Serino protease das sementes da planta Leucaena leucocephala: caracterização e aplicação biotecnológica

Abstract

The application of enzymes in biotechnological processes allows for a significant reduction in costs with a consequent reduction in energy consumption. In this context, proteases are the most produced biomolecules and represent 60 % of all enzymes sold in the world. The importance of using these molecules is due to factors such as high catalytic activity, selectivity, specificity, as well as production variability. Given the demand to produce studies aimed at obtaining enzymes with an industrial bias, we aimed to obtain and characterize a protease of biotechnological interest from the seeds of Leucaena leucocephala. Therefore, enzymatic extract from L. leucocephala seeds was prepared in Tris-HCl buffer, 50 mM at pH 8.0 and, subsequently, tests were carried out on pH, temperature, inhibition, effects of different substrates, metal ions, organic solvents and detergents on the activity enzymatic. The optimal enzyme activities were determined at 40º C and pH 9.0. In stability tests, the enzyme remained stable over a wide range of pH (5.0 to 9.0) and temperatures (40º C to 60º C). Furthermore, the enzyme's catalytic activity improved in the presence of metal ions (Na+, K+, Mn2+, Mg2+ and Ca2+). Furthermore, the enzyme has demonstrated stability and compatibility with detergents and organic solvents. This highlights the biotechnological potential of this enzyme in various industrial processes. When the enzyme extract was subjected to different chromogenic substrates conjugated with p-nitroaniline, the hydrolysis showed specificity for BApNA. Furthermore, enzyme activity was inhibited by PMSF (95.80 %) followed by Benzamidine (84.22 %). Therefore, the enzyme belongs to the class of serine proteases. In a senescence test, the enzyme demonstrated more significant enzymatic activity at the end of seed development. In relation to biotechnological application, the enzyme exhibited milk coagulating activity 13 Um/mL, in powdered milk, and this activity was more efficient in pasteurized whole milk 83 mU/mL, at a temperature of 50º C. The protein profile regarding proteolytic activity in skimmed milk powder was analyzed on a SDS-PAGE polyacrylamide gel. And according to the result, it was possible to observe a decrease in the protein profile of the milk due to the increase in incubation time. This result was also demonstrated by measuring residual proteins, using the Bradford reaction, where we observed that both experiments corroborate the assumption that the vegetable enzyme provides hydrolysis of milk proteins. The aqueous extract of L. leucocephala seeds was subjected to toxicity testing using a bioindicator. It was possible to demonstrate that no toxic environment was generated after 24 hours of exposure of the crustaceans, Artemia salina, to different concentrations of the aqueous extract. Through this research, it was possible to describe a new milk coagulant. Furthermore, the enzyme demonstrated stability in the presence of detergents and organic solvents, demonstrating the potential use of this enzyme in the industrial production of detergents and bioactive peptides, respectively.