GLUCOSE ISOMERASES

20250051751

18/718823

December 13, 2022

The invention relates to glucose isomerase enzymes (glucose isomerases) with improved enzymatic activity at low pH values and tolerance of calcium ions in the enzymatic reactions.

C-LEcta GmbH (Leipzig, DE)

1. A glucose isomerase comprising an amino acid sequence having at least 80% identity to SEQ ID NO: 1 and comprising at least one substitution selected from the group consisting of V187C, V187T, F242C, P313S, and A47S, wherein the glucose isomerase has at least one of characteristics (A), (B), (C), (D) and (E), or any combination thereof, wherein characteristic (A) is an increased enzymatic activity in the absence of Ca2+ (A0) in comparison to SEQ ID NO:1 with respect to the conversion of fructose into glucose or vice versa; characteristic (B) is an increased enzymatic activity in the presence of Ca2+ (ACa2+) in comparison to SEQ ID NO: 1 with respect to the conversion of fructose into glucose or vice versa; characteristic (C) is an increased residual enzymatic activity in the presence of Ca2+ (RACa2+) in comparison to SEQ ID NO:1 with respect to the conversion of fructose into glucose or vice versa; characteristic (D) is an increased enzymatic activity at neutral and acidic pH (ApH) in comparison to SEQ ID NO: 1 with respect to the conversion of fructose into glucose or vice versa; and characteristic (E) is an increased enzymatic activity at low temperatures (ATemp) in comparison to SEQ ID NO:1 with respect to the conversion of fructose into glucose or vice versa.

2. The glucose isomerase of claim 1, wherein the at least one substitution is selected from the group of consisting of V187C and F242C.

3. The glucose isomerase according to claim 1, wherein the at least one substitution is V187C.

4. The glucose isomerase according to claim 1, wherein the amino acid sequence further comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight substitutions at a position selected from the group consisting of V187, Q300, K343, A342, M241, F242, P313, A47, and M309.

5. The glucose isomerase according to claim 4, wherein the amino acid sequence comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or at least nine substitutions selected from the group consisting of V187C, V187T, Q300Y, Q300S, P313S, P313V, K343R, A342S, A47S, M241V, F242C, and M309I.

6. The glucose isomerase according to claim 1, wherein the amino acid sequence has at least 80%, or at least 82%, or at least 84%, or at least 86%, or at least 88%, or at least 90%, or at least 92%, or at least 94%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99.9% identity to SEQ ID NO: 1.

7. The glucose isomerase according to claim 1, wherein the amino acid sequence has at least 80%, or at least 82%, or at least 84%, or at least 86%, or at least 88%, or at least 90%, or at least 92%, or at least 94%, or at least 96%, or at least 98%, or at least 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 3, SEQ ID NO: 11, and SEQ ID NO: 19.

8. The glucose isomerase according to claim 1, wherein the amino acid sequence has at least 80%, or at least 82%, or at least 84%, or at least 86%, or at least 88%, or at least 90%, or at least 92%, or at least 94%, or at least 96%, or at least 98%, or at least 100% identity to an amino acid sequence selected from the groups consisting of SEQ ID NO: 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73; or SEQ ID NO: 28, 29, 32, 36, 37, 38, 39, 40, 41, 42, 44, 45, 46, 47, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, and 73; or SEQ ID NO: 26, 32, 34, 35, 43, 44, 51, 62, 64, 66, 70, and 72; or SEQ ID NO: 27, 30, 33, 34, 37, 41, 42, 43, 44, 45, 47, 48, 49, 50, 51, 53, 54, 55, 56, 58, 59, 60, 61, 63, 64, 65, 66, 68, 69, 70, 71, 72, and 73.

9. The glucose isomerase according to claim 1, which is adapted to catalyze conversion of an aldose molecule into a ketose molecule, and/or a ketose molecule into an aldose molecule.

10. The glucose isomerase according to claim 1, which exhibits (A) an increased enzymatic activity in the absence of Ca2+ (A0) in comparison to SEQ ID NO:7 with respect to the conversion of fructose into glucose or vice versa; and/or (B) an increased enzymatic activity in the presence of Ca2+ (ACa2+) in comparison to SEQ ID NO:7 with respect to the conversion of fructose into glucose or vice versa; and/or (C) an increased residual enzymatic activity in the presence of Ca2+ (RACa2+) in comparison to SEQ ID NO:7 with respect to the conversion of fructose into glucose or vice versa; and/or (D) an increased enzymatic activity at neutral and acidic pH (ApH) in comparison to SEQ ID NO:7 with respect to the conversion of fructose into glucose or vice versa; and/or (E) an increased enzymatic activity at low temperatures (ATemp) in comparison to SEQ ID NO:7 with respect to the conversion of fructose into glucose or vice versa.

11. A process for conversion of an aldose molecule into a ketose molecule said method comprising: providing the glucose isomerase of claim 1, and contacting said aldose molecule with the glucose isomerase, wherein the aldose molecule is converted into the ketose molecule.

12. The process according to claim 11, wherein the aldose molecule is glucose.

13. The process according to claim 11, wherein the contacting is performed in a liquid reaction medium that is characterized by (i) a pH of in the range of 5.0 to 8.0; and/or (ii) a calcium ion concentration of 0 mM or in the range between 0.5 mM to 60 mM.

14. A method comprising providing: the glucose isomerase of claim 1, a liquid reaction medium having a pH of in the range of 5.0 to 8.0 and/or a calcium ion concentration of 0 mM or in the range between 0.5 mM to 60 mM, and (i) an aldose molecule, wherein the glucose isomerase coverts the aldose molecule in the liquid reaction medium into a ketose molecule and/or (ii) a ketose molecule, wherein the glucose isomerase coverts the ketose molecule in the liquid reaction medium into an aldose molecule.

15. The method according to claim 14, wherein the aldose molecule is glucose that is converted to the ketose molecule fructose.

16. The method according to claim 13, wherein the liquid reaction medium is characterized by: a pH of in the range of 5.3 to 8.0, or 5.5 to 8.0, or 5.8 to 8.0, or 6.0 to 8.0, or 6.4 to 8.0, or 6.6 to 8.0, or 6.9 to 8.0, preferably within the range of 5.0 to 7.5, or 5.3 to 7.5, or 5.5 to 7.5, or 5.8 to 7.5, or 6.0 to 7.5, or 6.4 to 7.5, or 6.6 to 7.5, or 6.9 to 7.5; preferably in the range of 5.0 to 7.0, or 5.3 to 7.0, or 5.5 to 7.0, or 5.8 to 7.0, or 6.0 to 7.0, or 6.4 to 7.0, or 6.6 to 7.0, or 6.9 to 7.0; and/or a calcium ion concentration in the range between 1 mM to 60 mM, or 2 mM to 60 mM, or 3 mM to 60 mM, or 4.0 mM to 60 mM, or 5 mM to 60 mM, or 10 mM to 60 mM, or 20 mM to 60 mM, or 30 mM to 60 mM, or 0.5 mM to 30 mM, or 1 mM to 30 mM, or 2 mM to 30 mM, or 3 mM to 30 mM, or 4.0 mM to 30 mM, or 5 mM to 30 mM, or 10 mM to 30 mM, or 20 mM to 30 mM, or 0.5 mM to 20 mM, or 1 mM to 20 mM, or 2 mM to 20 mM, or 3 mM to 20 mM, or 4.0 mM to 20 mM, or 5 mM to 20 mM, or 10 mM to 20 mM, or 0.5 mM to 10 mM, or 1 mM to 10 mM, or 2 mM to 10 mM, or 3 mM to 10 mM, or 4.0 mM to 10 mM, or 5 mM to 10 mM.

17. The glucose isomerase according to claim 1, wherein the characteristic (A), (B), (C), (D) and/or (E) is the increased enzymatic activity with respect to the conversion of fructose into glucose.

18. The glucose isomerase according to claim 9, wherein the ketose molecule is fructose and the aldose molecule is glucose.

19. The glucose isomerase according to claim 10, wherein the increased enzymatic activity in (A), (B), (C), (D) and/or (E) is with respect to the conversion of fructose into glucose.

20. The process according to claim 13, wherein the pH is of in the range of 5.3 to 8.0, or 5.5 to 8.0, or 5.8 to 8.0, or 6.0 to 8.0, or 6.4 to 8.0, or 6.6 to 8.0, or 6.9 to 8.0; and/or the calcium ion concentration is in the range of between 1 mM to 60 mM, or 2 mM to 60 mM, or 3 mM to 60 mM, or 4.0 mM to 60 mM, or 5 mM to 60 mM, or 10 mM to 60 mM, or 20 mM to 60 mM, or 30 mM to 60 mM.

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