title: Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15.
abstract: OBJECTIVE
  This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15.
  
  
  RESULTS
  The heparinase II gene from Bact. stercoris HJ-15 was identified by Southern blotting and the sequence was deposited in GenBank. The gene was cloned and overexpressed in Escherichia coli, and rHepII was purified using two simple ion-exchange column chromatography steps. Enzymatic properties and substrate specificities of rHepII were assessed and its kinetic constants were calculated. Heparin-like glycosaminoglycans (HLGAGs) were digested with rHepII under optimal reaction conditions, and the products were analysed by SAX-HPLC.
  
  
  CONCLUSIONS
  The heparinase II gene is 2322-bp long and consists of 773 amino acids. rHepII is most active in 50 mmol l(-1) sodium phosphate buffer with 75 mmol l(-1) NaCl (pH 7.4) at 32 degrees C, and the activity is stable at 4 degrees C for 15 days on storage. Acharan sulfate is the best substrate for rHepII, followed by heparan sulfate and heparin. The major degradation products were verified as highly sulfated disaccharides through SAX-HPLC analysis. It means that rHepII prefers iduronic acid over glucuronic acid on the HLGAG structure.
  
  
  CONCLUSIONS
  This study provides easy and certain means for obtaining large amounts of pure rHepII and also provides important information regarding the tendencies of this enzyme and its digested products. rHepII digests HLGAGs in a different manner than heparinases from Flavobacterium heparinum; therefore, we anticipate that rHepII will be a powerful tool for studies of GAGs and GAGs lyases.
authors: Department of Life and Nanopharmaceutical Sciences and Department of Pharmaceutical Science, Kyung Hee University, Hoegi, Dongdaemun-ku, Seoul, Korea.
authors: Y-J
authors: YJ
authors: Hyun
authors: K-S
authors: KS
authors: Lee
authors: D-H
authors: DH
authors: Kim
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D001439:Bacteroides / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000737:chemistry / Q000378:metabolism; D019763:Heparin Lyase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D001426:Bacterial Proteins; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; C106771:acharan sulfate; C427386:acharan sulfate lyase 2; D019763:Heparin Lyase