Exploring the effect of recombinant LPMOs from Thermothielavioides terrestris on Lignin modification

• Ano de defesa: 2024
• Autor(a) principal: Gutierrez, Solange Grace Barrios
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: Indulin modification; Modificação de Indulin; Monolignóis; Monolignols; Oxidase; Oxidase. Peroxidase; Peroxidase; TtLPMOs
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/97/97140/tde-12122024-111715/

The potential of LPMOs in nature has been demonstrated through the cleavage of various polysaccharides such as chitin, cellulose, and starch. However, their oxidative potential can also interact with the lignin structure, not only through electron transfer but also through the cleavage of its monomeric or dimeric fractions of low molecular weight. Therefore, this study focuses on comprehending the enzymatic and non-enzymatic mechanisms of fungal LPMOs on lignocellulosic biomass, as well as the expression and characterization of two recombinant LPMOs from Thermothielaviodes terrestris, to analysis of their oxidative potential on Indulin modification. The understanding of the role of LPMOs from fungi that degrade lignocellulosic biomass in nature and the recent discovery of their involvement in lignin degradation, through enzymatic and non-enzymatic mechanisms was described. The experimental phase demonstrated the successful cloning, expression, and purification yielded protein quantities of 5.6 mg/mL for TtLPMO9C and 2.3 mg/mL for TtLPMO9F. Characterization tests, including pH, optimum temperature, specific activity, and regioselectivity, were conducted to understand the working conditions. The best pH was found to be 7 and 8 at 50 °C and the enzymes were selective for C1 (TtLPMO9C), and C1/C4 (TtLPMO9F) in Avicel and PASC as substrates. The evaluation of the effect of the two LPMOs on lignin monomeric compounds and insoluble conifer lignin (Indulin) showed their ability to oxidize vanillyl alcohol and modify Indulin by decreasing the low molecular weight fraction, as analyzed by HPLC and SEC. This pioneering investigation underscores the potential of TtLPMO9C and TtLPMO9F in modifying Indulin, opening new ways to explore these enzymes in lignin valorization processes.