Use of a n-acetylneuraminate lyase derived from the bacterium aliivibrio salmonicida in the production of neuraminic acid and derivatives thereof
AssigneeUniversitetet I Tromsø—Norges Arktiske Universitet
InventorsInger Lin Uttakleiv RÆDER, Bjørn ALTERMARK, Man Kumari GURUNG, Arne Oskar SMALÅS
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Abstract
The present invention provides to methods for the production of neuraminic acid or derivatives thereof under alkaline conditions and to the use of neuraminate lyases showing improved characteristics under alkaline conditions in the production of neuraminic acid or its derivatives. More particularly, the present invention employs inter alia a N-acetylneuraminate lyase which has been isolated from the psychrophilic bacterium Aliivibrio salmonicida LFI1238 and has shown to have improved characteristics under alkaline conditions.
Claims24
A method for the production of neuraminic acid or a derivative thereof comprising:
- a) reacting mannosamine or a derivative thereof with pyruvate at an alkaline pH of at least about 9 in the presence of a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:
- 1 or a functional variant thereof;
- wherein the functional variant is a polypeptide comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 1.
The method according to claim 24, wherein the alkaline pH in step b) is in the range from about 9 to about 12.
The method according to claim 24, wherein step b) is performed at the same pH or within the same pH range as in step a).
The method according to claim 24, wherein step b) is carried out at a temperature ranging from about 45 to about 70° C.
The method according to claim 24, wherein step b) is carried out at the same temperature or within the same temperature range as in step a).
The method according to claim 24, wherein
- the derivative of glucosamine is GlcNAc, and wherein the derivative of neuraminic acid and the derivative of mannosamine are selected from the group consisting of:
- the derivative of neuraminic acid is Neu5Ac and the derivative of mannosamine is ManNAc;
- the derivative of neuraminic acid is legionaminic acid and the derivative of mannosamine is 2,4-diacetamino-2,4,6-trideoxymannose;
- the derivative of neuraminic acid is Neu5,7Ac2, Neu5,8Ac2, Neu5,9Ac2 or any mixture thereof and the derivative of mannosamine is 4-0-acetyl-2-N-acetylmannosamine, 6-0-acetyl-2-N-acetylmannosamine or any mixture thereof;
- the derivative of neuraminic acid is Neu5Gc and the derivative of mannosamine is N-glycolylmannosamine;
- and
- the derivative of neuraminic acid is 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid and the derivative of mannosamine is D-mannose.
The method according to claim 24, wherein
- the derivative of glucosamine is GlcNAc, and wherein the derivative of neuraminic acid and the derivative of mannosamine are selected from the group consisting of:
- the derivative of neuraminic acid is Neu5Ac and the derivative of mannosamine is ManNAc;
- the derivative of neuraminic acid is legionaminic acid and the derivative of mannosamine is 2,4-diacetamino-2,4,6-trideoxymannose;
- and
- the derivative of neuraminic acid is Neu5,7Ac2, Neu5,8Ac2, Neu5,9Ac2 or any mixture thereof and the derivative of mannosamine is 4-0-acetyl-2-N-acetylmannosamine, 6-0- acetyl-2-N-acetylmannosamine or any mixture thereof.
Use of a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:
- 1 or a functional variant thereof in the production of neuraminic acid or a derivative thereof at an alkaline pH of at least about pH 9;
- wherein the functional variant is a polypeptide comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 1.
A method for cleaving neuraminic acid or a derivative thereof comprising incubating neuraminic acid or a derivative thereof in the presence of a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:
- 1 or a functional variant thereof;
- wherein the functional variant is a polypeptide comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 1.
A method for quantitating neuraminic acid or derivative thereof in a sample, the method comprising:
- a′) incubating said sample in the presence of a polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:
- 1 or a functional variant thereof;
- wherein the functional variant is a polypeptide comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:
- 1;
- and
- b′) determining the amount of mannosamine or a derivative thereof and/or determining the amount of pyruvate.
Dependencies are inferred from the claim text.