Purification and characterization of x-prolyl-dipeptidyl aminopeptidase from Lactococcus lactis subsp. cremoris NRRL 634

Abstract

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.

Characterization of protease activities in a crude extract of germinated cacao

“…Results revealed the presence of at least three structural characteristics of Xaa-Pro-DAP peptidases in the enzymatic extract of T. cacao: (1) serine peptidase type; (2) cysteine peptidase type; and (3) a metallic character, apart from a CP and an APE. APE and CP enzymes have been reported as metallopeptidases (Biehl et al, 1991; Pérez-Guzmán, Cruz y Victoria, Cruz-Camarillo & Hernández-Sánchez, 2006, 2004b, Ramírez-Zavala, Mercado-Flores, Hernández-Rodríguez & Vila-Tanaca, 2004b. Table 3 shows the specificity of each of the tested inhibitors, as well as the inhibitory effect on the enzymes identified in this study, including the carboxypeptidase-and aminopeptidase-type metallopeptidases of different plant and microorganism sources.…”

Purification and characterisation of a leucine aminopeptidase from Lactococcus lactis subsp. lactis cultured in skim milk

“…With regard to inhibition by metal ions, the isolated LAP differs from the enzyme prepared by Niven (1991) in that the former is not inhibited by Mn 2þ and Zn 2þ , and from L. lactis X-prolyl aminopeptidase (Pérez-Guzmán, Cruz, Victoria, Cruz-Camarillo, & Hernández-Sánchez, 2006) in that the X-prolyl aminopeptidase is not adversely affected by metal chelators.…”