Cloning, expression and characterization of acharan sulfate-degrading heparin lyase II from Bacteroides stercoris HJ-15

• Published in: Journal of applied microbiology Vol. 108; no. 1; pp. 226 - 235
• Main Authors: Hyun, Y.-J, Lee, K.-S, Kim, D.-H
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 2010; Blackwell Publishing Ltd; Blackwell
• Subjects: acharan sulfate lyase; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacteroides; Bacteroides - enzymology; Bacteroides - genetics; Bacteroides stercoris HJ-15; Base Sequence; Biological and medical sciences; Chromatography, Ion Exchange; Cloning, Molecular; Escherichia coli; expression and characterization; Flavobacterium heparinum; Fundamental and applied biological sciences. Psychology; gene cloning; Gene Expression; glycobiology; Glycosaminoglycans - chemistry; Glycosaminoglycans - metabolism; Heparin Lyase - biosynthesis; Heparin Lyase - chemistry; Heparin Lyase - genetics; Heparin Lyase - isolation & purification; Microbiology; molecular cloning; Molecular Sequence Data; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Substrate Specificity - physiology; Polysaccharide-lyases; gene cloning; Enzyme; Applied microbiology; Lyases; Sulfates; expression and characterization; Bacteroidaceae; Bacteroides; Characterization; Degradation; acharan sulfate lyase; Heparin lyase; Bacteria; Bacteroides stercoris HJ-15; Molecular cloning; Carbon-oxygen lyases; glycobiology
• DOI: 10.1111/j.1365-2672.2009.04418.x
• ISSN: 1364-5072

This study focused on the cloning, expression and characterization of recombinant heparinase II (rHepII) from Bacteroides stercoris HJ-15. 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. 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°C, and the activity is stable at 4°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. 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.