Serino protease e inibidor de tripsina: purificação, caracterização e aplicação biotecnológica

Abstract

It is known that most commercialization involving the production of enzymes is centered on the sale of proteases and the success in the use of these molecules is due to factors such as high catalytic activity, selectivity to specific substrates, as well as the diversity of Production. In the same way that enzymatic reactions are useful in biotechnological applications, their inhibition; on the other hand, it has also been the subject of much scientific research and development. Protease inhibitors (PIs) are seen as molecules present in virtually all living organisms and play a significant role in many biological processes, through the regulation of the proteolytic functions of target enzymes. In view of the need to disseminate studies aimed at obtaining proteins with a commercial bias, with this work we seek to verify serine proteases and trypsin inhibitors of biotechnological interest, followed by their purification, characterization and application. The work was carried out following comparative methodologies with different precipitation strategies (saline solution, organic solvents and three-phase system), assisted by liquid chromatography (S-100 Sephacryl and DEAE-Sepharose). All experimental development was carried out at 4oC, in order to minimize structural protein disorders. As for the biotechnological application of the crude extract of the species C. stipularia and the isolated inhibitor of the species C. plumieri, the coagulant activity (with skimmed and whole milk) and the inhibitory test (in vitro) against the serine protease isolated from the intestine of the pest of insects T. castaneum. Through this work, it was possible to describe the obtaining of a new milk coagulant and the purification of the trypsin type from seeds of the species C. stipularia; with 45 kDa and optimal activities determined at 50°C and pH 8.0. When subjected to reaction with different substrates conjugated to p- nitroaniline, the hydrolase showed specificity only for BApNA. In the case of studies carried out with the species C. plumieri, it was possible to isolate the trypsin inhibitor with an estimated mass of 50kDa; which showed high inhibitory specificity for the isolated protease of T. castaneum, which shows a probable usefulness of this molecule in combating the insect pest used in the research.