Synthetic peptides as substrate for assaying the proteolytic activity of Lactobacillus helveticus.
Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including alpha(s)1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro alpha(s)1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.
Caira
Simonetta
S
Istituto di Scienze dell'Alimentazione del C.N.R., 83100 Avellino, Italy. scaira@isa.av.cnr.it
Ferranti
Pasquale
P
Gatti
Monica
M
Fornasari
Maria Emanuela
ME
Barone
Francesca
F
Lilla
Sergio
S
Mucchetti
Germano
G
Picariello
Gianluca
G
Chianese
Lina
L
Neviani
Erasmo
E
Addeo
Francesco
F
D000596:Amino Acids / Q000378:metabolism; D000626:Aminopeptidases / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology / Q000378:metabolism; D002611:Cheese / Q000382:microbiology*; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000201:enzymology; D005516:Food Microbiology; D006868:Hydrolysis; D007778:Lactobacillus / Q000201:enzymology*; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D010455:Peptides; D010447:Peptide Hydrolases; D000626:Aminopeptidases