Two cell-wall-associated aminopeptidases from Lactobacillus helveticus and the purification and characterization of APII from strain ITGL1.
Lactobacillus helveticus ITGL1 is able to hydrolyse many amino-acyl and dipeptidyl-p-nitroanilides. Analysis of heat inactivation kinetics, metal ion and protease inhibitor effects, and the subcellular location of aminopeptidase activities in both the parental strain and mutants deficient in lysyl-p-nitroanilide hydrolysis, led to the characterization of two cell-wall-associated aminopeptidases, APII and APIV. APII, which catalysed L-lysine p-nitroanilide hydrolysis, was purified about 28-fold to homogeneity from cell-wall extracts of L. helveticus ITGL1 and characterized. The purified enzyme appeared to be monomeric, with a molecular mass of 97 kDa. Aminopeptidase activity was greatest at pH 6.5 and 50 degrees C. APII was completely inhibited by bestatin, chelating agents such as EDTA or 1,10-phenanthroline and the divalent cations Zn2+ and Cu2+. The activity of the EDTA-treated enzyme was restored by Co2+, Ca2+ or Mn2+. Although APII was able to degrade several dipeptides and tripeptides with hydrophobic N-terminal amino acid (Leu, Ala), it was inactive on peptides containing Pro or Gly, and may thus contribute to the development of cheese flavour by processing bitter peptides.
Laboratoire de Microbiologie et Génétique Moléculaire, Centre de Génétique Cellulaire et Moléculaire (CNRS UMR 106), Université Claude Bernard-Lyon I, Villeurbanne, France.
B
B
Blanc
P
P
Laloi
D
D
Atlan
C
C
Gilbert
R
R
Portalier
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification*; D002451:Cell Compartmentation; D002473:Cell Wall / Q000201:enzymology* / Q000378:metabolism; D004795:Enzyme Stability; D007778:Lactobacillus / Q000201:enzymology*; D007931:Leucyl Aminopeptidase / Q000032:analysis; D008239:Lysine / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D009113:Muramidase / Q000378:metabolism; D013379:Substrate Specificity
C072567:lysine 4-nitroanilide; D009113:Muramidase; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; D008239:Lysine