Study of peptidases involved on proteolytic activity ofLactobacillus plantarum= Studio di peptidasi coinvolte nell'attività proteolitica diLactobacillus plantarum

The proteolytic activity of lactic acid bacteria and their ability to hydrolyse toxic gluten peptides have been evaluated. In this thesis, we focus on the ability ofL. plantarumto use gluten as a nitrogen source, and define the enzymes responsible for this catalysis. The WCSF1 reference strain and two sourdough’s isolated strains was evaluated respect to their growth and proteolysis. A proteolytic activities characterisation and analysis of the residual gluten in the media are performed to evaluate which part of the gluten is hydrolysed. An ‘in-silico’ screening is also performed, and the genes identified cloned and expressed in an Escherichia coli system. Measurement of the enzymatic activities is performed, partial characterization of Lp_2953. X-prolyl-dipeptidyl-aminopeptidase and prolyl endopeptidase are the families of enzymes involved in gluten hydrolysis. Four different genes are identified as coding for putative prolyl-endopeptidases. One of these is listed in theL. plantarumdatabase as a ‘putative esterase’. Two enzymes show high activities against the chromogenic substrates. Partial characterisation of Lp_2953 demonstrates the specificity of this enzyme for the butyric-acid-derived ester, and a lower activity as an iminopeptidase. Even though these enzymes cannot hydrolyse the 33-mer peptide, they show interesting properties.