title: A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171.
abstract: 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.
authors.affiliation: Snow Brand European Research Laboratories B. V., Zernikepark 6, 9747 AN, Groningen, The Netherlands.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sasaki
authors.affiliation: Snow Brand European Research Laboratories B. V., Zernikepark 6, 9747 AN, Groningen, The Netherlands.
authors.forename: Boukje W
authors.initials: BW
authors.lastname: Bosman
authors.affiliation: Snow Brand European Research Laboratories B. V., Zernikepark 6, 9747 AN, Groningen, The Netherlands.
authors.forename: Paris S T
authors.initials: PST
authors.lastname: Tan
mesh_terms: 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
chemical_list: D009842:Oligopeptides; D000626:Aminopeptidases