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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Purification and Characterization of Heparin Lyase I from Bacteroides stercoris HJ-15

  • Kim, Wan-Seok (Natural Products Research Institute, College of Pharmacy, Seoul National University) ;
  • Kim, Byung-Taek (Microbiology and Immunology Lab., College of Pharmacy, Kyung Hee University) ;
  • Kim, Dong-Hyun (Microbiology and Immunology Lab., College of Pharmacy, Kyung Hee University) ;
  • Kim, Yeong-Shik (Natural Products Research Institute, College of Pharmacy, Seoul National University)
  • Published : 2004.11.30
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Abstract

Heparin lyase I was purified to homogeneity from Bacteroides stercoris HJ-15 isolated from human intestine, by a combination of DEAE-Sepharose, gel-filtration, hydroxyapatite, and CM-Sephadex C-50 column chromatography. This enzyme preferred heparin to heparan sulfate, but was inactive at cleaving acharan sulfate. The apparent molecular mass of heparin lyase I was estimated as 48,000 daltons by SDS-PAGE and its isoelectric point was determined as 9.0 by IEF. The purified enzyme required 500 mM NaCl in the reaction mixture for maximal activity and the optimal activity was obtained at pH 7.0 and $50^{\circ}C$. It was rather stable within the range of 25 to $50^{\circ}C$ but lost activity rapidly above $50^{\circ}C$. The enzyme was activated by $Co^{2+}$ or EDTA and stabilized by dithiothreitol. The kinetic constants, $K_m$ and $V_{max}$ for heparin were $1.3{\times}10^{-5}\;M$ and $8.8\;{\mu}mol/min{\cdot}mg$. The purified heparin lyase I was an eliminase that acted best on porcine intestinal heparin, and to a lesser extent on porcine intestinal mucosa heparan sulfate. It was inactive in the cleavage of N-desulfated heparin and acharan sulfate. In conclusion, heparin lyase I from Bacteroides stercoris was specific to heparin rather than heparan sulfate and its biochemical properties showed a substrate specificity similar to that of Flavobacterial heparin lyase I.

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References

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