title: Expression of heparinase I of Bacteroides stercoris HJ-15 and its degradation tendency toward heparin-like glycosaminoglycans.
abstract: 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 Ni(2+)-affinity column chromatography. The purified but denatured enzyme was then successfully refolded by dialysis against 50 mM Tris-HCl (pH 7.0) containing 1mM DTT and CaCl(2). BSrhepI was most active in 50mM Tris-HCl buffer containing 300 mM NaCl, 10 mM CaCl(2), and 1 mM DTT (pH 7.0) at 37°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.
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Dongdaemun-ku, Seoul 130-701, Republic of Korea.
authors.forename: Yang-Jin
authors.initials: YJ
authors.lastname: Hyun
authors.forename: Il-Hoon
authors.initials: IH
authors.lastname: Jung
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000737:chemistry* / Q000378:metabolism*; D006493:Heparin / Q000737:chemistry*; D019763:Heparin Lyase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013552:Swine
chemical_list: D006025:Glycosaminoglycans; D006493:Heparin; D019763:Heparin Lyase; D002118:Calcium; D004229:Dithiothreitol