CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Moringa oleifera Lam. à uma planta originÃria do Noroeste da Ãndia, bem adaptada Ãs regiÃes tropicais. De suas sementes foi isolada uma nova proteÃna ligante à quitina, a Mo-CBP3, com propriedades coagulantes e atividade antifÃngica contra o fitopatÃgeno Fusarium solani. As proteÃnas foram extraÃdas da farinha delipidada de sementes com o tampÃo Tris-HCl 0,05 M, pH 8,0, contendo NaCl 0,15 M. O teor mÃdio de proteÃna da farinha correspondeu a 216,44 mgP/gF. O extrato total foi fracionado em albuminas e globulinas por diÃlise contra Ãgua seguida de centrifugaÃÃo. As albuminas foram concentradas com sulfato de amÃnio a 90% de saturaÃÃo. A F0-90% foi submetida à cromatografia de afinidade em coluna de quitina, previamente equilibrada com o tampÃo de extraÃÃo. Foram obtidos um pico nÃo retido e dois picos retidos, correspondentes Ãs proteÃnas ligantes à quitina (CBP). O primeiro destes foi eluÃdo com soluÃÃo de N-acetil-D-glucosamina 0,1 M (PNAG), e o segundo, com Ãcido acÃtico 0,05 M, pH 3,0 (PAC). PNAG foi aplicado em coluna de troca catiÃnica, Resource S, acoplada a um sistema de FPLC, equilibrada com tampÃo acetato de sÃdio 0,05 M, pH 5,2. Dos picos obtidos, o terceiro correspondeu à proteÃna Mo-CBP3 - eluÃda com 0,5 M de NaCl no tampÃo de equilÃbrio. O teor protÃico mÃdio calculado para a proteÃna purificada Mo-CBP3 foi de 1,17 mgP/gF, representando um rendimento final de 0,54% das proteÃnas do extrato total. A massa molecular aparente por SDS-PAGE foi de 18,0 kDa sem o agente redutor e, de 9,0 kDa, na presenÃa deste. O resultado sugere que Mo-CBP3 seja uma proteÃna dimÃrica formada de subunidades idÃnticas, unidas por pontes dissulfeto. A massa molecular de Mo-CBP3, por cromatografia de exclusÃo molecular, foi de 14,34 kDa, e o pI de 10,8. Trata-se de uma glicoproteÃna com 2,5% de carboidratos, que nÃo apresenta atividade hemaglutinante ou quitinÃsica. Sua seqÃÃncia NH2-Terminal obtida foi CPAIQRCCQQLRNIQPPCRCCQ, com 22 aminoÃcidos, confirmando sua caracterÃstica bÃsica. Mo-CBP3 mostrou-se tÃo eficiente quanto o AlK(SO4)2 na capacidade de coagular matÃria em suspensÃo na Ãgua. Mo-CBP3 foi fungicida para esporos de Fusarium solani a 0,1 mg/mL. O aquecimento da proteÃna a 98 ÂC, por 1 h, e a prÃ-incubaÃÃo com o aÃÃcar N-acetil-D-glucosamina, nÃo reverteram sua aÃÃo. Mo-CBP3 mostrou-se capaz de retardar o crescimento micelial do fungo ainda na menor dose testada, 0,05 mg/mL. Mo-CBP3 à inativa contra o oomiceto Pythium oligandrum, que apresenta celulose no lugar da quitina na parede celular. A proteÃna foi, ainda, capaz de inibir cerca de 80% da acidificaÃÃo do meio, por esporos de F. solani, induzida por glicose, o que sugere a influÃncia de Mo-CBP3 sobre as bombas de prÃtons (H+ATPases) presentes na membrana celular dos esporos deste fungo. / Moringa oleifera Lam. is native from Northwest India, well adapted to tropical
regions. From its seeds it was isolated a new chitin binding protein, Mo-CBP3, which
has coagulant properties and antifungal activity against the phytopathogen Fusarium
solani. Proteins were extracted from defatted seeds flour by 0.05 M Tris-HCl buffer, pH
8.0, containing 0.15 M NaCl. The average protein content of the flour was 216.44
mgP/gF. The crude extract was fractionated in albumins and globulins by dialysis and
centrifugation. Albumins were concentrated by 90% ammonium sulfate saturation. This
fraction was applied into a chitin column, previously equilibrated with the same buffer.
An unadsorbed and two adsorbed peaks were obtained. The first adsorbed peak was
eluted with 0.1 M N-acetyl-D-glucosamine (PNAG), and the second one, with 0.05 M
acetic acid, pH 3.0 (PAC). PNAG was applied into a cation exchange column, Resource
S, equilibrated with 0.05 M sodium acetate buffer, pH 5.2. The third peak corresponded
to Mo-CBP3 â eluted with 0.5 M NaCl in equilibrium buffer. The protein content of Mo-
CBP3 was 1.17 mgP/gF. It represents a final yield of 0.54% of crude extract proteins.
Apparent molecular mass by SDS-PAGE was 18.0 kDa in the absence of β-ME, and
9.0 kDa, in its presence. Results suggest that Mo-CBP3 is a dimeric protein, made of
identical subunits, linked by disulfide bonds. By molecular exclusion chromatography,
calculated molecular mass was 14.34 kDa, pI 10.8. Mo-CBP3 is a glycoprotein with
2.5% of carbohydrates, which has not hemagglutinating or chitinase activities. Its NH2-
terminal sequence was CPAIQRCCQQLRNIQPPCRCCQ, with 22 amino acids,
conffirming its basic character. Mo-CBP3 was as efficient as AlK(SO4)2 in the capacity
of coagulating suspended material in water. Mo-CBP3 (0.1 mg/mL) was fungicide to
Fusarium solani spores. Heat treatment of the protein at 98 ÂC, du ring 1 h, and pre
incubation with N-acetyl-D-glucosamine, did not reverse its action. Mo-CBP3 was able
to retard the mycelial growth of the fungus even at the lowest tested dose of 0.05
mg/mL. Mo-CBP3 was inactive against the oomycete Pythium oligandrum, which has
cellulose in spite of chitin in cell wall. Protein was also able to inhibit about 80% of
medium acidification, induced by glucose, by F. solani spores, that suggests the
influence of Mo-CBP3 over the proton pumps (H+ATPases) present in cellular
membranes of F. solani spores.
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:4133 |
| Date | 10 December 2009 |
| Creators | Juliana Menezes Gifoni |
| Contributors | Ilka Maria Vasconcelos |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em BioquÃmica, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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