US5198355A
Expired / Lapsed
US
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Purification of glycosaminoglycan degrading enzymes with a sulfated polysaccharide
Assignee: Seikagaku Corp
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Filing: 1989-08-24
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Granted: 1993-03-30
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Expires: 2009-08-24
Abstract
Abstract Glycosaminoglycan degrading enzymes are fractionated and purified by chromatographically treating a solution containing the enzymes with an insoluble sulfated polysaccharide carrier. The enzymes are adsorbed onto the carrier and then subsequently desrobed from the carrier.
Inventors
- 👤 Hiroshi Kikuchi
- 👤 Ken-ichi Maeyama
- 👤 Keiichi Yoshida
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 17:51
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/88; C12N9/14; C12N9/16
Claims Analysis
Total Claims: 16
Claim 1
Independent Claim
68 words
A method of purifying glycosaminoglycan degrading enzymes by fractionating the glycosaminoglycan degrading enzymes into individual glycosaminoglycan degrading enzymes which comprises chromatographically treating a solution containing the glycosaminoglycan degrading enzymes with an insoluble sulfated polysaccharide carrier whereby said glycosaminoglycan degrading enzymes are adsorbed onto said insoluble sulfated polysaccharide carrier, produced by sulfination of a polysaccharide whereafter said adsorbed glycosaminoglycan degrading enzymes are desorbed from said insoluble sulfated polysaccharide carrier.
Claim 2
Independent Claim
68 words
A method of purifying glycosaminoglycan degrading enzymes by fractionating the glycosaminoglycan degrading enzymes into individual glycosaminoglycan degrading enzymes which comprises chromatographically treating a solution containing the glycosaminoglycan degrading enzymes with an insoluble sulfated polysaccharide carrier whereby said glycosaminoglycan degrading enzymes are adsorbed onto said insoluble sulfated polysaccharide carrier, produced by sulfination of a polysaccharide whereafter said adsorbed glycosaminoglycan degrading enzymes are desorbed from said insoluble sulfated polysaccharide carrier.
Claim 3
Dependent Claim
28 words
A method as claimed in claim 1, wherein the glycosaminoglycan degrading enzymes include at least two enzymes selected from the group consisting of a lyase, glycuronidase and sulfatase.
Claim 4
Dependent Claim
42 words
A method as claimed in claim 1, wherein the insoluble sulfated polysaccharide carrier is prepared by sulfating one or more hydroxyl groups on the sugar chain of the polysaccharide selected from the group consisting of cellulose, dextran, xylan, mannan, amylose and chitin.
Claim 5
Dependent Claim
32 words
A method as claimed in claim 1, wherein the insoluble sulfated polysaccharide carrier is prepared by crosslinking a water-soluble polysaccharide and then sulfating one or more hydroxyl groups on the sugar chain.
Claim 6
Dependent Claim
28 words
A method as claimed in claim 1, wherein the insoluble sulfated polysaccharide carrier has a sulfate group content ranging from 0.1 to 1% by weight on dried basis.
Claim 7
Independent Claim
107 words
A method of purifying a lyase, glycuronidase and/or sulfatase which comprises loading a hydroxyapatite or DEAE-cellulose column with a lyase-, glycuronidase-and/or sulfatase-containing solution, performing elution with a phosphate buffer on a linear sodium chloride concentration gradient, then applying the resulting eluate containing the lyase, glycuronidase and/or sulfatase as a major solute component to a column containing an insoluble sulfated polysaccharide carrier produced by sulfination of a polysaccharide and recovering a sulfatase solution as the effluent or pass-through fraction and/or eluting the lyase and the glycuronidase with an aqueous sodium chloride solution by linear sodium chloride concentration gradient and recovering the lyase and the glycroronidase as separate fractions.
Claim 8
Independent Claim
107 words
A method of purifying a lyase, glycuronidase and/or sulfatase which comprises loading a hydroxyapatite or DEAE-cellulose column with a lyase-, glycuronidase-and/or sulfatase-containing solution, performing elution with a phosphate buffer on a linear sodium chloride concentration gradient, then applying the resulting eluate containing the lyase, glycuronidase and/or sulfatase as a major solute component to a column containing an insoluble sulfated polysaccharide carrier produced by sulfination of a polysaccharide and recovering a sulfatase solution as the effluent or pass-through fraction and/or eluting the lyase and the glycuronidase with an aqueous sodium chloride solution by linear sodium chloride concentration gradient and recovering the lyase and the glycroronidase as separate fractions.
Claim 9
Dependent Claim
54 words
A method as claimed in claim 5, wherein the insoluble sulfated polysaccharide carrier is prepared by sulfating one or more hydroxyl groups on the sugar chain of cellulose, dextran, xylan, mannan, amylose or chitin or by crosslinking a water-soluble polysaccharide for insolubilization and then sulfating one or more hydroxyl groups on the sugar chain.
Claim 10
Dependent Claim
24 words
A method as claimed in claim 6, wherein the lyase is a chondroitin sulfate lyase, dermatan sulfate lyase, heparan sulfate lyase or heparin lyase.
Claim 11
Dependent Claim
15 words
A method as claimed in claim 8, wherein the chondroitin sulfate lyase is chondroitinase ABC.
Claim 12
Dependent Claim
14 words
A method as claimed in claim 8, wherein the heparan sulfate lyase is heparitinase.
Claim 13
Dependent Claim
13 words
A method as claimed in claim 8, wherein the heparin lyase is heparinase.
Claim 14
Dependent Claim
42 words
A method as claimed in claim 6, wherein the glycuronidase is an enzyme capable of cleaving off the uronic acid of the nonreducing terminal unsaturated sugar chain at the hexosaminide cleavage site resulting from the action of a lyase on a glycosaminoglycan.
Claim 15
Dependent Claim
17 words
A method as claimed in claim 6, wherein the sulfatase is chondro-4-sulfatase, chondro-6-sulfatase, glucosamine-N-sulfatase, glycuronate-2-sulfatase or glucosamine-3-sulfatase.
Claim 16
Dependent Claim
28 words
A method as claimed in claim 6, wherein the insoluble sulfated polysaccharide carrier has a sulfate group content ranging from 0.1 to 1% by weight on dried basis.
References Cited (Backward Citations)
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1996-03-29 | 1996-09-28 | 1997-03-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-03-29 | 2000-09-28 | 2001-03-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-03-29 | 2004-09-28 | 2005-03-30 | $7700.00 | Window Closed / Lapsed |