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Master's Dissertation
DOI
10.11606/D.91.2005.tde-21062005-134055
Document
Author
Full name
Luciana Maria Fedatto
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Piracicaba, 2004
Supervisor
Committee
Mui, Tsai Siu (President)
Carmona, Eleonora Cano
Gallo, Luiz Antonio
Title in Portuguese
Caracterização de proteases extracelulares produzidas por Xylella fastidiosa de citros e videira.
Keywords in Portuguese
bactéria patogênica
eletroforese
enzima extracelular
enzima proteolítica
fruta cítrica
inibidor de enzima
proteína
uva
Abstract in Portuguese
Xylella fastidiosa é uma bactéria patogênica encontrada em várias plantas. Esta bactéria secreta proteases extracelulares detectadas em gel de eletroforese, sendo a gelatina usada como substrato co-polimerizado. Três principais bandas protéicas foram detectadas com massa molar (MM) de 122, 84 e 65 kDa produzidas pelo isolado de citros (X0) e duas bandas de aproximadamente 84 e 65 kDa de isolado de videira (9713). Estas bactérias produziram zonas de hidrólise em meio sólido contendo gelatina, caseína e hemoglobina. Os resultados usando a gelatina como substrato foram os melhores para a atividade das proteases. A atividade enzimática das proteases de X. fastidiosa de citros e videira foi completamente inibida por PMSF e parcialmente inibida por EDTA, podendo ser visualizado em gel de eletroforese nativo. A temperatura ótima de atividade protéica foi de 30oC e o pH ótimo de 7,0. Além das proteases secretadas por este fitopatógeno, quitinase e β-1,3-glucanase foram também detectadas no sobrenadante das culturas. Os resultados sugeriram que estas proteases produzidas pela X. fastidiosa de citros e videira pertencem ao grupo das serina e metalo proteases.
Title in English
Characterization of extracellular proteases produced by Xylella fastidiosa from citrus and grapevines.
Keywords in English
citric fruit
eletrophoresis
enzyme extracellular
enzyme inhibitor
grape
phytopathogenic bacteria
protein
proteolytic enzymes
Abstract in English
Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a co-polymerized substrate. Three major protein bands were produced by strain X0 (citrus) with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. SDS-PAGE activity gel indicated that the protease activities of X. fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30oC with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and β-1,3-glucanase activities were also detected in cultures of X. fastidiosa (citrus). From these results, it is suggested that these proteases produced by strains of X. fastidiosa from citrus and grape, belong to the serine- and metallo-protease group.
 
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Publishing Date
2005-06-28
 
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