A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171.
An aminopeptidase with a very broad substrate specificity was purified to homogeneity from Lactobacillus helveticus SBT 2171 by FPLC. The enzyme was purified 144-fold from a cell-free extract with a yield of 16%. The purified enzyme appeared as a single band on an SDS-PAGE gel. It had a molecular mass of 95 kDa and an isoelectric point of 4.9. The enzyme hydrolysed a large range of naphthylamide- and nitroanilide-substituted amino acids, as well as several di-, tri- and oligopeptides. It also exhibited significant proline-iminopeptidase-like activity, since it hydrolysed several proline-containing peptides. Prolyl-p-nitroanilide was hydrolysed with a low affinity (Michaelis-Menten constant 0.6 mM) and a Vmax of 2.5 mumol min-1 (mg protein)-1 while lysyl-p-nitroanilide was hydrolysed with a high affinity [Km 0.003 mM; Vmax 37.5 mumol min-1 (mg protein)-1]. The aminopeptidase activity, which was optimal between pH 6.0 and 8.0 and at 50 degrees C, was very stable at 30 degrees C for more than 7 d. The activity lost by treatment with the thiol-blocking reagents could be restored with beta-mercaptoethanol, while Co2+ and Mn2+ restored the activity of the EDTA-treated enzyme. Immunological experiments with antibodies raised against the aminopeptidases from Lactococcus lactis and Lb. helveticus clearly showed that both aminopeptidases are at least immunologically different from each other.
Sasaki
Masahiro
M
Snow Brand European Research Laboratories B. V., Zernikepark 6, 9747 AN, Groningen, The Netherlands.
Bosman
Boukje W
BW
Snow Brand European Research Laboratories B. V., Zernikepark 6, 9747 AN, Groningen, The Netherlands.
Tan
Paris S T
PST
Snow Brand European Research Laboratories B. V., Zernikepark 6, 9747 AN, Groningen, The Netherlands.
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D003611:Dairy Products / Q000382:microbiology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007120:Immunochemistry; D007526:Isoelectric Point; D007700:Kinetics; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature
D009842:Oligopeptides; D000626:Aminopeptidases