Recombinant heparinase I was cloned from Bacteroides stercoris HJ-15 (BSrhepI), overexpressed in Escherichia coli, and intensively characterized. The complete gene of BSrhepI was identified by Southern blotting, and was overexpressed as an inclusion body. The inclusion body was solubilized with 4 M guanidine-HCl, and the denatured BSrhepI was easily purified using Ni2+-affinity column chromatography. The purified but denatured enzyme was then successfully refolded by dialysis against 50 mM Tris-HCl (pH 7.0) containing 1 mM DTT and CaCl2. BSrhepI was most active in 50 mM Tris-HCl buffer containing 300 mM NaCl, 10 mM CaCl2, and 1 mM DTT (pH 7.0) at 37 degrees C. This enzyme digested not only heparin, but also heparan sulfate. Through comparative HPLC- analysis of each degraded product of heparin and heparan sulfate by digestion with BSrhepI or flavobacterial heparinase I, we verified that BSrhepI has a broader spectrum of substrate specificities than other reported heparinases. (c) 2012 Elsevier Ltd. All rights reserved.