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 PAZ JUAN; HUGO A. PÉREZ; BUSTOS, ANA YANINA; LEDESMA, ANA ESTELA

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica.; 2022

Sociedad Argentina de Biofísica

The proteolytic system of lactic acid bacteria (LAB)comprises cell envelope proteinases (CEPs)that cleave casein into peptides;intracellular peptidases degrade peptides toamino acids, and specific transport proteins. Recently, CEPs have beenwidely used in differentbiotechnological applications, including the development of new nutraceutical compounds with important biopharmaceuticalpotential. Thus, the objective of this work wasto evaluate the CEPs and peptidase activity of four strains of Lactiplantibacillus plantarum andone strain of Lentilactobacillus parabuchneri, autochthonousof Santiago del Estero,through spectrophotometric, and spectrofluorometric assays.Besides, molecular dockinganalysis was employed to predict the affinity and types of interactions betweenthe CEPs from the selected LAB with αS1-, β- and k-casein. Proteolytic activity against bovinemilk 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 wasmeasured as emission value in spectrofluorometer using excitation wavelength at 280 nm. The three-dimensionalmodels were obtained by modelingthrough the I-TASSER server and the docking analysis was performed using the HADDOCK2.4 server. Our resultsshowed that all strains have proteolytic activityagainst casein, both in the cell free extract as well as in the crude proteinase extracts, showed an increase in amino acidsconcentration in a proportion of a  2 and 17 times after incubation, respectively. Fluorescence studiesreveal an increasein fluorescence emissionat 340 nm, corresponding to the exposureof amino acids tyrosinedue to proteolysis of casein. Thesefindings suggest that the lacticstrains present activityof both CEPs and intracelular peptidases. Regarding the computational studies,the predictions indicatedthat the proteases evaluated showed a higher affinity to β-casein compared with αS1 and k ones. β-casein attachment site is located near thecatalytic site, with Van der Waals and hydrophobic interactions enhancingthe binding of the ligand tothe receptor. Our results allowus to increase our knowledge about the proteolytic system of BAL strains thathave not been explored so far and also demonstrate that these enzymes couldhave an important biotechnological potential