Heterologous expression and characterization of carbohydrate active enzymes identified in the saliva of the insect Bradysia hygida

• Grant number: 22/07358-7
• Support Opportunities: Scholarships in Brazil - Master
• Start date: November 01, 2022
• End date: June 30, 2024
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Richard John Ward
• Grantee: Vitor Kunes Lopes
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 14/50884-5 - INCT 2014: National Institute of Science and Technology of Bioethanol, AP.TEM

Abstract

Biorefinery a key aspect of the bio-based circular economy, which aims to transform biomass into chemical products of industrial interest and to obtain clean energy. The use of carbohydrate active enzymes (CAZymes) in biomass degradation for this purpose is gaining prominence due to its many benefits and advantages, and the search for enzymes from various sources is a dynamic and expanding área of research. Despite their wide distribution, a large number of species distributed over 30 orders and diverse feeding habits, insect CAZymes are poorly explored. A recent -omics study of the insect Bradysia hygida identified a group of ²-glucosidases in the genome and transcriptome that are also present in the salivary proteome. Subsequently, enzymatic activity assays confirmed the presence of this set of enzymes in the insect's saliva. As part of a Scientific Internship project, the group of ²-glucosidase and b-glucansase sequences were analyzed and classified and compared with the sequences present in the NCBI and the CAZy databases, resulting the confirmation of 9 target enzymes active against the ²-glucan substrate in the proteome. In order to evaluate the biotechnological potential of these ²-glucanases, the current project proposes the cloning, heterologous expression and purification of five of these enzymes, followed by the characterization of their biophysical and biochemical properties. With this, we aim to obtain innovative results relating to the discovery and characterization of enzymes of biotechnological interest.