Polymer grafting by polysaccharide synthases

• US 20050124046A1
• Filed: 07/16/2002
• Published: 06/09/2005
• Est. Priority Date: 04/02/1998
• Status: Active Grant

• Current Assignee: Paul L. Deangelis
• Original Assignee: Paul L. Deangelis
• Inventors: DeAngelis, Paul L.
• Granted Patent: US 7,060,469 B2
• Time in Patent Office: Days
• US Class Current: 435/84
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates

Abstract

The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate synthase from Pasteurella multocida.

Claims

1. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase has an amino acid sequence encoded by a nucleotide sequence capable of hybridizing under standard conditions to a nucleotide sequence encoding the hyaluronic acid synthase as set forth in SEQ ID NO;

1; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

2. The method of claim 1, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

3. The method of claim 1, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

4. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence capable of hybridizing under standard conditions to the hyaluronic acid synthase nucleotide sequence as set forth in SEQ ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

5. The method of claim 4, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

6. The method of claim 4, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

7. The method of claim 4, wherein the hyaluronic acid synthase is isolated from a source capable of producing the hyaluronic acid synthase, wherein the source capable of producing the hyaluronic acid synthase lacks the ability to incorporate GlcUA or GlcNAc.

8. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence essentially as set forth in SEQ ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

9. The method of claim 8, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

10. The method of claim 8, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

11. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase has an amino acid sequence encoded by a nucleotide sequence capable of hybridizing under standard conditions to the nucleotide sequence encoding hyaluronic acid synthase as set forth in SEQ ID NO;

1; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

12. The method of claim 11, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

13. The method of claim 11, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

14. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence capable of hybridizing under standard conditions to the hyaluronic acid synthase nucleotide sequence as set forth in SEQ ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

15. The method of claim 14, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

16. The method of claim 14, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

17. A method for elongating a functional acceptor, comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof;

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is encoded by a nucleotide sequence essentially as set forth in SEQ. ID NO;

2; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor.

18. The method of claim 17, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

19. The method of claim 17, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

20. A method of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine, and wherein the hyaluronic acid synthase has an amino acid sequence encoded by a nucleotide sequence capable of hybridizing under standard conditions to the nucleotide sequence encoding hyaluronic acid synthase as set forth in SEQ ID NO;

1, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcUA and UDP-GlcNAc in the presence of the functional acceptor so as to create the glycosidic bond between the functional acceptor and at least one of GlcUA and GlcNAc.

21. The method of claim 20, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

22. The method of claim 20, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

23. A method of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA and GlcNAc and wherein the hyaluronic acid synthase is encoded by a nucleotide sequence capable of hybridizing under standard conditions to the hyaluronic acid synthase nucleotide sequence as set forth in SEQ ID NO;

2, wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcUA and UDP-GlcNAc in the presence of the functional acceptor so as to create the glycosidic bond between the functional acceptor and at least one of GlcUA and GlcNAc.

24. The method of claim 23, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

25. The method of claim 23, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

26. A method of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc wherein the functional acceptor has at least two sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine and further wherein the hyaluronic acid synthase has an amino acid sequence encoded by a nucleotide sequence essentially as set forth in SEQ ID NO;

2, and wherein the functional acceptor is attached to a substrate selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcUA and UDP-GlcNAc in the presence of the functional acceptor so as to create the glycosidic bond between the functional acceptor and at least one of GlcUA and GlcNAc.

27. The method of claim 26, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

28. The method of claim 26, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, thermosensitive alloys and combinations thereof.

29. A method of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA and GlcNAc and wherein the hyaluronic acid synthase has an amino acid sequence encoded by a nucleotide sequence capable of hybridizing under standard conditions to the nucleotide sequence encoding hyaluronic acid synthase as set forth in SEQ ID NO;

1, and wherein the functional acceptor is attached to a substrate wherein the substrate is selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcUA and UDP-GlcNAc in the presence of the functional acceptor so as to create the glycosidic bond between the functional acceptor and at least one of GlcUA and GlcNAc.

30. The method of claim 29, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyauronic acid and chondroitin.

31. The method of claim 29, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

32. A method of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine and wherein the hyaluronic acid synthase is encoded by a nucleotide sequence capable of hybridizing under standard conditions to the hyaluronic acid synthase set forth in SEQ ID NO;

2, and wherein the functional acceptor is attached to a substrate wherein the substrate is selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcUA and UDP-GlcNAc in the presence of the functional acceptor so as to create the glycosidic bond between the functional acceptor and at least one of GlcUA and GlcNAc.

33. The method of claim 32, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

34. The method of claim 32, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.

35. A method of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc, comprising the steps of:

providing a hyaluronic acid synthase capable of making a glycosidic bond between a functional acceptor and at least one of GlcUA and GlcNAc wherein the functional acceptor has at least three sugar units selected from the group consisting of GlcUA, GlcNAc, and hexosamine and further wherein the hyaluronic acid synthase has an amino acid sequence encoded by a nucleotide sequence essentially as set forth in SEQ ID NO;

2, and wherein the functional acceptor is attached to a substrate wherein the substrate is selected from the group consisting of silica, silicon, glass, polymers, organic compounds, metals and combinations thereof; and

incubating the hyaluronic acid synthase with at least one of UDP-GlcUA and UDP-GlcNAc in the presence of the functional acceptor so as to create the glycosidic bond between the functional acceptor and at least one of GlcUA and GlcNAc.

36. The method of claim 35, wherein the functional acceptor is a sugar acceptor selected from the group consisting of hyaluronic acid and chondroitin.

37. The method of claim 35, wherein the metal substrate is selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.