Evaluation of the activity of cell envelope proteinases and intracellular peptidases of strains of Lactiplantibacillus plantarum and Lentilactobacillus parabuchneri by in vitro and computer assays

CAROL, JUAN J.; PEREZ, HUGO; BUSTOS, ANA Y.; LEDESMA A E

Congreso; L Reunión Anual SAB; 2022

The proteolytic system of lactic acid bacteria (LAB) comprises cell envelope proteinases (CEPs) taht cleave casein into peptides, intracellular peptidases degrade peptides to amino acids, and specific transport proteins. Recently, CEPs have been widely used in different biotechnological applications, including the development of new nutraceutical compounds with important biopharmaceutical potential. Thus, the objective of this work was to evaluate the CEPs and peptidase activity of four strains of Lactiplantibacillus plantarum and one strain of Lentilactobacillus parabuchneri, autochthonous of Santiago del Estero, through spectrophotometric, and spectrofluorometric assays. Besides, molecular docking analysis was employed to predict the affinity and types of interactions between the CEPs from the selected LAB with αS1-, β- and 01D 705-casein. Proteolytic activity against bovine milk casein was evaluated both in cell-free and crude proteinase extracts.Free amino acids were quantified by o-ftalaldehyde (OPA) assay recording the absorbance at 340 nm, while released tyrosine was measured as emission value in spectrofluorometer using excitation wavelength at 280 nm. The three-dimensional models were obtained by modeling through the I-TASSER server and the docking analysis was performed using the HADDOCK 2.4 server.Our results showed that all strains have proteolytic activity against casein, both in the cell free extract as well as in the crude proteinase extracts, showed an increase in amino acids concentration in a proportion of a 2 and 17 times after incubation, respectively. Fluorescence studies reveal an increase in fluorescence emission at 340 nm, corresponding to the exposure of amino acids tyrosine due to proteolysis of casein. These findings suggest that the lactic strains present activity of both CEPs and intracelular peptidases. Regarding the computational studies, the predictions indicated that the proteases evaluated showed a higher affinity to β-casein compared with αS1 and 01D 705 ones.β-casein attachment site is located near the catalytic site, with Van der Waals and hydrophobic interactions enhancing the binding of the ligand to the receptor. Our results allow us to increase our knowledge about the proteolytic system of BAL strains that have not been explored so far and also demonstrate that these enzymes could have an important biotechnological potential.