Rapid purification, characterization and substrate specificity of heparinase from a novel species of Sphingobacterium.
A type of heparinase (heparin lysase, no EC number) was isolated from the periplasmic space of a novel species of Sphingobacterium by three-step osmotic shock. It was further purified to apparent homogeneity by a combination of SP-sepharose and Source 30S chromatographies with a final specific activity of 17.6 IU/mg protein and purification factor of 13-fold. MALDI-TOF mass spectrum of the purified heparinase gave a molecular mass of 75,674 Da of the native enzyme. Peptide mass spectrum showed poor homogeneity with the database in the peptide bank. Inhibition of the enzyme activity by N-acetylimidazole indicated that tyrosine residues were necessary for enzyme activity. K(m) and V(max) of the heparinase for de-o-sulfated-N-acetyl heparin were 42 micro M and 166 microM/min/mg protein, respectively. The heparinase showed similar activity on both heparin and heparan sulfate, except for the heparin from bovine lung. The heparinase exhibited only 8.3% of the activity when de-N-sulfated heparin was used as the substrate, but N-acetylation of the de-N-sulfated heparin restored the activity to 78.4%. Thus modification of N-site in heparin structure was favorable for heparinase activity. On the other hand, de-o-sulfation in heparin showed positive effects on the heparinase activity, since the enzyme activity for N-acetyl-de-o-sulfated heparin was increased by 150%. Based on the present findings, the sphingobacterial heparinase differed from flavobacterial and other reported heparinases in molecular mass, composition, charge properties, active site, substrate specificities and other important characteristics, suggesting that it a novel heparin lysase distinct from those from other sources.
Yapeng
Chao
C
Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080 China.
Ningguo
Gao
G
Xiulan
Cheng
C
Jing
Yang
Y
Shijun
Qian
Q
Shuzheng
Zhang
Z
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D019763:Heparin Lyase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D009997:Osmotic Pressure; D011134:Polysaccharides / Q000378:metabolism; D045249:Sphingobacterium / Q000145:classification / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine
D000596:Amino Acids; D011134:Polysaccharides; D019763:Heparin Lyase