Biochemical and biophysical analysis of lignin-related compounds effect on the activity of cellobiohydrolases

• Grant number: 22/01756-0
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: September 05, 2022
• End date: May 14, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Fernando Segato
• Grantee: Bianca Oliva
• Supervisor: Peter Westh
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil
• Institution abroad: Technical University of Denmark (DTU), Denmark
• Associated to the scholarship: 19/06663-8 - Biochemical and functional studies of cellobiohydrolases from basidiomycetes of white-rot fungi with biotechnological potential, BP.DR

Abstract

The use of high-solid loadings in biorefineries is a requirement to consolidate and improve the production of 2G bioethanol. In this process an increase in residual lignin and lignin-related compounds (LRC) contents have a negative effect on the enzymatic saccharification of plant biomass. In preliminary analyses from the current PhD project, the negative effect of LRC on the activity of several cellobiohydrolases (CBHs) was confirmed, however, further investigation regarding the inhibition mechanisms and the interaction of these compounds in CBHs are needed. In this research proposal (BEPE), the inhibitory effect of LCR on CBHs will be assessed using kinetic assays and biophysical methods. We will use recombinant CBHs from Phanerochaete chrysosporium (PcCel7C and PcCel7D) and CBHs containing different substrate affinities and structural mutations that may effect the inhibition mechanisms. The enzymes will be provided by Prof. Peter Westh from DTU, which has a close collaboration with the Danish company Novozymes (Novozymes A/S, Bagsværd, Denmark). The purified enzymes will be kinetically characterized in presence of LRCs to elucidate the inhibition mechanisms and explore if it is influenced by the properties of different CBHs. The CBH from the cellulolytic model fungus Trichoderma reesei (TrCel7A) will be used in the experiments as a control. Furthermore, the protein stability and complexation kinetics will be evaluated by, respectively, differential scanning calorimetry and tryptophan fluorescence quenching during the addition of LRC. These biophysical assays will provide information about the nature of the interaction between the enzyme and the LRCs, which, along with the three dimensional (3D) structures of CBHs, the catalytic domain or carbohydrate binding module, and its kinetic data, will help to highlight patterns of LRC-tolerant enzymes and outline strategies for rational engineering of enzymes for improved tolerance to lignin in biomass. (AU)