Purification, Kinetic Characterization of Lactococcus lactis X-prolyl Dipeptidyl Aminopeptidase Recombined in E. coli

TANG Song-shan,WU Jie, XIE Yan-fei, MING Xin, HU Zhuo-yi,LIU Jing-jing *

Chinese Journal of Biochemistry and Molecular Biology ›› 2003, Vol. 19 ›› Issue (02) : 144-150.

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Chinese Journal of Biochemistry and Molecular Biology ›› 2003, Vol. 19 ›› Issue (02) : 144-150.
Article

Purification, Kinetic Characterization of Lactococcus lactis X-prolyl Dipeptidyl Aminopeptidase Recombined in E. coli

  • TANG Song-shan,WU Jie, XIE Yan-fei, MING Xin, HU Zhuo-yi,LIU Jing-jing *
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Abstract

The recombinant x-prolyl dipeptidyl aminopeptidase (PepX) from Lactococcus lactis,a tool enzyme responsible for the maturation of the neuropeptides or active polypeptides, was purified to homogeneity by means of cell disruption, washing, centrifugation, dialysis, single ion exchange column chromatography on DEAE-cellulose 52. The specific activity of the PepX was enriched about 14.37 folds and the yield of total activity was 55.56%. PepX was composed by single polypeptide chain with molecular weight 89 kD determined by SDS-PAGE and gel column chromatography. In the study of the kinetics and inhibition of PepX against the substrate Gly-Pro-p-NA, Michaelis-Menton equation on PepX against the substrate was 1/V=0.048/[S]+0.2566 (r=0.994). K m constant of 0.1871 mmol/L and V max of 3.897 μmol/L.min -1 were calculated. PepX activity was inhibited by PMSF, trypsin inhibitor and some metal cations such as Mn 2+, Ba 2+, Cu 2+ and Zn 2+, but stimulated by Mg 2+. Further experiment showed when Cu 2+ or Zn 2+ concentration was increased to 3.726 mmol/L, the PepX activity was significantly inhibited. Low final concentrations of EDTA (≤0.6212 mmol/L) had no influence on the enzyme activity. The inhibition of PMSF showed that the active site includes a serine residue. The results suggest that PepX is a metal-independent serine peptidase.

Key words

X-prolyl dipeptidyl aminopeptidase / enzyme purification / kinetic characterization

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TANG Song-shan,WU Jie, XIE Yan-fei, MING Xin, HU Zhuo-yi,LIU Jing-jing *. Purification, Kinetic Characterization of Lactococcus lactis X-prolyl Dipeptidyl Aminopeptidase Recombined in E. coli[J]. Chinese Journal of Biochemistry and Molecular Biology, 2003, 19(02): 144-150

References

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