TY - JOUR
T1 - Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634
AU - Pérez-Guzmán, Alfredo E.
AU - Cruz Y Victoria, Teresa
AU - Cruz-Camarillo, Ramón
AU - Hernández-Sánchez, Humberto
PY - 2006/9
Y1 - 2006/9
N2 - 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.
AB - 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.
KW - Lactococcus lactis
KW - Pep-XP
KW - Peptidases
KW - Purification enzymes
KW - X-prolyl-dipeptidyl aminopeptidase
UR - http://www.scopus.com/inward/record.url?scp=33748313273&partnerID=8YFLogxK
U2 - 10.1007/s11274-006-9140-6
DO - 10.1007/s11274-006-9140-6
M3 - Artículo
SN - 0959-3993
VL - 22
SP - 953
EP - 958
JO - World Journal of Microbiology and Biotechnology
JF - World Journal of Microbiology and Biotechnology
IS - 9
ER -