microbiology, Vol 142, 799-808, Copyright © 1996 by Society for General Microbiology
A new, broad-substrate-specificity aminopeptidase from the dairy organism Lactobacillus helveticus SBT 2171
M Sasaki, BW Bosman and PS Tan
Snow Brand European Research Laboratories, Groningen, Netherlands.
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.
