Changes in ionic strength conditions release proteinase prtl from the cell envelope of Lactobacillus delbrueckii ssp. lactis CRL 581

BROWN, L.; VILLEGAS, J.M.; SAVOY DE GIORI, G.; HEBERT, E.M.

Congreso; IX Congreso de Microbiología General SAMIGE; 2013

Lactobacillus delbrueckii ssp. lactis CRL 581 is a thermophilic lactic acid bacterium (LAB), used as a starter culture for the manufacture of several fermented dairy products. This strain possess a specialized proteolytic system that consists of a cell envelope-associated proteinase, named PrtL, transport systems to allow uptake of the resulting peptides, and several intracellular peptidases, which degrade peptides to amino acids. PrtL has an essential role in bacterial growth and contributes to the development of the organoleptic properties of hard cheeses and the release of bioactive health-beneficial peptides from milk proteins.The method generally used to purify proteinases from LAB consists in release them from the cell surface by washes with calcium-free buffer. However, alternative methods, including treatment with lysozyme and cleavage of the C-terminal membrane anchor region, have been used. PrtL is resistant to most classical methods of extraction from the cell envelope. Since PrtL appears to be attached to the cell through ionic interactions (mainly due to an abundance of Lys residues in the C-terminal region), we developed a procedure using high ionic strength conditions to release PrtL from the cell. Cells grown in a chemical defined medium were resuspended in Tris?HCl (pH 7.5) or in the same buffer containing 0.5 M, 1 M and 3 M NaCl and incubated at room temperature for 30 min Then, samples were centrifuged and cell and supernatant fractions were subjected to enzymatic and SDS?PAGE analyses. At 0.5 M NaCl, PrtL activity remained mainly present in the cell fraction. However, a nearly complete loss of cell-bound PrtL activity was observed at 1 M and 3 M NaCl, accompanied by its appearance  in the supernatant. The total proteinase activity released by high ionic strength (plus that remaining in the final pellet) exceeded the initially determined activity. Results were corroborated by SDS-PAGE, where considerable amounts of enzyme were achieved in the supernatant fraction after incubation under high ionic strength. The obtained results were compared with the proteinases from Lactococcus lactis CRL 1195 and Lactobacillus helveticus CRL 1062.Considering the industrial importance and the potential beneficial health properties of PrtL, studies on its activity and stability under conditions usually present in fermented milk products or cheese environment (e.g., low pH, high NaCl concentration), could help to clarify the involvement of this enzyme in proteolysis during later stages of ripening.