A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171
1996
Saski, M. | Bosman, B.W. | Tan, P.S.T.
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 prolineiminopeptidase-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 micromol min-1 (mg protein)-1 while lysyl-p-nitroanilide was hydrolysed with a high affinity [Km 0.003 mM; Vmax 37.5 micromol 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.
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