Caracterização bioquímica de um inibidor de protease da larva do inseto Tribolium castaneum

Abstract

Protease inhibitors are substances that have the ability to regulate the action of proteases, blocking their catalytic activity. Many of these inhibitors are produced naturally by the body with the aim of maintaining homeostasis. Protease inhibitors have been the subject of study due to their biotechnological potential in pest control, the food industry, research into new medicines and also to increase understanding of inhibition mechanisms. The present work aimed to investigate the inhibitory activity and biochemical characteristics of a protease inhibitor present in the insect Tribolium castaneum and test its antifungal activity against a phytopathogenic fungus. The first step was to identify in which phase of the insect's life cycle there is a greater production of the inhibitor, then to improve the purity of the sample, the extract was subjected to purification techniques, such as saline precipitation and two-phase and three-phase aqueous systems. After choosing the best technique, the sample was subjected to resistance tests to pH and temperature, the influence of metallic salts and stabilizers, and the action against the phytopathogenic fungus Amphobotrys ricini was also tested. The study revealed the presence of the inhibitor in the larval, pupa and adult stages, with emphasis on the larval stage, which demonstrated more than 40% inhibitory activity. The most effective method to reduce contaminants in the sample was saline fractionation, followed by a three-phase aqueous system with isopropanol, resulting in an increase in inhibitory activity to 84.20%. The inhibitor activity remained stable at temperatures from 40 to 70ºC, but maintained its activity even at high temperatures, and also remained stable over a wide range of pHs (5-10). Furthermore, the inhibitor activity was influenced by different metal salts and different stabilizers, with BSA and Gelatin which increased inhibitory activity by around 20%. Finally, a decrease in the growth rate of the phytopathogenic fungus Amphobotrys ricini was observed. These results highlight the potential use of the endogenous protease inhibitor from the insect T. castaneum, and may also contribute to the understanding of the properties of insect inhibitors and mechanisms of enzyme regulation.