Tesis sobre el tema "Gel filtration chromatography"

Size exclusion chromatography (SEC) was applied for purification of psychoactive biomolecules from plants. These molecules are in the same molecular weight range, but do not necessarily share other chemical properties, that makes the SEC technique efficient. By applying SEC as a first purification step much of the co-extractives from the plants can easily be removed. Large amounts of target substance can be obtained with little effort if the system is automated. Combining SEC with a second purification step, consisting of normal phase chromatography, provides high purity of the target substance.

Both known and unknown psychoactive biomolecules can easily be purified using the purification method developed in this Master's Thesis. Purifications that previously required long time and much "hands-on" can be completed much faster and with less manual work.

The method developed was tested on cannabis, coffee and 'Spice' with good results.

Tripeptidyl peptidase II (TPPII) is an exopeptidase which cleaves tripeptides from theN-terminus of peptides. The exact functional role of TPPII is still a matter of investigation. Itis believed that the enzyme is primarily involved in intracellular protein degradation, where itcooperates with the proteasome and other peptidases to degrade proteins into free aminoacids. These amino acids can subsequently be used in the production of new proteins. The aimof this work was to express murine wild type TPPII using E. coli and thereafter purify theenzyme from the bacterial lysate. Methods used for the purification included protein andnucleic acid precipitation, anion exchange chromatography, hydrophobic interactionchromatography and gel filtration. The presence of TPPII was determined using activityassay, western blot and SDS-PAGE. Despite the fact that some modification is still needed,the purification yielded a total of 34μg TPPII with a purity of approximately 60%. Thispurified enzyme can be used for future functional characterization.

Bacterioferritin, an iron storage protein having a 24-subunit quaternary structure, was used as a model for the study of host-guest interactions and guest encapsulation, making use of its spherical cage-like structure. A hexahistidine-affinity tag fused to the C-terminus of each bacterioferritin subunit was constructed. The C-terminus of each subunit points toward the inside of the cavity, while the N-terminus is exposed on the surface of the protein. The hexaHistag was able to form strong interactions with a nickel-nitrilotriacetic acid linked dye molecule (guest) and this interaction was used in attempts to develop a principle to control guest molecule encapsulation within the spherical cavity of the 24-mer bacterioferritin protein molecule. The procedure involved (1) subunit dissociation under acidic pH, (2) affinity controlled dye-Histag binding with exposed C-terminal hexahistidine residues and (3) reassociation of the subunits at neutral pH. The encapsulation conditions involving step 1 and 3 were studied preliminarily using laser light scattering to measure size (hydrodynamic radius) of the protein particle with apoferritin as a model system as it resembles the size and structure of bacterioferritin. In order to encapsulate guest molecules, the emptied shell of bacterioferritin was generated by site-directed mutagenesis resulting in ferroxidase- as well as heme-free bacterioferritin mutants (E18A/M52L/E94A), and these mutants were used to examine protein stability before conducting encapsulation experiments. However, wild-type bacterioferritin possessed highest stability in maintaining its multisubunit structure; hence, it was used for the encapsulation studies. It was found that 100% bacterioferritin with hexahistidine tag at the C-terminus, and a combination of 60% bacterioferritin with hexahistidine tag at the C-terminus and 40% bacterioferritin without hexahistidine tag at the C-terminus yielded similar amounts of encapsulated guest molecules. This suggested that all hexahistidine at the C-terminus were not equally available for dye molecule binding.

Des proteines, capables de faciliter des mouvements intermembranaires de phospholipides, ont ete isolees a partir de feuilles d'epinard. Ces proteines, appelees proteines de transfert de phospholipides, ont ete purifiees par les techniques classiques de chromatographie (filtration sur gel, echangeurs d'ions) ou par les techniques plus resolutives et plus rapides de chromatographie liquide a haute performance (colonnes echangeuses d'ions ou en phase inverse). Nous avons verifie la purete des fractions par electrophorese en presence de sds

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The induction of resistance in plants to pathogens is an alternative method of disease control, wich involves activation of plant resistance mechanisms such as induction of phytoalexins. The elicitors molecules are able to induce and activate those responses, and therefore, techniques have sought to isolate and characterize fractions with elicitor character. The study aimed to purify, through ion exchange chromatography and gel filtration chromatography, eliciting molecules from pathogenic nematodes, and test them in phaseolin induction in beans hypocotyls beans and gliceolin in soybean cotyledons. The buffer solution Tris HCl 0.05 M (pH 6.8) was used as control and the acibenzolar-S-methyl (50 mg a.i. L-1) and Saccharomyces cerevisiae (20 mg mL-1) were used as induction standard treatments. Ion exchange chromatography (IEC) and gel filtration chromatography (GC) were performed to separate fractions with eliciting power from 500 female nematodes of Meloidogyne incognita and Meloidogyne javanica. For purification of elicitors from Meloidogyne javanica, through IEC, six glycidic fractions and six glycoproteins were obtained. These were purified on GC, obtaining sixty-three fractions. They have been classified according to their nature, as twenty-six glycidic and thirty-seven glycoprotein with molecular weights ranging from 29.19 to 2989.25 kDa. Regarding the elicitors purification of Meloidogyne incognita through IEC, nine glycidic and five glycoprotein fractions were obtained. From these fractions, a total of fifty-eight fractions was obtained through GC, twenty-five glycidic and thirty-three glycoprotein with molecular weights ranging from 37.42 to 200.32 kDa. From the fractions purified from Meloidogyne javanica eight had inducing potential of phaseolin. For gliceolin fifteen fractions showed inducing effect. Regarding the fractions purified from Meloidogyne incognita, no fraction has inductive potential of phaseolin superior to the standard treatment. However, twenty-two fractions suppressed phytoalexin inducing activity. For gliceolin ten fractions induced the same, whereas, twenty-three fractions suppressed the induction of gliceolin. Chromatography was efficient in the purification of elicitors compounds. Compounds with suppressing characteristics of gliceolin and phaseolin were checked in bioassays. For those fractions obtained through IEC, and then submitted to GC that did not induce phytoalexin, it is suggested that molecules need to act together to have elicitor effect and thus induce defense response in the plant
A indução de resistência em plantas contra patógenos é um método de controle alternativo de doenças, e que envolve a ativação dos mecanismos de resistência da planta, como a indução de fitoalexinas. As moléculas eliciadoras possuem a capacidade de induzir e ativar tais repostas, e assim sendo, técnicas têm buscado isolar e caracterizar frações com caráter eliciador. O trabalho teve por objetivo purificar, por cromatografia de troca iônica cromatografia de filtração em gel, moléculas eliciadoras a partir de nematoides fitopatogênicos, e testá-las na indução de faseolina em hipocótilos de feijoeiro e gliceolina em cotilédones de soja. O tampão Tris HCl 0,05 M (pH 6,8) foi utilizado como tratamento controle e o acibenzolar-S-metil (50 mg i.a. L-1) e o Saccharomyces cerevisiae (20 mg mL-1) foram utilizados como tratamento padrão de indução. Cromatografia de troca iônica (CTI) e cromatografia de filtração em gel (CFG) foram realizadas para separar frações com poder eliciador a partir de quinhentas fêmeas de nematoides de Meloidogyne incognita e Meloidogyne javanica. Para a purificação de eliciadores a patir de Meloidogyne javanica, por CTI, foram obtidos seis frações glicídicas e seis glicoproteicas. Estas, por sua vez, foram purificadas em CFG, sendo obtidos no total sessenta e três frações. As mesmas foram classificadas de acordo com sua natureza, sendo vinte e seis glicídicas e trinta e sete glicoproteicas, com massas moleculares variando de 29,19 a 2.989,25 kDa. Em relação a purificação de eliciadores de Meloidogyne incognita por CTI, foram obtidos nove frações glicídicas e cinco glicoproteicas. A partir destas, foram obtidos por CFG um total de cinquenta e oito frações, sendo vinte e cinco glicídicas e trinta e três glicoproteicas, com massas moleculares variando de 37,42 a 200,32 kDa. Das frações purificadas a partir de Meloidogyne javanica oito apresentaram potencial indutor de faseolina. Para gliceolina quinze frações mostraram efeito indutor. Em relação as frações purificadas a partir de Meloidogyne incognita, nenhuma fração apresentou potencial indutor de faseolina superior ao tratamento padrão. Entretanto, vinte e duas frações suprimiram a atividade de indução de fitoalexina. Para gliceolina dez frações induziram a mesma, enquanto que, vinte e três frações suprimiram a indução da gliceolina. A cromatografia foi eficiente na purificação de compostos eliciadores. Compostos com características supressoras de gliceolina e faseolina foram verificadas nos bioensaios. Para aquelas frações obtidas por CTI e posteriormente submetidas a CFG que não induziram fitoalexina, sugere-se que as moléculas necessitam atuar juntas para haver efeito eliciador e assim induzir a resposta de defesa no vegetal

Deux protéases alcalines P1 et P2 ont été isolées à partir d'homogénats de larves de Galleria mellonella. Les 2 enzymes ont été séparées par chromatographie sur échangeurs d'ions et purifiées ultérieurement par filtration sur gel. Les 2 protéases se distinguent par leur pH optimum d'action, leur poids moléculaire et leur sensibilité vis-a-vis de différents inhibiteurs de protéases. La répartition anatomique de l'activité protéolytique montre la présence de 2 protéases alcalines similaires à P1 et P2 dans le tube digestif, et d'une protéase similaire à P1 dans le tissu adipeux

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Induction of resistance involves the activation of plant defense mechanisms in response to treatment with biotic or abiotic elicitors. The application of plant extracts in order to induce resistance mechanisms is an interesting alternative to chemical control, however, besides the presence of inducers, can occur the presence of suppressors. This study aimed to partially purificate through gel filtration chromatography (GFC) and precipitation with ammonium sulfate (SA), compounds present in decoct of Adiantum capillus-veneris, capable to induce defense mechanisms in sorghum mesocotyls, including phytoalexins and peroxidase, polyphenoloxidase (PPO), phenylalanine ammonia-lyase (PAL) and chitinase. The decoct 1% was fractionated with concentrations of ammonium sulfate, 0-20%, 20-40%, 40-60%, 60-80% and 80-100% of SA and those fractions were subjected to GFC. We obtained nine protein peaks and one glucosic peak for decoct with molecular weights ranging from 0.61 to 0.01 KDa; to fraction 0-20% were obtained two protein and two glucosic peaks, with molecular weights lower than 0.01 KDa, and concentration of sugars ranging from 4.1 to 17.5 mg mL-1; to fraction 20-40% were obtained three protein peaks (0.98 to 111.5 KDa) and five glucosic peaks (11.3 to 73.7 mg mL-1); to fraction 40-60% were obtained two protein peaks (0.09 to 111.5 KDa) and two glucosic peaks (5.6 to 7.5 mg mL-1); to fraction 60-80% were obtained six protein peaks (lower than 0.02 KDa) and two glucosic peaks (16.5 to 51.3 mg mL-1); and to fraction 80-100% were obtained three protein peaks (lower than 0.09 KDa). Sorghum mesocotyl were treated with fractions from the GFC, and decoct, acibenzolar-S-methyl (ASM) (125 mg L-1 of a. i. as elicitor of reference) and sodium phosphate buffer 10 mM pH 6.0. After incubation of 96 h were measured the levels of phytoalexins in mesocotyls and the activity of defense-related enzymes in leaves. Treatment with peak II (0,09 KDa) induced phytoalexin 6.68% more than. Among the fractionn, 60-80% increased 76% compared to ASM. To peroxidase the peak IV (lower than 0,01 KDa) increased 21% the activity compared to control water, and 44% compared to ASM. For the fraction 0-20% the protein peak II (lower than 0,01 KDa) increased 39% the activity in relation to the fraction 0-20% and 19% in relation to decoct. The fraction, 80-100% increased 89% compared to, ASM. For the PPO the peak VI (lower than 0,01 KDa) from decoct decreased 88% the activity compared to ASM. For PAL the peak II (lower than 0,01 KDa) from fraction 0-20% was 91% higher than decoct. For chitinase 1% peak IV (lower than 0,01 KDa) from decoct was 68% higher than the ASM. It was possible to induce defense mechanisms in sorghum by the application of partially purified fractions from A. capillus-veneris, which can allow to obtain new molecules and development alternative methods to control plant diseases
A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. A aplicação de extratos vegetais visando à indução de mecanismos de resistência é uma alternativa interessante ao controle químico, entretanto, nestes extratos pode ocorrer além da presença de indutores, a presença de supressores. Este trabalho teve por objetivo a purificação parcial, por meio de cromatografia de filtração em gel (CFG) e precipitação com sulfato de amônio (SA), de compostos presentes em decocto de avenca (Adiantum capillus-veneris), eficientes na indução de mecanismos de defesa em mesocótilos de sorgo, incluindo as fitoalexina deoxiantocianidinas e as proteínas peroxidase, polifenoloxidase, fenilalanina amônia-liase e quitinase, buscando selecionar frações potencialmente eficientes na indução de resistência em plantas. Decocto (EA 1%) de A. capillus-veneris foi fracionado com concentrações de sulfato de amônio de 0-20%, 20-40%, 40-60%, 60-80% e 80-100% e esses cortes foram submetidos à cromatografia de filtração em gel (CFG). Foram obtidos nove picos protéicos e um pico glicídico para EA 1% com massas moleculares variando de 0,61 à 0,01 KDa; no corte 0-20% foram obtidos dois picos protéicos e dois glicídicos, com massas moleculares menores que 0,01 KDa, e concentração de açúcares redutores variando de 4,1 a 17,5 µg mL-1; no corte 20-40% três picos protéicos (111,5 à 0,98 KDa) e cinco glicídicos (11,3 a 73,7 µg mL-1 de açúcares); no corte 40-60% dois picos protéicos (111,5 à 0,09 KDa) e dois glicídicos (5,6 a 7,5 µg mL-1); no corte 60-80% seis picos protéicos (menor que 0,02 KDa) e dois glicídicos (16,5 a 51,3 µg mL-1); e no corte 80-100% três picos protéicos (menor que 0,09 KDa). Mesocótilos de sorgo foram tratados com as frações provenientes da CFG, além do decocto a 1%, acibenzolar-S-metil (ASM) (125 mg. L-1 do i.a. como elicitor de referência) e tampão fosfato de sódio 10 mM pH 6,0, totalizando 42 tratamentos. Após incubação por um período de 96 h, avaliou-se dos teores de fitoalexinas nos mesocótilos e análises bioquímicas dos folíolos. O tratamento pico II (0,09 KDa) do EA 1% mostrou-se eficiente na indução de fitoalexinas, sendo superior em 6,68% ao ASM. Entre os cortes, 60-80% permitiu incremento de 76% em relação ao ASM. Para peroxidase o pico IV (menor que 0,01 KDa) do EA 1% incrementou 21% a atividade em relação a testemunha água e 44% ao ASM. Para os precipitados 0-20% o pico protéico II (menor que 0,01 KDa) promoveu incremento de 39% na atividade em relação ao corte 0-20% e 19% para o EA 1%. O precipitado 80-100% foi superior 89% ao ASM. Para polifenoloxidase o pico protéico VI (menor que 0,01 KDa) do EA1% reduziu 88% a atividade em relação ao ASM. Para fenilalanina amônia-liase o pico protéico II (menor que 0,01 KDa) do corte 0-20% foi 91% superior ao EA 1%. Para quitinase o pico protéico IV (menor que 0,01 KDa) do EA 1% foi 68% superior ao ASM. Foi possível induzir mecanismos de defesa em sorgo pela aplicação de frações parcialmente purificadas de A. capillus-veneris, o que pode permitir a obtenção de novas moléculas e o desenvolvimento de métodos alternativos para controle de doenças em plantas

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
Full Text

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Induced resistance involves the activation of latent defense mechanisms in plants in response to the treatment with biotic or abiotic agents. The use of crude extracts aimed at inducing resistance mechanisms is an interesting alternative to chemical control, however, in this extracts can occurs besides de presence of inducers, the presence of suppressors. This work objectived a partial purification, through gel filtration chromatography (GFC) and by ammonium sulphate (AS) precipitation, of compounds in basidiocarps crude extracts from Pycnoporus sanguineus efficient to the control of Asian rust in soybean by induction of resistance. Crude extracts from P. sanguineus were submited to gel filtration chromatography (GFC), and five proteins and one carbohydrate peaks were obtained, with molecular weight ranging from 1,82 to 5,18 KDa. It was made the fractionated protein purification from 100 mL of crude extract from P. sanguineus. The fractions obtained were: 0-20%, 20-40%, 40-60%, 60-80% and 80-100% of ammonium sulphate. Incised soybean cotyledons were treated with fractions from GFC and AS precipitation, crude extract 20% (EB 20%), Saccharomyces cerevisiae (25 mg mL-1) and water. After incubation during 20 h, was made biochemical analyses to verify the phytoalexins content and peroxidases, polyphenoloxidases, β-1,3 glucanases and phenylalanine ammonia-lyase activities. The treatments from GFC, protein fractions III (3,44 KDa), IV (2,79 KDa) and V (1,82 KDa), and carbohydrate fraction, fractions 40-60%, 60-80% and 80-100%, obtained by ammonium sulphate precipitation, crude extract (CE) of basidiocarps at 20%, besides control treatments fungicide (tebuconazole, 0,5 g a.i. L-1) and water were used to evaluate the antimicrobial activity and induction of resistance in soybean against P. pacchirhizi in greenhouse. Soybean plants were treated and after three days were inoculated with the pathogen. Samples were collected 0, 1, 3, 6 and 9 days after the treatment for biochemical analyses and the severity was evaluated after 13 days. The data referring to evaluation of antimicrobial activity show that the fractions do not have inhibiting activity of germination of spores. As for severity, the treatments protein fraction III and CE 20% were efficient in the reduction of the number of injuries for cm2. For peroxidase the CE 20% presented tendency in reducing the enzymatic activity. Chitinases and polifenoloxidases did not presented statistical differences between the treatments. For β-1,3 glucanases there was local induction for the treatments with glicide fraction and protein fractions III and V, and fractions 40-60% and 60-80%, while CE 20% presented systemic induction for this enzyme. For phenylalanine ammonia-lyase the purified fractions of P. sanguineus reduced the enzymatic activity, except for CE 20%. It was possible to induce defense mechanisms in soybean against P. pachyrhizi for the application of partially purified fractions from P. sanguineus, indicating its possible use as an alternative method for controling this pathogen
A indução de resistência envolve a ativação de mecanismos de defesa latentes existentes nas plantas em resposta ao tratamento com agentes bióticos ou abióticos. A aplicação de extratos brutos visando à indução de mecanismos de resistência é uma alternativa interessante ao controle químico, entretanto, nestes extratos pode ocorrer além da presença de indutores, a presença de supressores. Este trabalho teve por objetivo a purificação parcial, por meio de cromatografia de filtração em gel (CFG) e precipitação com sulfato de amônio (SA), de compostos presentes em extrato bruto de Pycnoporus sanguineus, eficientes no controle de ferrugem asiática em soja por indução de resistência. Extrato bruto de P. sanguineus foi submetido à cromatografia de filtração em gel, sendo obtidos cinco picos protéicos e um pico glícido, com pesos moleculares variando de 1,82 a 5,18 KDa. Foi efetuada a precipitação fracionada das proteínas presentes em 100 mL de extrato bruto de basidiocarpos de P. sanguineus. As frações obtidas foram: 0-20%, 20-40%, 40-60%, 60-80% e 80-100% de SA. Cotilédones de soja incisados foram tratados com as frações provenientes da CFG e precipitação com SA, além dos tratamentos extrato bruto a 20% (EB 20%), Saccharomyces cerevisiae (25 mg mL-1) e água. Após incubação pelo período de 20 h efetuou-se análises bioquímicas dos cotilédones para verificar os teores de fitoalexinas, peroxidases, polifenoloxidases, β-1,3 glucanases e fenilalanina amônia-liase. Os tratamentos obtidos por CFG frações protéicas III (3,44 KDa), IV (2,79 KDa) e V (1,82 KDa), e glícida, além das frações 40-60, 60-80 e 80-100% obtidos por saturação com SA foram selecionados para avaliação de atividade antimicrobiana e de indução de resistência em soja contra P. pacchirhizi em casa de vegetação, utilizou-se também extrato bruto (EB) 20% de basidiocarpo de P. sanguineus e as testemunhas fungicida tebuconazole (0,5 g i.a. L-1) e água. Visando avaliar o controle da doença ferrugem asiática e indução de enzimas relacionadas à defesa, montou-se ensaio em casa de vegetação onde plantas foram tratadas e após três dias inoculadas com o patógeno. Amostras foram coletadas 0, 1, 3, 6 e 9 dias após os tratamentos para análises bioquímicas e a severidade foi avaliada após 13 dias. Os dados referentes à avaliação de atividade antimicrobiana mostram que as frações não possuem atividade inibidora da geminação de esporos. Quanto à severidade, a fração III e o EB 20% foram eficientes na redução do número de lesões por cm2. Para peroxidases, o EB 20% apresentou tendência em reduzir a atividade enzimática. Quitinases e polifenoloxidases não apresentaram diferenças estatísticas entre os tratamentos. Para β-1,3 glucanases houve indução local pelas frações glícida e protéicas III e V, e pelas frações 40-60% e 60-80% em relação ao EB 20%, sendo que o mesmo apresentou indução sistêmica para essa enzima. Para fenilalaniana amônia-liase as frações purificadas de P. sanguineus apresentaram tendência em reduzir a atividade enzimática, com exceção para EB 20%. Foi possível induzir mecanismos de defesa em soja contra P. pachyrhizi pela aplicação de frações parcialmente purificadas de P. sanguineus, o que pode permitir o desenvolvimento de métodos alternativos para controle desse patógeno

En el presente trabajo de tesis se evalúa la posible contribución de otros compuestos al desarrollo del olor sexual mediante la aplicación de técnicas de Head Space dinámico y cromatografía de filtración de gel. El estudio se efectuó en canales de cerdos enteros previamente clasificadas con concentraciónes bajas de escatol (<0,10 µg /g) y androstenona (0,50 µg/ g) , aunque presentaban , según las respuestas de un panel sensorial, olor sexual. La selección de muestras de grasa clasificadas sin olor sexual permitió la comparación estadística de los resultados obtenidos. El análisis de los compuestos volátiles mostró una mayor abundancia de aldehidos (hexanal, heptanal), ácidos grasos de cadena corta (hexanoico, heptanico nonanoico), 1,4-diclorobenceno y estireno en las muestras de grasa con defecto organoléptico, ue puede, debido a los atributos sensoriales de estas sustancias y sus bajos umbrales de detección, favorecer el desarrollo de aromas desagradables asociados al olor sexual por los miembros de un papel sensorial. El análisis de las fracciones obtenidas por filtración de gel mediante cromatografía de gases acoplada a la espectometría de masas permitió la identificación de la 4-fenil-3-buten-2-ona en las muestras clasificadas con olor sexual y concentraciones bajas de escatol y androstenona, que junto a los resultados obtenidos en el estudio sensorial, indican una influencia de este compuesto en el olor sexual. Por una parte la 4-fenil-3-buten-2-ona presentaría una acción sinérgica con la androstenona al potenciar su detección por parte de los panelistas a concentraciones inferiores de 0,01µg/g. Además, el olor a naftalina que presenta la 4-fenil-3-buten-2-ona puede confundir a los miembros del panel debido a que es uno de los descriptores sensoriales que se asocia con la presencia de escatol en las muestras de grasa evaluadas. Así mismo, la baja transferencia a la fase vapor del escatol y, especialmente, de la androstenona determinada mediante técnicas de Head Space estático, indica que la concentración de cada uno de los compuestos en la fase vapor no se corresponde con la concentración real en las muestras de grasa . Según estos resultados, las respuestas del panel test pueden estar influenciadas por la poca volatilidad de los compuestos implicados en el olor sexual y por la presencia en la fase vapor de otros compuestos aromáticos de mayor volatilidad.
La validación de métodos de análisis para la determinación de indol-escatol y androstenona-androstenoles en grasa dorsal porcina mediante cromatografía líquida en fase normal y cromatografía de gases acoplada a la espectrometría de masas, respectivamente, fue otro de los objetivos planteados del presente estudio. La simplificación del tratamiento de las muestras de grasa fue uno de los aspectos analíticos que se tuvo en mayor consideración con la finalidad de ofrecer una mayor capacidad de muestras en un intervalo de tiempo corto.

Le traitement des eaux usées urbaines constitue une phase importante de la gestion globale des ressources en eau. Le bioréacteur à membranes est une option de choix pour répondre à cette demande. Ce procédé de traitement présente plusieurs avantages par rapport aux procédés classiques à boues activées mais reste confronté à l’un des problèmes majeurs de la filtration membranaire : le colmatage de la membrane. De nombreuses études ont mises en évidence l’impact néfaste sur la filtration de composés contenus dans le liquide interstitiel des boues activées définis comme des colloïdes ou des composés solubles. Cependant dépendant des nombreuses conditions opératoires rencontrées dans les bioréacteurs à membranes, les résultats sont encore contradictoires. Ainsi une meilleure identification des composés problématiques et des mécanismes de colmatage est nécessaire afin de mieux optimiser les conditions opératoires et les opérations de maintenances. C’est pourquoi cette étude s’attache à identifier les principaux composés colmatants contenus dans la fraction non particulaire d’échantillons de boues activées réelles, à étudier la modification des dépôts de filtration induite par l’ajout de particules synthétiques submicroniques et à suivre la formation des dépôts in situ par mesure de différence de potentiel électrique transmembranaire. Les résultats soulignent l’important rôle dans le colmatage membranaire jouer par les biopolymères organiques, plus précisément la structure de type protéines des ces composés. Il a été montré que ces composés induisent des dépôts très résistants, réversibles et très compressibles. De plus, nous avons montré que l’ajout de particules submicroniques au sein de ces dépôts à pour principal impact de modifier la compressibilité de ces dépôts et d’augmenter la rétention indépendamment des interactions prenant lieu entre les particules et les matières organiques. Cette étude ouvre donc sur la possibilité de réduire les doses de composés chimiques utilisés pour limiter le colmatage en prenant en compte les effets de structuration des dépôts. De plus les résultats montrent, que pour des matrices organiques relativement simples, la possibilité de suivre la formation de dépôts colmatants par mesure de potentiel électrique. Cependant de plus amples expériences doivent être réalisées afin d’interpréter le signal électrique lors de la filtration de fluides biologiques complexes
Direct treatment of activated sludge in membrane bioreactor frequently results in low net fluxes and frequent maintenance operations. The water separation by membrane is largely determined by the quality of the activated sludge (AS) and by the mechanisms involved in fouling. Many research studies have focused on the understanding of fouling mechanisms and on the development of processes to reduce fouling in order to achieve a stable operation. This thesis investigates the role of fine “inert” particles (melamine and latex) on structuration of a fouling layer formed during membrane filtration of waters rich in natural organic matters (waste waters). Two cases were investigated: (i) the fine particles were mixed with AS supernatant and then filtered on a virgin microfiltration membrane. And secondly (ii) filtration was performed on a dynamic filter previously made of the fine particles. In order to characterise fouling layer properties during filtration a new experimental method was developed by measuring the electrical potential across the membrane as an in situ measurement. Results emphasized the important role in reversible fouling of macromolecular proteins present in the biopolymer of the water phase of the sludge. It was found that fouling layer induced by these macromolecules is highly compressible. Moreover results show that the addition of particles into biofluid diminishes the fouling layer compressibility and improve its removal by backwash. It was found that OM fouling resistance increase when latex particles are present whereas the resistances are lower when melamine is added. Thus even small interactions (small compared to classical adsorbent particles such as activated carbon) between particles and OM can improve the filterability of the biofluids. Results show that melamine particles are more prone to interact with organic matter than latex, and lead to less flux decline. However dynamic filter made of melamine leads to more flux decline and compressible organic matter layer but is easily removable. Add to this the measurement of electrical potential during fouling layer formation show, in simple case (filtration of fine particles alone), the possibility to characterise fouling layer properties in terms of surface charge and thickness

Etude de la relation entre le pouvoir impregnant des brais et leurs proprietes physico-chimiques. Definition d'un indice de filtrabilite. Caracteristique de l'aptitude des brais a l'impregnation. Etude de l'evolution des proprietes de filtration des brais en fonction des traitements thermiques. Etude des mecanismes responsables de la filtrabilite par fractionnement et caracterisation des fractions du brai

Notre but a été d'élaborer un protocole expérimental pour la purification rapide des enzymes de synthèse des catécholamines. La chromatographie liquide haute performance (CLHP) par filtration sur gel et échange d'ions a permis de réduire considérablement la durée d'analyse avec un rendement accrue et un gain d 'activité spécifique des enzymes. Les durées d'analyse courtes. Par l'emploi de la CLHP. Ont évité l'hydrolyse des protéines souvent observée en chromatographie classique et due à la présence de certaines protéases. Plusieurs méthodes chromatographiques ont été employées afin d'éliminer les protéines non spécifiques. L'activité spécifique de l'aromatique L-amino acid decarboxylase dans des fractions de médulJo-surrénale de bovin. Initialement de 4 nanomoles de dopamine formée/min. /mg de protéine, est accrue à 120 par chromatographie d'échange d'ions sur une colonne DEAE­ sephacel. L'activité spécifique de l'enzyme est encore augmentée d'environ 7 fois par chromatographie de filtration sur gel sur une bi-colonne TSK 3000 SW. Enfin, l'emploi de la chromatographie d'interactions hydrophobes améliore cette activité spécifique de 12 fois. Les poids moléculaires des enzymes déterminés par chromatographie de filtration sur gel : 290000 pour la tyrosine hydroxylase, 360000 pour la dopamine bêta-hydroxylase, 37000 pour la phéniléthanolamine N-méthyltransférase et 110000 pour l'aromatic L-amino acid decarboxJiase, est proche de ceux estimés par d'autres méthodes classiques. La chromatographie de perméation de gel, en utilisant des phases mobiles de faible force ionique et en évitant totalement les solvants organiques ou les détergents, a permis une conservation optimale de l'activité biologique.

L'objectif de ce travail est d'expliquer la formation des pms refractaires et de caracteriser ces produits quantitativement et qualitativement lors du traitement d'une eru par boues activees. La premiere partie presente un bilan des connaissances sur des pms a la dco residuelle de boues activees. La deuxieme partie a permis d'evaluer la dco soluble produite dans la dco soluble residuelle de boues activees traitant une eru en mode continu. La troisieme partie presente la cinetique de la formation des pms lors de la degradation du glucose par boues activees en sbr. Dans la quatrieme partie et dans la cinquieme partie, la caracterisation quantitative et qualitative des pms et l'interpretation de matieres organiques dans une eru traite par boues activee. Toutes les experiences realisees ont demontre l'importance des pms formes lors du traitement d'une eau residuaire urbaine par boues activees

A Oiss{!y'tation $uhm'itted in fulfilment of the r~;~airements, for' the degree of f·1aster· oi~'j Science at the University of the ~titwatersrand ' Juhanoasbul"g January 1991
An attempt was made to separate ribosomal subunits by gel filtl'ation on Trisacryl GF2000 and Sepharose 4B. Trisacryl GP~2000,a sYllthet'ic gol, , separated rat .ljver rlbosonal subunits orl 'f~'135,em column with a resolution of ""Or3, resulting 'in <'1" 60 rJ impm''ity 'of each of the \~ subunits. subunits" were not resolved. Sepharose 4131 an agarose based gol, separated " the subunits by adsorpttcn chromatography rathr.r than 'i>.V () Q ':) At 4°C, the 405 .subunf ts were eluted with a kd () ruO,:~9( but the GOrf' $ubud"its adsorbed to th~.'g,~Jl and were eluted I; /) when the temperature of "the column was increas~d to 250C..3SoC. (' ,. ('-. This edsorpt ion phenonenon seems to b{~ tl propert~ of a 11 agllrose -: \') ~\:; based gels studied here, includ'ing Sepharose 2.B and .seph'ato~eoBt arid is exc 1us iva to !,mamm'i'lan r ibosOIntrt subuni ts • Anal,ys1S 0'(" the , u subunits by in vitro r14C]polypheny1alanine sy'nthes'is showed OCI " ,,'c. /\ I 7', " diff'erence 'in the act lvtt ies of dbosoma'J ~ubunH~ p~epdrf#d by ;/'/ ' grndient centrifugation or by 5epharos,e cni"OmatogNPPY;' Analy~ii ~~of " (~ (( the subunits by ~crylamidec'ugarose coU)po5it~) gs1s resulted ,in the '/ resolution ,.:n"f subunits isolated fr'oill lower organisms in o II non-denaturlnq !Jcl systems and SI,lbut1its from m«mm~nan'tissue in II /'\';1 Ga'! f'iltrat'ion does tyffer a 5t!i:l;~'ble metrKld'¥or the pr~p;n~¥tiol1of <::) . I \) \\ I) ribosoma 1 'subunits I but only' if 'the act~orption JH'OrJ&~"t'Jo,s of ,) (,) ':' o ,1) ,{

碩士
國立中興大學
生物化學研究所
88
A purification process was developed to obtain highly pure rVP2H particles, formed by a structural protein (VP2) of infectious bursal disease virus (IBDV) with additional six histidine residues at its C-terminus, for the study of its antigenicity and conformational structure and the development of an efficient subunit vaccine against IBDV infection. The expressed VLPs mimic th authentic structure of the critical epitopes in the native viruses and are capable of inducing a stronger immunological response than does the free form of rVP2H. Those particles in the inected High-FiveTM cell lysates were partially purifed employing immobilized metal-ion (Ni＋2) affinity chromatography (IMAC). This step recovered about 85 percent of immunogeric rVP2H proteins but did not separate the rVP2H particles from the free rVP2H proteins or the degraded products of rVP2H. A further separation of the particulate form of rVP2H from the free form of rVP2H was achieved using either gel filtration chromatography or CsCl density gradient ultracentrifugation. The latter method is suitable for the purification of small amount of rVP2H particles and the particle's buoyant density determine using this method was very close to 1.27 g/cm3. However, when large quantities of the rVP2H particles are needed, it is more efficient to purify the particles by using gel filtration chromatography than the conventional ultracentrifugation method. The separation of the particulate form from the free form of rVP2H proteins enables the evaluation of their own immunogenicity to induce the virus-meutralizing antibodies leading to the protection of chickens from IBDV infection. In addition, the abundant quantities of pure rVP2H particles coupled with the uniform dimensions of the particles will facilitate an examination of higher order structure of the immunogenic particles and, therefore, the design of better vaccines against the virus will evolve.

Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC 3.2.1.45). Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery.
Graduate

The gram-negative bacteria Escherichia coli, a neutralophile, is remarkable in its defenses against acid stress. Of interest to our laboratory is the inducible lysine decarboxylase (LdcI) system, an acid resistance system which renders acid resistance to E. coli in mild acid stress (~pH 5). It was found that this enzyme forms an extremely large (~3.3 MDa) and tight complex (Kd ~ 0.56 μM) with a MoxR AAA+ ATPase named Regulatory ATPase Variant A (RavA). The cryo-EM structure at 14 Å was determined. Through size-exclusion chromatography (SEC) experiments, the binding sites on both LdcI and RavA have been determined. It is proposed that the complex can form through both charged and hydrophobic interactions. In the course of these studies, unexpected observations led to the characterization of the LARA domain of RavA as an amyloid protein under in vitro conditions. The physiological significance of this observation is still under investigation.