Purification and characterization of a novel heparinase from Bacteroides stercoris HJ-15.
A novel type of heparinase (heparin lyase, no EC number) has been purified from Bacteroides stercoris HJ-15, isolated from human intestine, which produces three kinds of heparinases. The enzyme was purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chromatographies with a final specific activity of 19.5 mmol/min/mg. It showed optimal activity at pH 7.2 and 45 degrees C and the presence of 300 mM KCl greatly enhanced its activity. The purified enzyme activity was inhibited by Cu(2+), Pb(2+), and some agents that modify histidine and cysteine residues, and activated by reducing agents such as dithiothreitol and 2-mercaptoethanol. This purified Bacteroides heparinase is an eliminase that shows its greatest activity on bovine intestinal heparan sulfate, and to a lesser extent on porcine intestinal heparan sulfate and heparin. This enzyme does not act on acharan sulfate but de-O-sulfated acharan sulfate and N-sulfoacharan sulfate were found to be poor substrates. The substrate specificity of this enzyme is similar to that of Flavobacterial heparinase II. However, an internal amino acid sequence of the purified Bacteroides heparinase shows significant (73%) homology to Flavobacterial heparinase III and only 43% homology to Flavobacterial heparinase II. These findings suggest that the Bacteroidal heparinase is a novel enzyme degrading GAGs.
College of Pharmacy, Kyung Hee University, Seoul 130-701, Korea.
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Kim
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Kim
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Kim
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Linhardt
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Kim
D000596:Amino Acids / Q000032:analysis; D001439:Bacteroides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D019763:Heparin Lyase / Q000737:chemistry* / Q000302:isolation & purification; D006497:Heparan Sulfate / Q000737:chemistry; D006801:Humans; D007422:Intestines / Q000382:microbiology; D007700:Kinetics; D013379:Substrate Specificity
D000596:Amino Acids; D019763:Heparin Lyase; D006497:Heparan Sulfate