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Doctoral Thesis
DOI
10.11606/T.91.2007.tde-07022008-155823
Document
Author
Full name
Lígia Aíra de Medeiros
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2007
Supervisor
Committee
Molina, Silvia Maria Guerra (President)
Azevedo, Ricardo Antunes de
Carmona, Eleonora Cano
Chagas, Eliana Pereira
Monteiro, Regina Teresa Rosim
Title in Portuguese
Xilanases de Penicillium chrysogenum: produção, purificação, caracterização e aplicação no pré-branqueamento de polpa celulósica de pseudocaule de bananeira frutífera
Keywords in Portuguese
Banana
Branqueamento
Enzimas
Penicillium
Polpa de celulose
Purificação
Resíduos agrícolas.
Abstract in Portuguese
O objetivo desse trabalho foi estudar o potencial de xilanases presentes no filtrado de cultura e de xilanases purificadas de Penicillium chrysogenum no processo de branqueamento de polpa celulósica de pseudocaule de bananeiras frutíferas. Inicialmente, estabeleceu-se o meio e o tempo de cultivo ótimos para a produção de xilanase pelas linhagens IFO-4626 e M-85 de Penicillium chrysogenum. Ambas as linhagens apresentam, além da alta atividade de xilanase, alta atividade de pectinases e baixa atividade de celulases, características que contribuem para seu emprego no processamento de polpa e(ou) fibras celulósicas. Para a linhagem IFO-4626, a melhor produção de xilanase foi obtida no meio Ferreira após 60h de cultivo, usando como indutor xilana de oat spelt. O desempenho da linhagem M-85 só se iguala ao da IFO-4626 no tempo 72h, no meio Haas. Como fontes de carbono, palha de cana-de-açúcar e fibra de coco podem substituir a xilana de oat spelt na produção de xilanase pela linhagem IFO-4626. Xilose pode induzir a síntese de xilanase quando nenhuma fonte de xilana é adicionada ao meio. A inibição da atividade de xilanase foi observada nos meios com xilana e glicose (1%) ou galactose (1%). Nos experimentos de purificação da xilanase foi usado o filtrado de cultura obtido após 72h de cultivo da linhagem IFO-4626 no meio Ferreira. Dois picos com atividade de xilanase foram obtidos após a eluição em uma coluna de troca aniônica DEAE-Sephacel. A xilanase I, com massa molecular de 12,6 kDa, Km = 12,14 mg/mL e Vmáx = 7,75 U/µg de proteína, não se ligou à resina e a xilanase II, com massa molecular de 20 kDa, Km = 39,32 mg/mL e Vmáx = 1579,62 mg/mL, foi eluída com 100 mM de NaCl. Tanto as xilanases presentes no filtrado de cultura como as xilanases I e II, apresentaram boa estabilidade térmica a 40°C e 50°C e em valores de pH de 3,5 a 9,0. Porém, além de não possuírem boa estabilidade a 60°C, suas temperaturas e pH ótimos de reação são baixos. As xilanases presentes no filtrado de cultura foram ativadas pela presença de Fe+2, Mn+2, Ca+2 e ditiotreitol (DTT) e inibidas pela presença de Zn+2, dodecil sulfato de sódio (SDS), Pb+2 e Hg+2. A atividade da xilanase I foi estimulada por DTT, Ca+2 e Mn+2 e inibidas por Cu+2, Zn+2, SDS e Hg+2. Já a xilanase II foi inibida por Hg+2 e ativada por diversos íons: Mn+2, Co+2, Fe+2, Ba+2, Ca+2, Cu+2, Mg+2 e por NH4+ e DTT. As xilanases presentes no filtrado de cultura da linhagem IFO-4626 e as xilanases purificadas I e II favoreceram a liberação de cromóforos a 237nm e de açúcares redutores. Porém, as enzimas presentes no filtrado de cultura mostraram-se mais adequadas para liberação de cromóforos com absorção em diferentes comprimentos de onda. Ou seja, constatou-se que as enzimas presentes no filtrado de cultura possuem melhor potencial do que as xilanases purificadas I e II, para favorecer o posterior branqueamento da polpa kraft de pseudocaules de bananeiras frutíferas.
Title in English
Xylanases from Penicillium chrysogenum: production, purification, characterization, and their application to pre-bleaching cellulosic pulp of banana tree
Keywords in English
Chromophores.
Enzymatic bleaching
Enzyme purification
Penicillium chrysogenum
Xylanase
Abstract in English
The aim of this work was to study the potential of xylanases present in culture filtrate of Penicillium chrysogenum and of this xylanases purified in favor of the pre-bleaching of pseudo-stem cellulosic pulp of banana trees. Early, it was established the optimal media and time of culture to production of xylanases by IFO-4626 and M-85 Penicillium chrysogenum strains. Both strains have presented such characteristics as to contribute to their use in pulp and/or cellulosic fibers industrial process, besides the high activity of pectinases and the low activity of cellulases. To the IFO-4626 strain, the best production of xylanases using as inductor oat spelt xylan was obtained in the Ferreira medium, after 60h of culture. The performance of M-85 strain only was equal to that at the 72 hours in the Haas medium. As carbon sources, both sugar-cane straw and coconut fiber can substitute the oat spelt xylan in the production of xylanase by IFO-4626 strain. Xylose can induce the synthesis of xylanase when no xylan source was added to the culture medium. The inhibition of activity of xilanase was observed in medium with xylan plus glycose (1%) or galactose (1%). In the essays about xylanase purification, it was used the culture filtrate of IFO-4626 strain, obtained after 72 hours of culture in Ferreira medium. Two peaks with xylanase activity were obtained after the elution in an anionic-exchange column DEAE-Sephacel. The xylanase I, showed molecular mass of 12.6 kDa, Km = 12.14mg/mL and Vmax = 7.75 U/µg of protein, and it was not bind to the resin, and the xylanase II, with molecular mass of 20 kDa, Km = 39.32 mg/mL and Vmáx = 1579.62 mg/mL, was eluted with NaCl 100 mM. The xylanases in the culture filtrate, and the xylanases I and II, have presented good stability at 40°C and 50°C, and in pH values 3.5 to 9.0, despite of having no good stability at 60°C, their optimal temperatures and pH of reaction are low. The xylanases present in culture filtrate were activated by the presence of Fe+2, Mn+2, Ca+2 and dithiothreitol (DTT) and inhibited by the presence of Zn+2, sodium dodecyl sulphate (SDS), Pb+2 and Hg+2. The xylanase I activity was stimulated by DTT, Ca+2, and Mn+2, and inhibited by Cu+2, Zn+2, SDS, and Hg+2. On the other hand, the xylanase II was inibited by Hg+2, and it was activated by several ions, like as: Mn+2, Co+2, Fe+2, Ba+2, Ca+2, Cu+2, Mg+2, and by NH4+ and DTT. The xylanases from IFO-4626 strain, present in the culture filtrate, and the purified xylanases I and II favored the release of chromophores at 237 nm, and release of reducing sugars. Although, the enzymes present in the culture filtrate show better adequacy than the purified ones in order to release chromophores, they show absorption in different wave lengths. In other words, it was verified that the enzymes present in the culture filtrate have the best potential to favor the further bleaching of the banana tree pseudo-stem kraft pulp.
 
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Publishing Date
2008-02-13
 
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