Cloning, Expression of Aspartic Peptidase from Rhizomucor miehei in Komagataella phaffii (Pichia pastoris): Purification, Biochemical Characterization and Subsite Mapping

Abstract

The prospect of using microbial enzymes in industrial processes has increased considerably in the 21st century. This trend of enzyme application increases as new enzymes are discovered and have significant potential to meet the needs of industrial sectors. The enzyme market is expected to reach a value of US$14.7 billion by 2025, registering a compound annual growth rate of 6.7% in value terms. Microbial peptidases constitute the main group of enzymes used industrially, responsible for about 2/3 of the total marketed enzymes. The study of fungal peptidase is a relevant topic worldwide, as it involves physiological studies that help in understanding how some pathogenic fungi interact with the hosts, in the field of biotechnology, with the use of enzymes from filamentous fungi in various industrial sectors. Currently, fungal enzymes are the most used, for example, in the food, pharmaceutical, biofuel, and other industries. The importance in determining the specificity of peptidases, that is, the mapping of subsites helps in the rational design of inhibitors, makes it possible to determine the cleavage in proteins and to obtain peptides with biological activity. This project aims to clone the gene that encodes the aspartic peptidase of the filamentous fungus Rhizomucor miehei in the expression system in Komagataella phaffii, carry out the biochemical characterization and mapping of the subsites. In this way, it will be possible to verify a possible application for aspartic peptidase, after evaluating the results obtained.