3-epimerase
The disclosure provides a method of producing allulose by contacting a protein having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6 with a fructose substrate, wherein the protein has allulose 3-epimerase activity, and at least partially purifying the allulose. The disclosure also provides a method of producing allulose by providing a vector comprising a nucleic acid molecule having a polynucleotide sequence encoding a protein having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, wherein the protein has allulose 3-epimerase activity, synthesizing the protein having allulose 3-epimerase activity, contacting fructose with the protein having allulose 3-epimerase activity, and partially purifying the allulose produced.
1. A method of producing allulose comprising:
(a) contacting a protein with a fructose substrate under conditions such that the fructose substrate is converted into allulose, wherein the protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, wherein the protein has allulose 3-epimerase activity, and
(b) at least partially purifying the allulose.
2. The method according to claim 1 , wherein the protein is present in an isolated host cell transformed with a vector comprising a nucleic acid molecule encoding the protein.
3. The method according to claim 1 , wherein the protein is in isolated form.
4. The method according to claim 1 , wherein the conditions comprise maintaining the protein and the fructose substrate at a temperature between 25° C. and 75° C.
5. The method according to claim 1 , wherein the conditions comprise maintaining the protein and the fructose substrate between pH 4 and pH 10.
6. The method according to claim 1 , wherein the conditions comprise maintaining the fructose substrate concentration between 75% and 95% (W/V).
7. A method of producing allulose comprising the steps of:
i) providing a vector comprising a nucleic acid molecule having a polynucleotide sequence encoding a protein comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6, wherein the protein has allulose 3-epimerase activity;
ii) synthesizing the protein having allulose 3-epimerase activity encoded by the polynucleotide sequence;
iii) contacting fructose with the protein having allulose 3-epimerase activity and maintaining the fructose and the protein under conditions to permit the conversion of fructose to allulose; and
iv) at least partially purifying the allulose produced in step iii).
8. The method according to claim 1 , wherein the protein having allulose 3-epimerase activity comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6.
9. The method according to claim 7 , wherein the protein having allulose 3-epimerase activity comprises an amino acid sequence having at least 95% sequence identity to the amino sequence SEQ ID NO: 6.
10. The method according to claim 7 , wherein step ii) comprises transforming competent host cells with the vector of step i) and, optionally, culturing the transformed host cells.
11. The method according to claim 10 , wherein step iii) comprises mixing the transformed host cells with the fructose.
12. The method according to claim 10 , wherein step iii) comprises culturing the transformed host cells to produce the protein having allulose 3-epimerase activity, isolating the protein having allulose 3-epimerase activity from the host cells, and mixing the protein having allulose 3-epimerase activity with the fructose.
13. The method according to claim 12 , wherein the protein having allulose 3-epimerase activity is immobilized on a substrate after isolation of the protein.
14. The method according to claim 7 , wherein, in step ii), the protein having allulose 3-epimerase activity is synthesized by in vitro translation and subsequently isolated.
15. The method of claim 1 , wherein the protein comprises an amino acid sequence having 90% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
16. The method of claim 15 , wherein the protein comprises an amino acid sequence having 90% to 95% sequence identity to the amino acid sequence of SEQ ID NO: 6.
17. The method of claim 7 , wherein the protein comprises an amino acid sequence having 90% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
18. The method of claim 17 , wherein the protein comprises an amino acid sequence having 90% to 95% sequence identity to the amino acid sequence of SEQ ID NO: 6.
19. The method of claim 8 , wherein the protein comprises an amino acid sequence having 95% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
20. The method of claim 9 , wherein the protein comprises an amino acid sequence having 95% to 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.