Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese

SIMPLE SUMMARY: Lactic acid bacteria are nutritionally fastidious microorganisms typically used in the production of fermented dairy foods. The lactic acid bacteria’s proteolytic system is crucial for their growth in milk, and plays a paramount role in developing the organoleptic and healthy properties of fermented dairy foods. Cell-envelope proteinases are the first component of this system, responsible for the degradation of caseins into short peptides. In the present work, the cell-envelope proteinases of two highly proteolytic Lacticaseibacillus casei strains, previously isolated from ripened Parmigiano Reggiano cheese, were characterized from a genetic and biochemical point of view. Two different prt genes existed in the genomes of both strains, but only one, named prtR1, was expressed. PrtR1 proteins extracted from both strains displayed the highest activity at 40 °C and pH 7. Interestingly, PrtR1 extracted from Lacticaseibacillus casei PRA205 retained some residual activity at 5 °C and at pH 4. These important biotechnological features can be exploited in the production of fermented dairy products. Peptidomic analysis revealed that both proteinases were able to release β- and αS1-casein-derived bioactive peptides, suggesting that Lacticaseibacillus casei can be a source of new proteinases that can be exploited for the formulation of dairy beverages or hydrolysates enriched in bioactive peptides. ABSTRACT: In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca(2+) ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1′ subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.