Cloning and Expression of Serine Peptidase from Fusarium oxisporum in Komagataella phaffii (Pichia pastoris): Purification, Biochemical Characterization and Inhibition Assays

• Grant number: 22/16549-0
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2023
• End date: April 30, 2024
• Field of knowledge: Biological Sciences - Microbiology - Applied Microbiology
• Principal Investigator: Hamilton Cabral
• Grantee: Murilo Sousa do Couto Rosa
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The study on fungal peptidase is considered a relevant topic worldwide, as these enzymes are involved in several physiological processes in different organisms, so the understanding of how some pathogenic fungi interact with the hosts, using these enzymes can open new opportunities for therapies, in the design of inhibitors for peptidases involved in pathogenicity associated with filamentous fungi. Another field in which peptidases are important is in the field of biotechnology, with the use of enzymes from filamentous fungi in various industrial sectors. Microbial peptidases constitute the main group of enzymes used industrially, responsible for about 2/3 of the total marketed enzymes. Enzymes of microbial origin, in addition to being robust, have significant potential in waste management and, consequently, in the development of a safer environment. The importance in determining the specificity of peptidases, that is, the mapping of subsites helps in the rational design of inhibitors and makes it possible to understand the mode of action of peptidases on target proteins, for example. It also helps to determine possible commercial application, as it predicts the cleavage point of the peptide bond between the amino acids present in peptides and proteins. This project aims to clone the synthetic gene that encodes the serine peptidase of the filamentous fungus Fusarium oxysporum in the expression system in Komagataella phaffi, in addition to the expression, purification and performance of the biochemical characterization of the peptidase. Due to the need for greater knowledge about the regulation of the activity of this peptidase, inhibition assays will be performed on a library of synthetic compounds. In this way, at the end of this project we hope to have an overview of the biochemical characteristics of this peptidase, in addition to possible compounds that regulate the activity of this enzyme, so that further studies can be carried out.