Evaluation of interaction sites in the recombinant cellobiohydrolase Cel7D from Phanerochaete chrysosporium with low molecular weight phenolic compounds

• Grant number: 22/09945-7
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2022
• End date: September 30, 2023
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Fernando Segato
• Grantee: João Vicente Zanotto
• Host Institution: Escola de Engenharia de Lorena (EEL). Universidade de São Paulo (USP). Lorena , SP, Brazil
• Associated research grant: 21/06679-1 - Enzymatic oxidation of sugarcane bagasse 2: exploring the interaction between LPMOs and its redox partner CDHs aiming for the development of more efficient enzymes for insertion into an engineered cell factory, AP.BIOEN.JP2

Abstract

The use of biomass from the sugar and alcohol sector, such as sugarcane bagasse and straw for the production of biofuels and chemical compounds still represents a challenge for the development of biorefinery in Brazil. In view of its large-scale production sugarcane residues have a great energy potential and still are inefficiently exploited. The enzymatic hydrolysis step has been used to transform the polymeric sugars available in the cell wall of this by-product into its fermentable monomers. In this step, the degradation of cellulose into glucose has been elucidated with the participation of several classes of enzymes which Exo-1,4 ²glucanases(cellobiohydrolases or CBHs) are the protagonists since it constitutes about 70 to 80% of protein mass in the commercial cocktails. These CBHs show processive activity and cleave the cellulose chain from available reducing and non-reducing regions producing cellobiose as a product. However, the enzymes present in commercial cocktails are inhibited by the interaction with the phenolic compounds generated in the pre-treatment process of lignocellulosic, which remain present in the reaction medium during enzymatic hydrolysis. Cocktails used industrially are composed of enzymes from ascomycetes of the genus Trichoderma, which are inhibited by the presence of phenolics. In this way, it was found that basidiomycete enzymes, such as Phanerochaete chrysosporium, may show adaptations that allow being less inhibited by lignin phenolic compounds since these organisms naturally colonize highly lignified substrates being able to degrade lignin and cellulose simultaneously. Using heterologous expression systems, it becomes possible to produce enzymes from different sources in a large scale, therefore, they make possible the studies of CBHs which still are little explored. The interaction sites of CBHs with phenolic compounds are not yet elucidated, so the crystallization of these enzymes in the presence of these inhibitors becomes a strategy to identify important amino acids during inhibition and collaborate in the engineering of these proteins to soften this inhibition.(AU)