Characterization of the cell wall associated proteinase activity from Lactobacillus delbrueckii strains

Abstract

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented dairy products. During milk fermentationprocess, the proteolytic system of LAB plays a key role because it enables these bacteria to grow in milk. LAB are fastidious microorganisms thatrequire an exogenous source of amino acids or peptides, which are provided by the proteolysis of casein, the most abundant protein in milk andthe main source of amino acids. The proteolytic system of LAB consists of a cell envelope-associated proteinase (CEP), amino acid and peptidetransport systems and various intracellular peptidases. The CEP is the key enzyme of the system and it is responsible for casein initial degradation.In the present work, the goal is to characterize the proteinase activity of 36 L. delbrueckii strains belonging to the CERELA culture collectionconsidering the major economic importance of these species as dairy starters. All strains were subjected to genotyping using the rep-PCR techniqueto group those isolates corresponding to clones of the same strain. One representative of each profile group was selected to further characterizetheir CEP enzymes. The strains were grown in a chemically defined medium (CDM) and their proteolytic activities were evaluated by two methods:the degradation of the chromogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide; and by the degradation profiles of alphaand beta-casein by SDS-PAGE. Results from the hydrolysis of alpha- and beta-casein degradation evidenced six types of caseinolytic cleavagespecificity. Since proteolytic activity is repressed under high peptide content, we next study the inhibitory effect of peptides concentration on theCEP activity by growing bacterial cells in CDM plus Casitone. The proteolytic activity was repressed in the presence of peptides; however thestrength of repression was strain-dependent. Finally, the release of these CEPs from the cell envelope was observed after treatment with 2 M NaCl.These results contribute to enlarge the limited knowledge on thermophilic lactobacilli CEP and are important from an industrial point of viewsince during the manufacture of hard cheeses, high concentrations of NaCl are present, and CEPs would remain active either bound to the cell orreleased, maintaining the beneficial health effects of the fermented milk products.