Prospecção e identificação de proteínas do extrato de Phaseolus vulgaris L.

Abstract

Proteins are extremely important biomolecules for biological systems. Studying them leads to a better understanding of how life and its various forms work in such an elaborate way. These researches also open space for the application of these proteins in activities carried out by humanity. Science has already had great success in applying proteins biotechnologically, such as insulin and DNA polymerase. Several sources for these proteins can be cited, and plants are among them. Plant proteins have been and still are studied extensively and have unexplored biotechnological potential. In this context, legumes are a plant family of extensive distribution, abundance and diversity that has the characteristic of storing high protein content in its seeds. With that in mind, the aim of this study was to extract and identify proteins/enzymes from mature seeds of Carioca Beans, Phaseolus vulgaris, with biotechnological potential. For this purpose, seeds obtained commercially were crushed and subjected to solubilization to extract proteins from the testa and cotyledons. The crude extract was dialyzed and applied to a DEAE-Sephacel anion exchange column for chromatography. Three peaks were eluted with increasing KCl concentration. 12% SDS-PAGE was performed with the fractions and extract to analyze the protein profile and excise the bands present in the gel. The excised bands were reduced and alkylated so that their peptides could be extracted. Mass spectrometry was the method used to sequence the extracted peptides and the proteins were identified using the MASCOT program, using the Swissprot database. In addition, the extracts from 7 brands of Carioca beans were tested for L-asparaginase activity. 8 bands could be excised for identification, resulting in 4 identified proteins and their subunits: Lipoxygenase, Phaseolin, Phytoagglutinin and α-Amylase Inhibitor. Among the tested beans, only one brand showed minimal L-asparaginase activity, even when the test conditions were maximized. It is concluded that it was possible to identify 4 proteins from Carioca bean seeds, each one with its biotechnological potential that can still be explored. In addition, the null asparaginase activity still gives us learning and ideas for possible strategies to purify it and better characterize it.