Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634 |
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Authors: | Alfredo E. Pérez-Guzmán Teresa Cruz y Victoria Ramón Cruz-Camarillo Humberto Hernández-Sánchez |
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Affiliation: | (1) Departamento de Graduados e Investigación en Alimentos, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Carpio y Plan de Ayala, CP 11340, México, DF, México;(2) Departamento de Microbiología, ENCB, IPN, México, DF, México |
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Abstract: | Summary An X-prolyl-dipeptidylaminopep tidase (Pep-XP) was purified from the crude intracellular extract of Lactococcus lactis subsp. cremoris NRRL 634 by ion exchange and gel filtration chromatographies. The enzyme was purified 80-fold with a recovery of 6%, and appeared as a single band with a molecular weight of about 80 kDa on polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS-PAGE). The peptidase showed its maximal activity on arginyl-proline-p-nitroanilide at pH 7.0 and at a temperature of 45 °C, although there was a good activity of Pep-XP in the pH range of 5.5–7.0 and temperatures between 40 and 50 °C. The Michaelis constant (K m) and the maximum reaction velocity (V max) values were 0.92 mM and 7.9 U/mg protein min, respectively. The activity of Pep-XP was completely inhibited by phenylmethanesulphonyl fluoride, an inhibitor of serine peptidases, and metal chelators had little effect on enzyme activity. The purified enzyme hydrolyzed synthetic substrates whose structure is X-Pro-Y like Lys-Pro-pNA, but did not hydrolyse Pro-pNA or azocasein, showing that the enzyme did not have aminopeptidase or endopeptidase activities. |
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Keywords: | L actococcus lactis peptidases purification enzymes Pep-XP X-prolyl-dipeptidyl aminopeptidase |
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