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US 5,290,695 AGranted
Heparitinase, process for producing the same and bacteria producing the same
AssigneeSeikagaku Kogyo Kabushiki Kaisha
InventorsKiyoshi Morikawa, Hirofumi Miyazono, Hiroshi Maruyama, Keiichi Yoshida
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Abstract
Disclosed are novel enzymes, heparitinase T-I, heparitinase T-II, heparitinase T-III and heparitinase T-IV, which degrade heparan sulfate and/or heparin, a process for producing thereof by cultivating a novel Bacillus circulans HpT 298 having an ability of producing these enzymes and a novel Bacillus circulans HpT 298.
Claims1
Enzymes consisting of heparitinase T-I, heparitinase T-II, heparitinase T-III and heparitinase T-IV, having the following properties:
- (A) action:
- each enzyme is a lyase which degrades glucosaminide linkages of heparin or heparin sulfate, and cleaves them to form double bonds between 4-position and 5-position carbon atoms of uronic acids,
- (B) substrate specificity:
- heparitinase T-I and heparitinase T-II do not degrade heparin and degrade heparin sulfate, and unsaturated disaccharides formed as degraded products are non-sulfate compound (.DELTA.DiHS-OS) and a small amount of uronic acid-glucosamine-N-sulfate (.DELTA.DiHS-NS);
- heparitinase T-III does not degrade heparin and degrades heparin sulfate, and unsaturated disaccharides formed as degraded products are non-sulfate compound (.DELTA.diHS-OS) and uronic acid-glucosamine-N-sulfate (.DELTA.diHS-NS);
- heparitinase T-IV degrades heparin and heparin sulfate, and unsaturated disaccharides formed as degraded products are uronic acid-glucosamine-N-sulfate (.DELTA.DIHS-OS), uronic acid-glucosamine-N,6-disulfate (.DELTA.DiHS-diN, 6S), uronic acid-2-sulfate-glucosamine-N-sulfate (.DELTA.DiHS-diU,NS) and uronic acid-2-sulfate-glucosamine-N,6-disulfate (.DELTA.DiHS-Tris),
- (C) optimum pH:
- ______________________________________ heparitinase T-I 5.5 to 6.5 heparitinase T-II 5.5 to 6.5 heparitinase T-III 7.0 to 8.0 heparitinase T-IV 7.5 to 8.0, ______________________________________
- (D) pH stability:
- ______________________________________ heparitinase T-I 4.5 to 9.5 heparitinase T-II 5.0 to 9.5 heparitinase T-III 5.0 to 9.5 heparitinase T-IV 5.0 to 10.0, ______________________________________
- (E) optimum temperatures at which the enzyme has the highest degradation rate:
- ______________________________________ heparitinase T-I 55.degree. C. heparitinase T-II 55.degree. C. heparitinase T-III 50.degree. C. heparitinase T-IV 40.degree. C. ______________________________________
- (F) heat stability temperature at which or lower than which the enzyme is stable and at higher than which the enzyme is degenerated due to heat and deactivated:
- ______________________________________ heparitinase T-I 50.degree. C. or lower heparitinase T-II 50.degree. C. or lower heparitinase T-III 45.degree. C. or lower heparitinase T-IV 40.degree. C. or lower, ______________________________________
- (G) inhibition and activation at ion concentration of 5 mM:
- ______________________________________ heparitinase T-I activated by Ca.sup.2+, Co.sup.2+, Mg.sup.2+ and Mn.sup.2+, and inhibited by Zn.sup.2+ heparitinase T-II activated by Ba.sup.2+, Ca.sup.2+, Co.sup.2+, Mg.sup.2+ and Mn.sup.2+, and inhibited by Zn.sup.2+ heparitinase T-III inhibited by Zn.sup.2+ heparitinase T-IV activated by Ba.sup.2+, Ca.sup.2+ and Mg.sup.2+, and inhibited by Co.sup.2+ and Zn.sup.2+. ______________________________________
13 more elements
Dependencies are inferred from the claim text.