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Doctoral Thesis
DOI
https://doi.org/10.11606/T.76.2022.tde-13102022-082756
Document
Author
Full name
Paula Miwa Rabêlo Higasi
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2022
Supervisor
Committee
Polikarpov, Igor (President)
Buckeridge, Marcos Silveira
Murakami, Mário Tyago
Nagem, Ronaldo Alves Pinto
Segato, Fernando
Title in Portuguese
Mono-oxigenase lítica de polissacarídeos TtLPMO9H: caracterização e mecanismos moleculares de ativação catalítica
Keywords in Portuguese
AA9
LPMO
Mono-oxigenase lítica de polissacarídeos
Abstract in Portuguese
A degradação da biomassa lignocelulósica é essencial para o aproveitamento dessa matéria-prima para a produção de biocombustíveis e compostos químicos. Apesar de aplicada há anos, a degradação enzimática dessa biomassa ainda envolve novidades, incluindo um tipo completamente distinto de enzimas, as mono-oxigenases líticas de polissacarídeos (LPMOs), cuja ação potencializa a atividade das hidrolases de glicosídeos que já são utilizadas. Descoberta há pouco mais de uma década, muitos aspectos básicos das LPMOs ainda são desconhecidos. No trabalho desenvolvido, uma LPMO recombinante do ascomiceto Thermothelomyces thermophilus, TtLPMO9H, foi estudada quanto a sua especificidade de substrato, régio-seletividade, estabilidade térmica e ativação. TtLPMO9H, pertencente à família AA9, teve atividade em celulose e hemicelulose, com ação nos carbonos C1 e C4 da cadeia polissacarídica e gerou celo-oligossacarídeos oxidados e não oxidados. Apresentou estabilidade térmica, e esta propriedade foi usada para sua purificação. A enzima teve atividade em ampla extensão de pHs, e a variação deste influenciou o perfil de produtos liberados por TtLPMO9H. Esta LPMO foi ainda ativada por diferentes sistemas, incluindo pequenas moléculas com e sem adição de H2O2, clorofilina e luz, múltiplas oxidoredutases, incluindo duas oxidases que nunca foram reportadas para a ativação de LPMO, e por H2O2.
Title in English
Lytic polysaccharide monooxygenase TtLPMO9H: features and molecular mechanisms of catalytic activation.
Keywords in English
AA9
LPMO
Lytic polysaccharide monooxygenase
Abstract in English
The degradation of lignocellulosic biomass is essential for the use of this raw material to produce biofuels and biochemical compounds. Although it has been applied for years, the enzymatic degradation of this biomass still offers novelties, including a completely different type of enzyme, the lytic polysaccharide monooxygenases (LPMOs), whose action potentiates the activity of the glycoside hydrolases that are already used. Discovered just over a decade ago, many basic aspects of LPMOs are still unknown. In the work developed, a recombinant LPMO of the ascomycete Thermothelomyces thermophilus, TtLPMO9H, was studied for its substrate specificity, regioselectivity, thermal stability and activation. TtLPMO9H, which belongs to the AA9 family, had activity on cellulose and hemicellulose, acted on the C1 and C4 carbons of the polysaccharide chain and generated oxidized and non-oxidized cello-oligosaccharides. It showed thermal stability, and this property was used for its purification. The enzyme had activity over a wide range of pHs, and its variation influenced the profile of products released by TtLPMO9H. This LPMO was further activated by different systems, including small molecules with and without addition of H2O2, chlorophyllin and light, multiple oxidoreductases, including two oxidases that have never been reported to activate LPMO, as well as H2O2.
 
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Publishing Date
2022-10-13
 
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