Stabilization of dehydrogenases with stable coenzymes
View Patent ↗The present invention relates to a method for stabilizing an enzyme by storing the enzyme in the presence of a stable coenzyme. The present invention further relates to an enzyme stabilized with a stable coenzyme, and to the use thereof in test elements for detecting analytes.
1. A method for stabilizing a coenzyme-dependent dehydrogenase, the method comprising the step of:
mixing the coenzyme-dependent dehydrogenase with a stable artificial coenzyme, wherein the coenzyme-dependent dehydrogenase comprises a dehydrogenase selected from the group consisting of glucose dehydrogenase (E.C.1.1.1.47), lactate dehydrogenase (E.C.1.1.1.27, 1.1.1.28), malate dehydrogenase (E.C.1.1.1.37), glycerol dehydrogenase (E.C.1.1.1.6), alcohol dehydrogenase (E.C.1.1.1.1), alpha-hydroxybutyrate dehydrogenase, sorbitol dehydrogenase, and L-amino-acid dehydrogenase (E.C.1.4.1.5);
and the stable artificial coenzyme is a compound of formula (II)
wherein
A=adenine,
T=in each case independently O,
U=in each case independently OH,
V=in each case independently OH or a phosphate group;
W=CON(R) 2 or COR, with R=in each case independently H or C 1 -C 2 -alkyl,
X 1 , X 2 =in each case independently O,
Y=O or CH 2 , AND
Z is a carbocyclic or heterocyclic 5-membered ring of the general formula (III)
where a single bond is present between R 5′ and R 5″ , with
R 4 =in each case independently H,
R 5 =CH 2 ,
where R 5′ =NH, CH 2 , or CHOH, and
R 5″ =CR 4 2 or CHOH, and
R6, R6′=in each case independently CH or CCH 3 ; and
storing the mixture of the coenzyme-dependent dehydrogenase stabilized with the stable artificial coenzyme—for a period of at least 2 weeks at a temperature of at least 20° C.;
wherein the stored coenzyme-dependent dehydrogenase retains at least 50% of its initial enzyme activity in the presence of the stable artificial coenzyme.
2. The method according to claim 1 , wherein the coenzyme-dependent dehydrogenase comprises glucose dehydrogenase.
3. The method according to claim 1 , in which R 5′ and R 5″ are each CHOH.
4. The method according to claim 1 , wherein the stable artificial coenzyme is carbaNAD of structure
5. The method according to claim 1 , wherein the coenzyme-dependent dehydrogenase stabilized with a stable artificial coenzyme is stored without a drying reagent.
6. The method according to claim 1 , wherein the coenzyme-dependent dehydrogenase stabilized with a stable artificial coenzyme is stored at a relative humidity of at least 50%.
7. The method according to claim 1 , wherein the storage of the coenzyme-dependent dehydrogenase stabilized with a stable artificial coenzyme takes place as dry substance.
8. The method according to claim 1 , wherein the storage of the coenzyme-dependent dehydrogenase stabilized with a stable artificial coenzyme takes place in liquid phase.
9. The method according to claim 1 , wherein the storage of the coenzyme-dependent dehydrogenase stabilized with a stable artificial coenzyme takes place on a test element.
10. The method according to claim 1 , wherein the mixture of the coenzyme-dependent dehydrogenase stabilized with the stable artificial coenzyme—for a period of at least 4 weeks.
11. The method according to claim 1 , wherein the mixture of the coenzyme-dependent dehydrogenase stabilized with the stable artificial coenzyme—for a period of at least 8 weeks.
12. The method according to claim 1 , wherein the mixture of the coenzyme-dependent dehydrogenase stabilized with the stable artificial coenzyme—at a temperature of at least 30° C.
13. The method according to claim 1 , wherein the stored coenzyme-dependent dehydrogenase retains at least 70% of its initial enzyme activity in the presence of the stable artificial coenzyme.