Purification and Characterization of a Novel Heparinase from Bacteroides stercoris HJ-15

Abstract

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, DEAF-cellulose, CM-Sephadex C-50, hydroxyapatite, and HiTrap SP chro-matographies with a final specific activity of 19.5μmol/min/mg. It showed optimal activity at pH 7.2 and 45°C and the presence of 300mM KCl greatly enhanced its activity. The purified enzyme activity was inhibited by Cu²⁺ Pb²⁺, 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 sub-strates. The substrate specificity of this enzyme is similar to that of Flavobacterial hepa-rinase 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.