Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581

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

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2015 vol. 99 p. 4277 - 4286

0175-7598

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1,925 amino acids and is a multidomain protein anchored to the cell via its W-domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37ºC and 40ºC and pH between 7 and 8. In optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar a- and ß-casein degradation pattern was observed with the purified and the cell-envelope bound proteinase. Finally, on the basis of its specificity towards caseins and, the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified at a new group of CEP. A broad knowledge about anchorage mechanism of proteins to the cell envelope and the development of efficient and commercially viable methods for proteinases extraction will open new possibilities of applications.