Purification and characterization of heparin lyase I from Bacteroides stercoris HJ-15.
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 degrees C. It was rather stable within the range of 25 to 50 degrees C but lost activity rapidly above 50 degrees 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 10(-5) M and 8.8 micromol/min.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.
Kim
Wan-Seok
WS
Natural Products Research Institute, College of Pharmacy, Seoul National University, Seoul 110-460, Korea.
Kim
Byung-Taek
BT
Kim
Dong-Hyun
DH
Kim
Yeong Shik
YS
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D001439:Bacteroides / Q000201:enzymology*; D004795:Enzyme Stability; D006493:Heparin / Q000378:metabolism; D019763:Heparin Lyase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000737:chemistry; D013379:Substrate Specificity; D013552:Swine
D001426:Bacterial Proteins; D006493:Heparin; D019763:Heparin Lyase; D006497:Heparan Sulfate