Novel Aspergillus fumigatus recombinant LPMOs: biochemical characteristics, and their participation in saccharification and photobiocatalysis processes

• Main Author: Mendoza, Josman Andrey Velasco
• Publication Date: 2022
• Format: Doctoral thesis
• Language: eng
• Source: Biblioteca Digital de Teses e Dissertações da USP
• Download full: https://www.teses.usp.br/teses/disponiveis/97/97140/tde-05102022-152812/
• topic: Aspergillus fumigatus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus nidulans. Photobiocatalysis; Enzymatic saccharification; Fotobiocatalise; LPMO; LPMO; Sacarificação enzimática

From the discovery of the oxidative mechanism that LPMOs use to promote the degradation of several polymers in nature, the interest of both academic and industrial sectors in these enzymes has drastically increased, allowing the technological developments of enzymatic cocktails that are already used in industrial processes to plant biomass conversion. The present work describes the AA9 family LPMOs from Aspergillus fumigatus var. niveus, enzymes that until the beginning of this thesis had not been characterized. After an accurate bioinformatic study, the enzymes were heterologous expressed in the fungus Aspergillus nidulans using recombinant DNA technology. Physical and biochemical characteristics such as 3D structure, optimal temperature, pH effect on enzyme activity, melting temperature and regioselectivity are consistently described for the three LPMOs (AfAA9A, AfAA9B and AfAA9C) allowing to know the enzymes to be applied in plant biomass bioconversion processes. The application of AfAA9A and AfAA9B were added to the Celluclast 1.5L commercial cocktail beind applied in the process of sugarcane bagasse saccharification and allowed an increase in sugar release of 20% when AfAA9B was present in the reaction and even more interesting, the lignin present in biomass was modified by the enzymatic action as observed by confocal scanning microscopy. On the other hand, photobiocatalysis experiments were carried out using AfAA9C using chlorophyllin/light systems as electron donor coupled or not with chemical reductants. AfAA9C showed the ability to oxidize polysaccharides being activated by the photosystem and a synergistic effect between the light-induced electron donor system and chemical reductants to increase enzyme activity was detected. The literature describes LPMOs as enzymes that have a type of selectivity for different chemical donors, the experiments performed in this work allowed to describe for the first time an apparent change in enzyme selectivity by chemical donor when coupled to the photosystem. The results obtained here show the potential of A. fumigatus LPMOs to boost the saccharification processes and participate in photobiocatalysis, but also open new perspectives for studying the structural characteristics of these proteins, the electron delivery process and the type of changes that are caused in lignin product by the enzyme action.