Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

Abstract

An aminopeptidase was purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that included diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, gel filtration, and high-performance liquid chromatography over an anion-exchange column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 95,000. The aminopeptidase was capable of degrading several peptides by hydrolysis of the N-terminal amino acid. The peptidase had no endopeptidase or carboxypeptidase activity. The aminopeptidase activity was optimal at pH 7 and 40°C. The enzyme was completely inactivated by the p-chloromecuribenzoate mersalyl, chelating agents, and the divalent cations Cu2' and Cd2+. The activity that was lost by treatment with the sulfhydryl-blocking reagents was restored with dithiothreitol or P-mercaptoethanol, while Zn2+ or Co2' restored the activity of the 1,10-phenantroline-treated enzyme. Kinetic studies indicated that the enzyme has a relatively low affinity for lysyl-p-nitroanilide (Km, 0.55 mM) but that it can hydrolyze this substrate at a high rate (Vmax, 30 ,umol/min per mg of protein).

Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

“…The 7.0 optimal pH of this enzyme is similar to that of other aminopeptidases from lactic bacteria, however, this enzyme is stable at a wide range of pH, from 4.0 to 8.0. The 45°C optimal temperature is high as compared to those reported for lactic bacteria, such as Lactococcus lactis and S. thermophilus that are of 37°C [21,30]; other aminopeptidases isolated from Lactobacillus depict optimal temperatures of around 40°C [29].…”

Debittering of a Tryptic Digest of Bovine p‐casein Using Porcine Kidney General Aminopeptidase and X‐Prolydipeptidyl Aminopeptidase from Lactococcus lactis subsp. cremoris AM2

“…Therefore, it was necessary to remove Pep X from the LAP activity (and vice versa) in order to study the debittering potential of LAP and Pep X separately. Phenyl Sepharose chromatography has previously been successfully employed in the purification of general aminopeptidase activities from Lactococci (Tan and Konings 1990;Niven 1991). Free proline was not detected in any of the LAP-and Pep X-treated digests, indicating that both purified enzyme preparations were free of prolidase and prolinase activities.…”

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

“…An aminopeptidase N (or PepN) has been purified from two different strains of I,. lactis subsp, cremoris [73,74] and the gene (pel)N) encoding this enzyme has been cloned from three different lactococcal strains [75][76][77]. This 93-95-kDa enzyme showed highest activity towards substrates with N-terminal lysine, leucine or arginine, considerably less activity with alanyl, phenylalanyl and methionyl substrates and negligible activity with glycyl, prolyl and glutamyl substrates ( [73]; i.J.…”

“…This enzyme, which may be membrane-associated (see below), is specific for N-terminal aspartyl and glutamyl residues. Thus it would complement the action of aminopeptidase N which does not readily hydrolyse glutamyl substrates [73,74]. 191 A third aminopeptidase (recently designated PepC) has been isolated from L. lactis subsp.…”

“…Several claims of a cell wall location for peptidase activity have been based oil the finding of peptidase activity released into the medium when cells are incubated in CaZ'-free buffer, i.e. using the same conditions as those used to rclcase the cell envelope proteinasc [40,67,73]. Demonstration of minimal releasc of intracellular marker enzymes such as /J-galactosidase or fructose bisphosphate aldolase under these same conditions is cited as evidencc thai the peptidasc activity detected has not been released by cell lysis.…”