Molecular and functional studies of fungal xyloglucanases and lichenases: applications in the hydrolysis of lignocellulosic biomass

Author(s)

Alex Graça Contato

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2023-06-07

Examining board members

Maria de Lourdes Teixeira de Moraes Polizeli; Rolf Alexander Prade; Wanderley Dantas dos Santos; Fernando Segato

• Advisor: Maria de Lourdes Teixeira de Moraes Polizeli

Abstract

The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Its degradation and conversion are attractive in biotechnology. Fractionation of the cell wall is not a simple process; however, microbial enzymes may be the solution. The first step was to prospect lignocellulolytic fungal enzymes with potential industrial application, produced through temporal analysis using jatoba and tamarind seeds, residues from the fruit pulp industry, as carbon sources. Enzymes were produced by eight isolated and identified filamentous fungi. The best producers regarding enzymatic activity were Thermothelomyces thermophilus and Trichoderma longibrachiatum. The optimal conditions for enzyme production were media supplemented with tamarind seeds, under agitation, for 72 h. Then, the saccharification of three sugarcane varieties (sugarcane bagasse, and SP80-3280 and Energy cane culms) pre-treated by autohydrolysis or chemically, in addition to the steam explosion sugarcane bagasse, was performed. Knowing the enzymatic production, the largest producer of xyloglucanases was chosen, since xyloglucan is the predominant hemicellulose of the primary cell wall of higher plants. In this way, the enzymes that cleave this polymer are very useful in the degradation and conversion of lignocellulosic biomass. Therefore, the aim was to optimize the production of xyloglucanase from T. longibrachiatum using a central composite rotatable design as well as immobilization on ionic supports such as MANAE, DEAE, CM-cellulose and PEI, since the immobilization process can be used to solve problems related to stability, in addition to the economic benefits brought by the possibility of its repeated use and recovery. This study described, for the first time, the immobilization of a fungal xyloglucanase using these supports. Based on these results, molecular biology experiments were performed. Therefore, a GH74 xyloglucanase with a molecular weight of 77 kDa was identified and its gene sequence was optimized for cloning and expression in A. nidulans A773. Then, the analysis of the secretome of the microorganisms secretome were performed. T. thermophilus cultivated by submerged fermentation with tamarind seeds revealed 80 distinct CAZymes, especially the GH16 family, known for producing lichenases, an enzyme of economic and industrial interest. The production of lichenase was confirmed through enzymatic assays. Meanwhile, the analysis of the T. longibrachiatum secretome cultivated by submerged fermentation with two lignocellulosic residues, sugarcane bagasse and tamarind seeds, in addition to a simulation of hemicellulose, revealed 206 distinct CAZymes, and in each condition there were particularities and differences, mainly in cultivation using the tamarind seeds that produced the highest number of hemicellulases. For this reason, this residue was chosen to perform the coculture of T. longibrachiatum with T. thermophilus, which were cultivated in a bioreactor and increased the protein production. These results suggest that the co-culture between these microorganisms has the potential to produce an enzymatic cocktail with high performance in the hydrolysis of materials in the sugar and alcohol industry. (AU)

• FAPESP's process: 17/25862-6 - Structural studies and functional properties of xyloglucanases and lichenases: applications on hydrolysis of lignocellulosic biomass
• Grantee: Alex Graça Contato
• Support Opportunities: Scholarships in Brazil - Doctorate