Cloning, overexpression, and characterization of recombinant heparinase III from Bacteroides stercoris HJ-15.
Recombinant heparinase III (rHepIII) from Bacteroides stercoris HJ-15 was cloned, expressed, and characterized. The full-length heparinase III gene from B. stercoris HJ-15 was identified by Southern blotting, and the sequence was deposited in GenBank. The heparinase III gene, which is 2,001-bp long, was cloned and overexpressed in Escherichia coli; highly active rHepIII was easily purified using only one step of immobilized Ni(2+) affinity column chromatography. Enzymatic properties and substrate specificities of rHepIII were assessed, and its kinetic constants were calculated. rHepIII was most active in 50 mM sodium phosphate buffer with 350 mM NaCl (pH 6.6) at 45 degrees C. Through amino acid modification studies and site-directed mutagenesis assay, cysteines and histidines were identified as crucial residues for enzymatic activity. Moreover, this enzyme digested not only heparan sulfate but also heparin and hyaluronic acid, and their degradation products were verified by strong anion exchange/high-performance liquid chromatography. These characteristics, including active residues and substrate specificities were interesting compared with those of existing heparinase III from other species. We anticipate that the convenience of purification and the characteristics of this enzyme will make it a powerful tool for studies of glycosaminoglycans and their lyases.
Hyun
Yang-Jin
YJ
Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, South Korea.
Lee
Jeong Hoon
JH
Kim
Dong-Hyun
DH
D019943:Amino Acid Substitution / Q000235:genetics; D001439:Bacteroides / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006493:Heparin / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006820:Hyaluronic Acid / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011133:Polysaccharide-Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004269:DNA, Bacterial; D006493:Heparin; D006497:Heparan Sulfate; D006820:Hyaluronic Acid; D011133:Polysaccharide-Lyases; D011993:Recombinant Fusion Proteins; C021349:heparitinsulfate lyase