Identificação de um inibidor de tripsina e purificação de uma tripsina símile presente nas sementes da espécie vegetal Chamaecrista nictitans (L.) moench

Abstract

The species Chamaecrista nictitans belongs to the family Fabaceae, is classified as an invasive plant, and can attack several types of plantations, however, studies report that its extract has antiviral properties, and act in the treatment of kidney stones. There is no study regarding the proteolytic activities present in this plant. Some molecules found in plants are the targets of several researches in the field of bioteconology. Protease inhibitors are compounds that inhibit enzymatic activity, among which the serinoprotease inhibitors form the class with the highest percentage of catolographic species, the Kunitz and Bowman-Birk families being the most studied. Some secondary metabolites may inhibit the action of proteases. Trypsins, synthesized by the pancreas, are in the class of hydrolases and are responsible for cleaving peptide bonds of Arg and Lys residues in the C-terminal regions. They are present in eukaryotes, prokaryotes and viruses, but their presence in plants is poorly described in the literature and has many biological and industrial applications. The main objective of the present work is to identify protease inhibitors and to carry out the purification of a trypsin similar to C. nitilans seeds, observing the importance of protease and trypsin inhibitors in plants and the biotechnological importance of this enzyme. For both, the identification of protein inhibitors and the partial purification of trypsin, present in the seeds of this species, through the use of ammonium sulfate precipitation and molecular exclusion chromatography were performed. A complex extract, characterized by numerous protein bands and the presence of a trypsin inhibitory activity, was observed through the polyacrylamide gels. From the fractionation, it was possible to solve a fraction with trypsin inhibitory activity and another with trypsin activity, in the fractions 20-40% and 40-60%, respectively, of the salt precipitation. The methods used to purify the enzyme obtained high recovery and considerable resolution. By inhibition zimography it was possible to notice that the inhibitor contained in the extract does not present protein characteristics, since it did not inhibit the hydrolysis of the casein present in the gel, suggesting to be a secondary nonprotein metabolite. A description of a trypsin inhibitory activity and partial purification of simian trpsin present in C. nictitans seeds using saline precipitation and molecular exclusion chromatography was obtained. This work presents a relevant scientific discovery and tends to contribute to future research on enzymes that present biotechnological applications from plants.