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.
Blanc
B
B
Laboratoire de Microbiologie et Génétique Moléculaire, Centre de Génétique Cellulaire et Moléculaire (CNRS UMR 106), Université Claude Bernard-Lyon I, Villeurbanne, France.
Laloi
P
P
Atlan
D
D
Gilbert
C
C
Portalier
R
R
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