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 pu rified from Bacteroides stercoris HJ-15 which was isolated from human intes tinal bacteria with glycosaminoglycan (GAG) degrading enzymes. These enzyme s were purified to apparent homogeneity by a combination of QAE-cellulose, DEAE-cellulose, carboxymethyl-Sephadex C-50, hydroxyapatite and HiTrap SP S ephadex C-25 column chromatography with the final specific activity of 50.5 and 76.7 mu mol.min(-1).mg(-1), respectively. Both acharan sulfate lyases are single subunits of 83 kDa by SDS/PAGE and gel filtration. ASL1 showed o ptimal activity at pH 7.2 and 45 degreesC. ASL1 activity was inhibited by C u2+, Ni2+ and Co2+, but ASL2 activity was inhibited by Cu2+, Ni(2+)and Pb-2 . Both enzymes were slightly inhibited by some agents that modify histidine and cysteine residues, but activated by reducing agents such as DL-dithiot hreitol and 2-mercaptoethanol. Both purified bacteroidal acharan sulfate ly ases acted to the greatest extent on acharan sulfate, and to a lesser exten ts on heparan sulfate and heparin. They did not act on de-O-sulfated achara n 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.