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| Author(s): |
Bianca Oliva
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Lorena. |
| Institution: | Universidade de São Paulo (USP). Ciência e Engenharia dos Materiais (IFS/EES) |
| Defense date: | 2024-03-14 |
| Examining board members: |
Fernando Segato;
Marco Antonio Seiki Kadowaki;
Adriane Maria Ferreira Milagres;
Viviane Isabel Serpa Muller
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| Advisor: | Fernando Segato; André Luis Ferraz |
| Abstract | |
The establishment of alternative and sustainable sources of energy is one of the major global demands. Lignocellulose, an industrial waste, still retains a significant carbon content that can be converted into ethanol and other products. However, the enzymatic cocktails necessary for the saccharification of this material still face challenges related to harsh industrial conditions and impurities in the reaction medium. Exploring enzymes from different sources may yield advantages that have yet to be observed. Basidiomycete fungi naturally degrade lignocellulosic materials with a high lignin content, a macromolecule that has adverse effects on enzymes in industrial processes. This work describes the cloning and expression of two cellobiohydrolases (PcCel7C and PcCel7D) from the basidiomycete Phanerochaete chrysosporium in Aspergillus nidulans. Biochemical characterization demonstrated that heterologous expression maintained the intrinsic properties of the recombinant enzymes, with higher endo-initiation activity and catalytic velocity on cellulose compared to the cellobiohydrolase (CBH) from the ascomycete Trichoderma reesei. In addition to deletions in the loops forming the catalytic tunnel of CBHs, PcCel7D also exhibit negatively charged residues exposed on the surface during the in silico analysis, resulting in a higher negative electrostatic charge compared to PcCel7C. In the presence of amorphous cellulose and soluble lignin-related compounds (LRCs), PcCel7D demonstrated greater tolerance to these deactivators. However, the study with other enzyme sources showed that thermostable enzymes displayed higher tolerance to LRCs, and the determination of the unfolding temperature revealed that the interaction mechanism between LRCs and cellulases involves structural destabilization and deactivation, contrary to what the literature suggests (inhibition). Intrinsic tryptophan fluorescence assays unveiled that residues near the active site are favored for interaction with LRCs. This study provides a biochemical analysis of basidiomycete CBHs in comparison to ascomycete CBHs and elucidate the type of interaction that occurs between LRCs and cellulases. This contributes to the rational development of more robust enzymes for industrial processes. (AU) | |
| FAPESP's process: | 19/06663-8 - Biochemical and functional studies of cellobiohydrolases from basidiomycetes of white-rot fungi with biotechnological potential |
| Grantee: | Bianca Oliva |
| Support Opportunities: | Scholarships in Brazil - Doctorate |