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Doctoral Thesis
DOI
10.11606/T.87.2014.tde-16052014-124535
Document
Author
Full name
Aline Ramos da Silva
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Rodrigues, Maria Filomena de Andrade (President)
Palma, Mauri Sergio Alves
Perpetuo, Elen Aquino
Vasconcellos, Marina Beatriz Agostini
Vasconcellos, Suzan Pantaroto de
Title in Portuguese
Biodegradação de hexaclorociclohexano utilizando microrganismos e enzimas desenhadas computacionalmente.
Keywords in Portuguese
Biodegradação ambiental
Enzimas
Pesticidas
Poluição do solo
Reatores bioquímicos
Abstract in Portuguese
Hexaclorociclohexano (HCH), pesticida organoclorado mundialmente utilizado, apresenta efeitos tóxicos à saúde humana e ao meio ambiente. Os microrganismos degradadores mais conhecidos são as Sphingomonas sp. Técnicas de biodegradação foram aplicadas em duas etapas. A primeira focou na biorremediação de solo contaminado, de Santo André SP, e foi realizada em biorreatores no Instituto de Pesquisas Tecnológicas (IPT). Experimentos nas fases sólida e semi-sólida apresentaram até 90% de degradação de HCH no solo. A segunda parte, na Universidade de Groningen (RuG), Países Baixos, focou no tratamento de soluções contaminadas usando enzimas selvagens e variantes desenhadas computacionalmente. Mutantes foram construídas, expressadas e purificadas. Ensaios de Thermofluor® mostraram que as variantes estavam enoveladas. Ensaios enzimáticos foram realizados em solução aquosa com b-HCH e dimetil sulfóxido (5%), sendo as amostras extraídas com acetato de etila e analisadas por cromatografia gasosa com detector de captura de elétrons. As variantes apresentaram atividade.
Title in English
Biodegradation of hexachlorocyclohexane using microorganisms and computationally designed enzymes.
Keywords in English
Biochemical reactors
Environmental degradation
Enzymes
Pesticides
Soil pollution
Abstract in English
Hexachlorocyclohexane (HCH) is an organochlorine pesticide used world-wide which shows toxic effects in human health and causes environmental problems. The most known HCH-degrading microorganisms are Sphingomonas sp. Biodegradation techniques were applied in this work, divided in two parts. The first one focused on the bioremediation of a contaminated soil, from Santo Andre - SP, in bioreactors at the Institute for Technological Research (IPT). Experiments were carried in solid and slurry phases, which could achieve around 90% of HCH degradation. The second part was developed at the University of Groningen (Rug), The Netherlands. Contaminated solutions were treated with wild-type enzymes and computationally designed variants. Mutants were constructed, expressed and purified. Thermofluor® assay showed that all variants were well folded. Enzymatic assays were carried in aqueous solution with b-HCH and dimethyl sulfoxide (5%). The samples were extracted with ethyl acetate and analysed by gas chromatography using an electron capture detector. The variants were actives.
 
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Publishing Date
2014-06-11
 
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