Identification and partial characterization of proteolytic activity of Enterococcus faecalis relevant to their application in the dairy industry

Abstract

Bacteria of the Enterococcus genus represent a group of lactic acid bacteria (LAB). They occur ubiquitous in many traditional fermented foods, especially in artisanal cheeses, playing a beneficial role in the development of cheese flavor. Several enterococcal strains are successfully used as pharmaceutical probiotics and some of them are able to produce bacteriocin and bioactive peptides. However, some Enterococcus strains can cause nosocomial infections, including endocarditis, urinary tract infections and bacteremia. Therefore, the questions on their safe use in foods are still valid. The main goals of the study were to investigate a proteolytic potential and to identify key enzymes of the proteolytic system in Enterococcus faecalis isolated from artisan Polish cheeses. An extracellular-secreted (E) and a cell envelope proteinase (CEP) were isolated and the enzymes' activities depending on bacterial growth phase were evaluated. CEP displayed a higher protease activity than E, and a CEP fraction has been purified 70-fold by a method that included precipitation, diafiltration and gel filtration chromatography. The molecular mass of the enzyme has been estimated by SDS-PAGE to be ~25 kDa. The maximum enzyme activity of the proteinase has been observed at pH 6.9 and 37°C. The enzyme was able to hydrolyze: casein, bovine serum albumin, α-lactalbumin, β-lactoglobulin, but not Leu-pNa. The results of zymography, SDS- PAGE and LC-MS-MS/MS data had allowed us to identify key enzymes of the proteolytic system of E. faecalis as coccolysin and glutamyl endopeptidase. To asses microbiological safety of the tested strain, evaluation of virulence factors presence and antibiotic susceptibility was also conducted.