Molecular engineering in the development of high-performance GH1 b-glucosidases for application in cellulolytic enzyme cocktails promoting efficient saccharification of cellulose from agro-industrial waste

• Grant number: 24/14795-0
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: January 01, 2025
• End date: February 28, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Maria de Lourdes Teixeira de Moraes Polizeli
• Grantee: Aline Pereira de Almeida
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

In Nature, the enzymatic degradation of cellulose into glucose is carried out by the synergistic action of cellulases. Among these, b-glucosidases play a crucial role in the cellulose depolymerization cascade, catalyzing the hydrolysis of cellobiose into glucose monomers. This action is essential to mitigate the inhibitory effect of cellobiose on the activities of endoglucanases and cellobiohydrolases. However, inhibition by glucose is the most observed phenomenon among b- glucosidases. Thus, highlighting the importance of studying glucose-tolerant b-glucosidases for the development of an optimized cellulolytic enzyme cocktail (CEC) that meets industrial requirements. In this context, this research proposes sustainable solutions for agricultural waste management, focusing on the production of fermentable sugars. To achieve this, the aim is to develop and investigate the biotechnological potential of new b-glycosidases from the GH1 family of the fungus Trichoderma RP698, created through protein engineering. These enzymes will be evaluated in the supplementation of thermostable CECs intended for the efficient saccharification of cellulose from agricultural waste. To this end, the proposed objectives include the creation of highly efficient, thermostable, and glucose-tolerant GH1 b-glucosidases using rational andirrational methodology and molecular engineering. The sensible method adopted will involve site-directed mutagenesis based on structural information from homologous enzymes and will focus on glucose tolerance. The irrational approach will be adopted for thermostability via directed evolution "in vitro" by standard techniques of random mutagenesis by epPCR and DNA shuffling.It is expected to obtain b-glucosidases with superior performance at high temperatures and glucose concentrations, in addition to structural and functional information about the biochemical factors that influence their activity. For this, biochemical and biophysical analyses will be carried out to understand the properties of the mutants. In this way, the most promising enzymeswill be selected, and their efficiency in CECs during the hydrolysis of pre-treated agro-waste will be evaluated. In the end, after mapping the best mutations, point mutagenesis will be carried out, obtaining a new and potential glucose thermotolerant ¿glucosidase GH1 that will encompass all the desired characteristics. With the success of this project, which will be associated with the National Institute of Bioethanol Science and Technology (INCT - 2024/50884-5), a significant scientific advance isexpected in the development of enzymes for the renewable energy industry, in addition to promoting sustainability and efficiency in the ecological use of agro-waste.