Cellobiose dehydrogenase
The present invention relates to cellobiose dehydrogenases (CDH) having glucose oxidation activity at a pH of 7.4 or above, modifications to modify the pH dependency of the enzymes activity, uses for these CDHs, in particular electrode sensors and electrochemical cells.
1. An electrode comprising an immobilized cellobiose dehydrogenase (CDH) of Corynascus thermophiles or a CDH comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:7.
2. The electrode of claim 1 , wherein the CDH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 7.
3. The electrode of claim 2 , wherein the CDH comprises an amino acid sequence SEQ ID NO: 7.
4. The electrode of claim 1 , wherein the CDH is a modified CDH of Corynascus thermophilus , comprising a flavin and a haem domain, wherein electron transfer from the flavin to the haem domain is increased by a modification as compared to wild type CDH of Corynascus thermophilus.
5. The electrode of claim 1 , wherein the CDH has glucose oxidation activity at a pH of 7.4 or above, and wherein the glucose oxidation activity is a glucose dehydrogenase activity.
6. The electrode of claim 1 , wherein the CDH has glucose oxidation activity at a pH of 7.4 or above, and wherein the activity is an electrocatalytic oxidation of glucose.
7. The electrode of claim 1 wherein the electrode has at least 10% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by a cyt c assay.
8. The electrode of claim 1 wherein the electrode has at least 14% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by the cyt c assay.
9. The electrode of claim 1 wherein the electrode has at least 20% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by the cyt c assay.
10. The electrode of claim 1 wherein the electrode has at least 30% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by the cyt c assay.
11. The electrode of claim 1 , wherein the CDH has glucose oxidation activity at a pH of 7.4 or above, and wherein the glucose oxidation activity at pH 7.4 is at least 0.5 U/mg cellobiose dehydrogenase.
12. The electrode of claim 1 , wherein the cellobiose dehydrogenase has a Km value for a glucose oxidation reaction at pH 7.4 of below 1M.
13. The electrode of claim 1 , wherein the cellobiose dehydrogenase is immobilized by adsorption or complex formation and/or wherein the immobilized cellobiose dehydrogenase is cross-linked to increase stability or activity.
14. The electrode of claim 13 , wherein the cellobiose dehydrogenase is immobilized by complex formation via an additional complexing linker, covalent or ionic linkage and/or wherein the immobilized cellobiose dehydrogenase is cross-linked by at least one bifunctional agent.
15. An electrochemical cell comprising an electrode of claim 1 as an anodic element and a cathodic element.
16. The electrochemical cell of claim 15 , comprising a glucose containing solution as anodic fluid.
17. The electrochemical cell of claim 15 comprising a solution of at least pH 6.0 as anodic fluid.
18. The electrochemical cell of claim 17 comprising a solution of at least pH 6.5 as anodic fluid.
19. The electrochemical cell of claim 18 comprising a solution of at least pH 7.2 as anodic fluid.
20. A method of detecting and/or quantifying glucose in a sample comprising:
providing the electrode of claim 1 ;
contacting a fluid sample having a pH of at least 6.5 with the electrode; detecting and quantifying oxidation of glucose in the sample by the electrode;
detecting and quantifying the glucose in the sample based on the amount of oxidation.
21. The method of claim 20 , wherein the oxidation is detected electrochemically.
22. The method of claim 20 , wherein the electrode has at least 10% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by a cyt c assay.
23. The method of claim 22 , wherein the electrode has at least 20% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by a cyt c assay.
24. The method of claim 23 , wherein the electrode has at least 30% glucose, lactose or cellobiose oxidizing activity at a pH of 7.4 as compared to maximal activity at a lower pH as determined by a cyt c assay.