Stabilization of dehydrogenases with stable coenzymes

Patent metadata

Field	Value
Publication number	US-9896666-B2
Application number	US-85965410-A
Country	US
Kind code	B2
Filing date	Aug 19, 2010
Priority date	Feb 19, 2008
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Horn Carina
• Heindl Dieter
• Gaessler Dietsche Claudia
• Hoenes Joachim
• Roche Diabetes Care Inc

Inventors

• Horn Carina
• Heindl Dieter
• Gaessler Dietsche Claudia
• Hoenes Joachim

Classifications

• C12N9/96 Primary

  Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates · CPC title

• C12N9/0006 Primary

  acting on CH-OH groups as donors (1.1) · CPC title

• C12Y101/01006

  Glycerol dehydrogenase (1.1.1.6) · CPC title

• C12N9/0016

  with NAD or NADP as acceptor (1.4.1) · CPC title

• C12Y101/01037

  Malate dehydrogenase (1.1.1.37) · CPC title