Purification and characterization of acharan sulfate lyases, two novel heparinases, from Bacteroides stercoris HJ-15.
Two novel acharan sulfate lyases (ASL1 and ASL2: no EC number) have been purified from Bacteroides stercoris HJ-15 which was isolated from human intestinal bacteria with glycosaminoglycan (GAG) degrading enzymes. These enzymes were purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, carboxymethyl-Sephadex C-50, hydroxyapatite and HiTrap SP Sephadex C-25 column chromatography with the final specific activity of 50.5 and 76.7 micromol.min-1.mg-1, respectively. Both acharan sulfate lyases are single subunits of 83 kDa by SDS/PAGE and gel filtration. ASL1 showed optimal activity at pH 7.2 and 45 degrees C. ASL1 activity was inhibited by Cu2+, Ni2+ and Co2+, but ASL2 activity was inhibited by Cu2+, Ni2+and Pb2. Both enzymes were slightly inhibited by some agents that modify histidine and cysteine residues, but activated by reducing agents such as DL-dithiothreitol and 2-mercaptoethanol. Both purified bacteroidal acharan sulfate lyases acted to the greatest extent on acharan sulfate, and to a lesser extents on heparan sulfate and heparin. They did not act on de-O-sulfated acharan sulfate. These findings suggest that the biochemical properties of these purified acharan sulfate lyases are different from those of the previously purified heparin lyases, but these enzymes belong to heparinase II.
Kim
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College of Pharmacy, Kyung Hee University, Seoul, Korea; Natural Products Research Institute, Seoul National University, Seoul, Korea.
Hong
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Kim
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Kim
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Kim
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D000595:Amino Acid Sequence; D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D006025:Glycosaminoglycans / Q000378:metabolism*; D019763:Heparin Lyase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013379:Substrate Specificity
D006025:Glycosaminoglycans; C106771:acharan sulfate; C427387:acharan sulfate lyase 1; C427386:acharan sulfate lyase 2; D019763:Heparin Lyase