to 2.44 ml/g sample), oil (from 2.38 to 2.81 ml / g sample) and gel concentrations (8-10%). Already seeds showed higher foaming (52.66 to 57.89%). / O feijão-caupi [Vigna unguiculata (L.) Walp] é uma importante leguminosa devido ao seu potencial nutricional, revelado principalmente pelo alto teor de proteínas. As proteínas apresentam destaque nas propriedades funcionais (tecnológicas) e o interesse por novas fontes proteicas a custos acessíveis, leva ao crescente estudo das leguminosas. Assim, o presente trabalho objetivou a obtenção de isolados proteicos de dois genótipos de feijão-caupí (Cauamé e Tumucumaque), bem como a caracterização bioquímica e funcional destes isolados e de suas sementes. Os resultados mostraram que as sementes dos genótipos estudados são boas fontes de proteínas (23,19 a 24,02%), fibras (17,75 a 19,78%) e apresentam baixo teor de lipídeos (1,67%). Os isolados proteicos obtidos apresentaram elevado teor proteico (> 90%) e rendimento (35,11 e 38,12%, respectivamente Tumucumaque e Cauamé). Tanto os isolados como as sementes não se mostraram tóxicos, mas apresentraram inibidores de tripsina (24,2 a 28,2 UI/ µg de proteína), quimiotripsina (26,66 a 32,17 UI/ µg de proteína) e lectina (80.000 a 320.000 UI/ µg de proteína). A digestibilidade dos isolados (63,23 a 63,81%) foi superior a das sementes (50,07 a 54,37%). A fração proteica predominante foi a globulina, com maior concentração nos isolados. A solubilidade das sementes e isolados apresenta-se em formato de U, com menor solubilidade no ponto isoelétrico. Os isolados proteicos apresentaram melhor capacidade de absorção de água (2,41 a 2,44 ml/g de amostra), de óleo (2,38 a 2,8 1ml/g de amostra) e formação de gel (8 a 10%). Já as sementes apresentaram maior formação de espuma (52,66 a 57,89%). Assim, pode-se concluir que os isolados proteicos obtidos desse genótipo são promissores para aplicação nutricional, por apresentarem elevado teor proteico (> 90%), razoável digestibilidade (>60%) e perfil de aminoácidos. Da mesma forma, são promissores para a aplicação tecnológica, uma vez que se destacaram nas propriedades de solubilidade, capacidade de absorção de água e de óleo e formação de gel. Assim, os isolados proteicos podem ser utilizados como ingredientes de produtos alimentícios, contribuindo com o aumento do valor nutricional dos alimentos e agregando valor aos genótipos melhorados do feijão-caupi.

THI1 é provavelmente uma proteína bifuncional, uma vez que está envolvida na biossíntese de tiamina e na estabilidade do DNA organelar, notadamente o mitocondrial. Interessantemente, a biossíntese de tiamina ocorre em compartimentos distintos em plantas (cloroplastos) e em leveduras (mitocôndrias). Ensaios de complementação funcional mostraram que o gene thi1 de Arabidopsis thaliana é capaz de complementar uma cepa mutante de levedura para o gene ortólogo. A proteína THI1 de Arabidopsis thaliana é codificada por um único gene. Uma análise detalhada da região N-terminal da proteína, responsável pela sua localização na células, revelou a presença de duas sequências de direcionamento adjacentes. Na extremidade N-terminal encontra-se um peptídeo de trânsito cloroplástico seguida por uma região capaz de formar uma &#945;-hélice anfifílica, tipicamente encontrada em pré-sequências de direcionamento mitocondriais. Com o objetivo de avaliar se a localização final de THI1 pode apresentar um tipo de regulação temporal ou espacial, foram obtidas plantas transgênicas expressando a proteína THI1 fundida a GFP ("green fluorescent protein"). Análises dessas plantas por meio de microscopia confocal revelaram que THI1 está presente majoritariamente em cloroplastos e raramente em mitocôndrias. Ao contrário do que acontece em Arabidopsis thaliana, em cana de açúcar foram encontrados pelo menos três isoformas/parálogos de thi1. O alinhamento da seqüência de aminoácidos dessas isoformas com a THI1 de Arabidopsis thaliana revelou alta similaridade, inclusive na seqüência de direcionamento. Com o intuito de avaliar o padrão de direcionamento dessas isoformas de cana de açúcar foram obtidas construções gênicas contendo ou a seqüência de direcionamento completa ou o peptídeo de trânsito cloroplástico ou a pré-seqüência mitocondrial, fundidas a GFP sob o comando do promotor 35S. A expressão transiente dessas construções em epiderme de cebola, revelou que no caso das construções contendo a seqüência de direcionamento completa ou o peptídeo de trânsito, o direcionamento ocorreu apenas para os cloroplastos. No caso das construções contendo somente a seqüência de direcionamento mitocondrial a GFP permaneceu difundida no citoplasma. Além do aspecto do direcionamento, THI1 foi avaliada sob o ponto de vista filogenético. As análises filogenéticas mostram que thi1 é raramente encontrado em bactérias mas é amplamente distribuído em Archaea. As distâncias genéticas indicam que provavelmente os eucariontes herdaram thi1 de Archaea. As poucas bactérias que possuem esse gene provavelmente obtiveram-no por meio de herança horizontal. / THI1 is probably a bifunctional protein, since it is involved in thiamin biosynthesis and organelar genome stability mainly the mitochondrial. Interestingly, the thiamin biosynthesis occurs at different compartments in plants (chloroplasts) and yeasts (mitochondria). Functional complementation assays showed that Arabidopsis thaliana thi1 gene is able to complement a yeast mutant strain for the hortolog gene. The Arabidopsis thaliana THI1 is encoded by a single copy gene. A detailed analysis of the THI1 N-terminal region, that is responsible for its targeting in cells, reveled the presence of two in tanden directing sequences. At N-terminal region there is a chloroplastic transit peptide followed by a region able to form an anfifilic &#945;-helix frequently present in mitochondrial presequences. Aiming to evaluate if the THI1 final localization could present a temporal or spacial regulation, transgenic plants expressing THI1 fused to GFP ("green fluorescent protein") where obtained. Confocal microscopy analysis of these transgenic plants showed that THI1 is mainly found in chloroplasts and barely found in mitochondrias. Different from what happens in Arabidopsis thaliana, in sugar cane where founded at least three thi1 isoforms/paralogs. The amino acids sequence alignment of these isoforms with the thi1 one, reveled high similarity including the targeting sequence. To evaluate the directing standard of these sugarcane isoforms, gene constructions made by the complete targeting sequence or the chloroplastic transit peptide or the mitochondrial presequence, fused to GFP under the guidance of 35S promotor, where obtained. A transient expression of these gene constructios in epidermal onion cells prove that in the case of the constructions containing either the complete targeting sequence or the chloroplastic transit peptide the directing occurred only to chloroplasts. On the other hand, the constructions containing the mitochondrial pre sequence, GFP were kept defused in the citoplasm. Besides the directing aspect, THI1 were evaluated under the filogenetic point of view. Filogenetic analysis showed that thi1 is rarely found in bacteria but is widely distributed in Archaea. The genetic distances pointed out that probably eucaryotes THI1 came from Archaea. This gene in a few bacterias probably were inherited by lateral transference.

A existência de uma proteína mitocondrial desacopladora em plantas, PUMP, foi demonstrada em 1995. A PUMP, como a proteína desacopladora de mitocôndrias de tecido adiposo marrom, UCP1, e outras proteínas homólogas descobertas posteriormente, aumenta a condutividade de membrana a H+. Nucleotídeos de purina, PN, inibem a atividade das proteínas desacopladoras e o mecanismo de condução de H+ depende da presença de ácidos graxos livres, FFA. A atividade e a expressão da PUMP são estimuladas pela exposição ao frio e mudam durante o amadurecimento de frutos. A expressão do gene da PUMP de Arabidopsis thaliana em E. coli permite a obtenção de AtPUMP. Neste trabalho, analisando a funcionalidade da AtPUMP incorporada em proteolipossomos, demonstramos que esta proteína é funcional. A incorporação de proteínas desacopladoras em proteolipossomos fornece um sistema modelo que permite analisar as suas propriedades funcionais e mecanísticas. A condutância a H+ em proteolipossomos contendo PUMP isolada de batata foi claramente ativada por FFA. Contudo, a inibição por PN não se mostrou reprodutível. A AtPUMP, reconstituída em proteolipossomos, foi ativada por FFA com Km\'s aparentes de: 42 &#181;M (ácido linoleico, LA), 55 &#181;M (ácido láurico) e 70 &#181;M (ácido palmítico), e inibida por PN com Ki\'s aparentes de: 0.8 mM (GDP), 0.85 mM (ATP), 0.98 mM (GTP) e 1.4 mM (ADP). O efluxo de H+ ativado por LA mediado por AtPUMP aumentou exponencialmente em função do potencial transmembrânico (&#916;&#968;). O coeficiente de partição (KP) entre a fase aquosa e proteolipossomos contendo AtPUMP para o LA de 64170, foi ~1,6 vezes superior que ao KP obtido para o LA em lipossomos. Em ensaios de ligação obtidos usando microcalorimetria de titulação isotérmica (ITC), determinou-se que a AtPUMP tem, provavelmente, dois sítios de ligação para o LA e que essa interação é exotérmica. Em ensaios de microcalorimetria com suspensão de mitocôndrias de batatas, determinou-se que há uma correlação linear entre o calor produzido e o oxigênio consumido (65,6 kcal/mol O2) quando a PUMP foi ativada por LA. Através dos resultados obtidos até então, concluo que a PUMP (AtPUMP) é um desacoplador mitocondrial presente em plantas. Os estudos apresentados aqui, de reconstituição em lipossomos, foram essenciais para a compreensão da regulação da atividade dessa proteína. Além disso, foram obtidas as primeiras medidas diretas de liberação de calor pela PUMP por microcalorimetria. / In 1995, a plant mitochondrial uncoupling protein, PUMP, was first described. PUMP, like the known uncoupling protein from brown adipose tissue, UCP1, increases the inner mitochondrial membrane permeability to H+. H+ transport is dependent on the presence of free fatty acids, FFA, and it is inhibited by purine nucleotides, PN. PUMP expression and activity are stimulated by cold exposure, which may vary during fruit ripening. By expressing a full length cDNA encoding the Arabidopsis UCP in E. coli, the recombinant AtPUMP was obtained. In this study, AtPUMP was incorporated in proteoliposomes and the functionality of the protein was demonstrated. The incorporation of uncoupling proteins in proteoliposomes constitutes a model that allows the functional and the mechanistic analysis of these proteins. The H+ conductance mediated by reconstituted potato PUMP was, undoubtedly, activated by FFA. However, the inhibition by PN was not reproducible. Reconstituted AtPUMP was activated by FFA and the apparent Km\'s were determined: 42 &#181;M (linoleic acid, LA), 55 &#181;M (lauric acid) and 70 &#181;M (palmitic acid). Reconstituted AtPUMP was inhibited by PN, and the apparent Kis were determined: 0.8 mM (GDP), 0.85 mM (ATP), 0.98 mM (GTP) and 1.4 mM (ADP). AtPUMP mediated H+ efflux rate, activated by LA, was exponentially dependent on membrane potential (&#916;&#968;). The partition coefficient (KP) between the aqueous phase and the membrane phase was determined for LA. The KP was 1.6 times higher for AtPUMP proteoliposomes than for liposomes. Using Isothermal Titration Microcalorimetry (ITC), we verified that there is a linear correlation between the heat produced and oxygen depletion (65,2 kcal/mol O2) in a suspension of potato mitochondria, when PUMP was activated by LA. Using ITC we also determined that LA might bind to two different sites in AtPUMP. Based on the results obtained, we concluded that PUMP (or AtPUMP) is a plant mitochondrial uncoupler. The reconstitution assays permitted the study of FFA and PN regulation. In addition, our results represent the first direct confirmation that fatty acids regulate heat release in plant mitochondria, a process that may play a role in cold adaptation, fruit ripening and flower blossoming.

Os isolados protéicos de soja são utilizados como ingredientes em diversos alimentos e sua utilização vêm aumentando juntamente com o aumento das pesquisas sobre os metabólitos secundários da soja, as isoflavonas. Alguns efeitos benéficos vem sendo associados às isoflavonas, entre estes a sua ação antioxidante, a redução ao risco de câncer, doenças cardiovasculares e osteoporose. O objetivo deste estudo foi o de otimizar as condições de extração das isoflavonas e de suas formas conjugadas a partir da farinha desengordurada de soja, visando o preparo de isolado protéico de soja. Os resultados mostraram que a obtenção de isolados protéicos de soja com teor aumentado de isoflavonas depende da utilização de condições brandas de centrifugação para a separação do precipitado isoelétrico, assim como da utilização de água acidificada na sua lavagem. A presença de isoflavonas no isolado resulta de três fatores, o primeiro referindo-se à associação entre isoflavonas e proteínas através de interações hidrofóbicas, eletrostáticas, e pontes de hidrogênio; o segundo à menor solubilidade das isoflavonas presentes na farinha desengordurada de soja no pH isoelétrico; e o último ao processo de carreamento (físico) das isoflavonas pelas proteínas insolubilizadas. / Soy protein isolates are used as ingredients in several food products and their use is increasing together with the increase of the researches on the secondary metabolites of soy, the isoflavones. Some beneficial effects have been associated to the isoflavones, among these their antioxidant action, prevention of cancer, cardiovascular diseases and osteoporosis. The objective of this study was to optimize the extraction conditions of the isoflavones from the defatted soy flour, seeking the preparation of soy protein isolates. The results showed that the obtention of soy protein isolates with increased content of isoflavones depends on the use of mild conditions of centrifugation for the separation of the isoelectric precipitate, as well as on the use of water acidified in the washing step. The presence of isoflavones in the isolates resulted from three factors, the first refers to the association between isoflavones and proteins through hydrophobic; and electrostatic interactions, and hydrogen bonding; the second to the decreased solubility of the isoflavones extracted from the defatted soy flour in the isoelectric pH; and the last to the carrying process (physical) of isoflavones by the precipitating proteins.

MSCAGR (Plant Production) / Department of Plant Production / Bambara groundnut (Vigna subterranea) is one of the most important legumes cultivated primarily for food by smallholder farmers in Africa. It is an affordable source of protein and contributes to income generation as well as soil fertility. Despite its importance, it is cultivated largely for subsistence purposes in South Africa. Growers use landraces. The agronomic performance of the traditional varieties depends on environmental factors prevailing in a particular area. In Limpopo and Mpumalanga Provinces, there is no adequate information regarding the performance of bambara groundnut germplasm. The objectives of the study were to (i) determine the agronomic performance of Bambara groundnut across three contrasting locations in Limpopo and Mpumalanga provinces over two cropping seasons (ii) determine the genotypic variation in the seed protein level among 42 bambara groundnut genotypes. Forty-two bambara groundnut genotypes were evaluated under three different environmental conditions (Syferkuil, Thohoyandou and Nelspruit) over two (2013/2014, 2014/2015) seasons in a 7 × 6 rectangular lattice design replicated three times. Eight agronomic traits including dry shoot weight (DSW), number of pods per plant (NPP), pod length (PL), number of seed per pod (NSP), pod weight per plant (PWT), seed weight per plant (SWT), 100 seed weight (100-SWT) and seed yield (SYLD) were measured. The results showed that there were significant genotype x location interactions which demonstrated that the prevailing agro-ecological conditions at the test locations were distinct from each other. Five genotypes (‘BGN-19‘, ‘BGN-11‘, ‘BGN-12‘, ‘BGN-4‘and ‘BGN-34‘) attained >25.0% seed yield advantage over the local check ‘BGN-39‘. The results also showed that light brown coloured genotypes attained relatively higher seed yield compared to the other seed colours types. The cultivar superiority index (CSI) showed that three genotypes (‘BGN-12‘, ‘BGN-19’ and ‘BGN-34’) were the most stable across the test locations and attained >900.0 kg/ha on average. There were significantly high positive correlations between PWT and each of the three other attributes (SWT, 100 SWT and SYLD). In terms of seed protein, the results showed a poor relationship between seed yield and protein levels. ‘BGN-12’ which produced the highest seed yield, attained the lowest percent seed protein while genotype. On average, the genotypes contained 21.72% protein. The highest and lowest seed protein quantities were attained by the genotypes ‘BGN-42’ (25.17%) and ‘BGN-12’ (19.89%) respectively. / NRF

A adubação equilibrada com nitrogênio, potássio e boro pode alterar a nutrição, produção e valor nutritivo das gramíneas forrageiras. Mediante as aplicações de combinações de doses de nitrogênio, potássio e boro objetivou-se avaliar os efeitos: a) nos aspectos morfológicos, na produção de massa seca e no estado nutricional da parte aérea; b) nas características morfológicas, produtivas e nutricionais das raízes e, c) nos componentes de parede celular e fracionamento de proteínas do capim-tanzânia. O experimento foi conduzido em casa de vegetação no período de janeiro a maio de 2010, utilizando quartzo moído como substrato. Cinco doses de nitrogênio (2, 10, 18, 26 e 34 mmol L-1) foram combinadas com cinco doses de potássio (0,2; 3,1; 6,0; 8,9 e 11,8 mmol L-1), que foram combinadas com cinco doses de boro (0; 45,45; 90,90; 136,35 e 181,80 mol L-1) em fatorial 53 fracionado, perfazendo 23 combinações de nitrogênio, potássio e boro, com quatro repetições. Realizaram-se três cortes das plantas de capim-tanzânia, nos seguintes períodos: 32 dias após o transplantio, 39 dias após o primeiro corte e 45 dias após o segundo corte. As doses de boro não interferiram nos aspectos produtivos e morfológicos da parte aérea e das raízes nem na composição de parede celular e frações proteicas. O boro somente influenciou a concentração de nitrogênio, potássio e boro da parte aérea. A interação doses de nitrogênio x doses de potássio foi significativa para as variáveis número de perfilhos, número de folhas, área foliar, valor SPAD e produção de massa seca da parte aérea. Com o pouco crescimento da planta ocorreu acumulação de nitrogênio, potássio e boro e diluição desses nutrientes nas folhas diagnósticas com a maior produção das plantas. A produção de massa seca das raízes, comprimento total e superfície total aumentaram com as combinações de doses de nitrogênio e potássio. As concentrações de nitrogênio, potássio e boro nas raízes foram influenciadas pela combinação das doses de nitrogênio com potássio. As concentrações de nitrato e amônio foram influenciadas positivamente pela combinação das doses de nitrogênio e potássio, tendo ocorrido maior concentração de amônio do que nitrato nas raízes. Com o aumento das doses de nitrogênio e potássio o comprimento específico e a superfície específica das raízes diminuíram. Os teores de FDN, FDA aumentaram e a relação FE+LM/CB diminuiu com as doses de potássio no segundo e terceiro cortes. O teor de PB no segundo corte aumentou com as doses de nitrogênio e diminuiu com as doses de potássio. As frações proteicas B1+B2 aumentaram e a fração B3 diminuiu no segundo e terceiro cortes com o aumento das doses de nitrogênio. A fração C para o terceiro corte diminuiu com as doses de nitrogênio. A combinação das doses de nitrogênio e potássio aumentou os teores da fração A para o terceiro corte. O suprimento de nitrogênio e potássio proporcionou aumento nos aspectos produtivos, morfológicos e nutricionais da parte aérea e das raízes, além de ter melhorado o valor nutritivo do capim-tanzânia. / The balanced fertilization with nitrogen, potassium and boron may change plant nutrition, yield and nutritive value of forage grass. Through the supply of combinations of nitrogen, potassium and boron it was aimed to evaluate the following effects on Tanzania guineagrass: a) plant morphology, dry matter production and nutritional status in the shoots; b) morphological, productive and nutritional aspects in the roots and c) cell wall components and protein fractions in the shoots. The experiment was conducted in a greenhouse from January to May 2010, using ground quartz as substrate. Five rates of nitrogen (2, 10, 18, 26 and 34 mmol L-1) were combined with five rates of potassium (0.2, 3.1, 6.0, 8.9 and 11.8 mmol L-1) and five rates of boron (0, 45.45, 90.90, 136.35 and 181.8 mol L-1) in 53 fractionated factorial design, which had 23 combinations of nitrogen, potassium and boron, with four replications. Three harvests of Tanzania guineagrass were made: 32 days after transplanting, 39 days after the first harvest and 45 days after the second harvest. Boron rates did not interfere with production and morphological aspects of shoots and roots and in cell wall composition and protein fractions. Boron only influenced the concentration of nitrogen, potassium and boron in the shoots. The interaction nitrogen rates x potassium rates was significant for the number of tillers, number of leaves, leaf area, SPAD value and shoots dry matter production. When plants had short growth, nitrogen, potassium and boron accumulated in plant tissues and those nutrients were diluted in plants with great growth. The dry mass of roots, total root length, total root area increased with the combination of nitrogen and potassium rates. The concentration of nitrogen, potassium and boron were influenced by the combinations of nitrogen and potassium rates. The concentrations of nitrate and ammonium were positively influenced by the combinations of nitrogen and potassium rates, and higher concentrations of ammonium than nitrate were found in root tissues. Root specific length and specific surface area decreased as rates of nitrogen and potassium increased. The NDF and ADF increased and EF+MB/SS ratio decreased with the potassium rates in the second and third harvests. The CP content of the second harvest increased with the nitrogen and decreased with potassium rates. Fractions B1+B2 increased and fraction B3 decreased for the second and third harvests with increasing nitrogen rates. The fraction C for the third harvest decreased with nitrogen rates. The combination of nitrogen and potassium increased the content of fraction A in the third harvest. Nitrogen and potassium supply provided to increase the productive changes, morphological and nutritional aspects in shoots and roots well as it improved the nutritive value of Tanzania guineagrass.

A adubação equilibrada com nitrogênio, potássio e boro pode alterar a nutrição, produção e valor nutritivo das gramíneas forrageiras. Mediante as aplicações de combinações de doses de nitrogênio, potássio e boro objetivou-se avaliar os efeitos: a) nos aspectos morfológicos, na produção de massa seca e no estado nutricional da parte aérea; b) nas características morfológicas, produtivas e nutricionais das raízes e, c) nos componentes de parede celular e fracionamento de proteínas do capim-tanzânia. O experimento foi conduzido em casa de vegetação no período de janeiro a maio de 2010, utilizando quartzo moído como substrato. Cinco doses de nitrogênio (2, 10, 18, 26 e 34 mmol L-1) foram combinadas com cinco doses de potássio (0,2; 3,1; 6,0; 8,9 e 11,8 mmol L-1), que foram combinadas com cinco doses de boro (0; 45,45; 90,90; 136,35 e 181,80 mol L-1) em fatorial 53 fracionado, perfazendo 23 combinações de nitrogênio, potássio e boro, com quatro repetições. Realizaram-se três cortes das plantas de capim-tanzânia, nos seguintes períodos: 32 dias após o transplantio, 39 dias após o primeiro corte e 45 dias após o segundo corte. As doses de boro não interferiram nos aspectos produtivos e morfológicos da parte aérea e das raízes nem na composição de parede celular e frações proteicas. O boro somente influenciou a concentração de nitrogênio, potássio e boro da parte aérea. A interação doses de nitrogênio x doses de potássio foi significativa para as variáveis número de perfilhos, número de folhas, área foliar, valor SPAD e produção de massa seca da parte aérea. Com o pouco crescimento da planta ocorreu acumulação de nitrogênio, potássio e boro e diluição desses nutrientes nas folhas diagnósticas com a maior produção das plantas. A produção de massa seca das raízes, comprimento total e superfície total aumentaram com as combinações de doses de nitrogênio e potássio. As concentrações de nitrogênio, potássio e boro nas raízes foram influenciadas pela combinação das doses de nitrogênio com potássio. As concentrações de nitrato e amônio foram influenciadas positivamente pela combinação das doses de nitrogênio e potássio, tendo ocorrido maior concentração de amônio do que nitrato nas raízes. Com o aumento das doses de nitrogênio e potássio o comprimento específico e a superfície específica das raízes diminuíram. Os teores de FDN, FDA aumentaram e a relação FE+LM/CB diminuiu com as doses de potássio no segundo e terceiro cortes. O teor de PB no segundo corte aumentou com as doses de nitrogênio e diminuiu com as doses de potássio. As frações proteicas B1+B2 aumentaram e a fração B3 diminuiu no segundo e terceiro cortes com o aumento das doses de nitrogênio. A fração C para o terceiro corte diminuiu com as doses de nitrogênio. A combinação das doses de nitrogênio e potássio aumentou os teores da fração A para o terceiro corte. O suprimento de nitrogênio e potássio proporcionou aumento nos aspectos produtivos, morfológicos e nutricionais da parte aérea e das raízes, além de ter melhorado o valor nutritivo do capim-tanzânia. / The balanced fertilization with nitrogen, potassium and boron may change plant nutrition, yield and nutritive value of forage grass. Through the supply of combinations of nitrogen, potassium and boron it was aimed to evaluate the following effects on Tanzania guineagrass: a) plant morphology, dry matter production and nutritional status in the shoots; b) morphological, productive and nutritional aspects in the roots and c) cell wall components and protein fractions in the shoots. The experiment was conducted in a greenhouse from January to May 2010, using ground quartz as substrate. Five rates of nitrogen (2, 10, 18, 26 and 34 mmol L-1) were combined with five rates of potassium (0.2, 3.1, 6.0, 8.9 and 11.8 mmol L-1) and five rates of boron (0, 45.45, 90.90, 136.35 and 181.8 mol L-1) in 53 fractionated factorial design, which had 23 combinations of nitrogen, potassium and boron, with four replications. Three harvests of Tanzania guineagrass were made: 32 days after transplanting, 39 days after the first harvest and 45 days after the second harvest. Boron rates did not interfere with production and morphological aspects of shoots and roots and in cell wall composition and protein fractions. Boron only influenced the concentration of nitrogen, potassium and boron in the shoots. The interaction nitrogen rates x potassium rates was significant for the number of tillers, number of leaves, leaf area, SPAD value and shoots dry matter production. When plants had short growth, nitrogen, potassium and boron accumulated in plant tissues and those nutrients were diluted in plants with great growth. The dry mass of roots, total root length, total root area increased with the combination of nitrogen and potassium rates. The concentration of nitrogen, potassium and boron were influenced by the combinations of nitrogen and potassium rates. The concentrations of nitrate and ammonium were positively influenced by the combinations of nitrogen and potassium rates, and higher concentrations of ammonium than nitrate were found in root tissues. Root specific length and specific surface area decreased as rates of nitrogen and potassium increased. The NDF and ADF increased and EF+MB/SS ratio decreased with the potassium rates in the second and third harvests. The CP content of the second harvest increased with the nitrogen and decreased with potassium rates. Fractions B1+B2 increased and fraction B3 decreased for the second and third harvests with increasing nitrogen rates. The fraction C for the third harvest decreased with nitrogen rates. The combination of nitrogen and potassium increased the content of fraction A in the third harvest. Nitrogen and potassium supply provided to increase the productive changes, morphological and nutritional aspects in shoots and roots well as it improved the nutritive value of Tanzania guineagrass.

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144