Stabilization of enzymes with stable coenzymes

Patent metadata

Field	Value
Publication number	US-9540702-B2
Application number	US-201213400209-A
Country	US
Kind code	B2
Filing date	Feb 20, 2012
Priority date	Aug 20, 2009
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

Methods for stabilizing an enzyme by storing the enzyme in the presence of a stabilized coenzyme are disclosed. In addition, an enzyme stabilized with a stabilized coenzyme as well as the use thereof in test elements for detecting analytes are also disclosed. Other aspects include unique compositions, methods, techniques, systems and devices involving enzyme stabilization.

First claim

What is claimed is:

1. A method for stabilizing an enzyme, which comprises:
storing the enzyme in a presence of a stabilized coenzyme, or a salt form thereof, or a reduced form thereof for a period of storage of at least 2 weeks at a temperature of at least 20° C., wherein the enzyme is selected from the group consisting of an alcohol dehydrogenase (EC 1.1.1.2), a glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and a diaphorase (EC 1.6.99.2), wherein the stabilized coenzyme is a compound of general formula (II):

in which
A=adenine or an analogue thereof;
T=in each case independently denotes O or S;
U=in each case independently denotes OH, SH, BH 3 − , or BCNH 2 − ;
V=in each case independently denotes OH or a phosphate group, or two groups which form a cyclic phosphate group;
W=COOR, CON(R) 2 , COR, or CSN(R) 2 in which R in each case independently denotes H or a C 1 -C 2 alkyl;
X 1 , X 2 =in each case independently denote O, CH 2 , CHCH 3 , C(CH 3 ) 2 , NH, or NCH 3 ;
Y=NH, S, O, or CH 2 ;
Z=a linear residue with 4-6 C atoms or a saturated or an unsaturated carbocyclic or heterocyclic 5-membered ring of general formula (III):

in which a single or double bond may be present between R 5′ and R 5″ , where
R 4 =in each case independently denotes H, F, Cl, or CH 3 ;
R 5 =CR 4 2 ;
R 5′ =O, S, NH, NC 1 -C 2 -alkyl, CR 4 , CHOH, or CHOCH 3 , and R 5″ =CR 4 2 , CHOH, or CHOCH 3 if there is a single bond between R 5′ and R 5″ ;
R 5′ =R 5″ =CR 4 if there is a double bond between R 5′ and R 5″ ; and
R 6 , R 6′ =in each case independently denote CH or CCH 3 ;
wherein the Z and the pyridine residue are not linked by a glycosidic bond, and
wherein enzyme activity decreases by less than 50% after the period of storage of at least 2 weeks at a temperature of at least 20° C. when compared to an initial value of enzyme activity.

2. The method of claim 1 , wherein the stabilized coenzyme comprises carbaNAD.

3. The method of claim 1 , wherein the period is at least four weeks.

4. The method of claim 1 , wherein the period is at least eight weeks.

5. The method of claim 1 , wherein the temperature is at least 25° C.

6. The method of claim 1 , wherein the temperature is at least 30° C.

7. The method of claim 1 , which includes storing the enzyme in the presence of the stabilized coenzyme with at least one of a relative air humidity of at least 50% and an absence of desiccants.

8. The method of claim 1 , which includes storing the enzyme in the presence of the stabilized coenzyme as a dry substance or in a liquid phase.

9. The method of claim 1 , which further includes storing the enzyme in the presence of light at a wavelength of 300 nm.

10. The method of claim 1 ,
wherein enzyme activity decreases by less than 50% after a period of storage of at least 2 weeks at a temperature of at least about 20° C. in a presence of light at a wavelength of ≧300 nm when compared to an initial value of enzyme activity.

11. The method of claim 10 , wherein the native coenzyme is one of a native nicotinamide adenine dinucleotide (NAD/NADH) compound and a native nicotinamide adenine dinucleotide phosphate (NADP/NADPH) compound.

Assignees

• Roedel Wolfgang
• Horn Carina
• Steinke Nelli
• Bucci Nadine
• Meier Thomas
• Schmuck Rainer
• Nagel Rolf
• Heindl Dieter
• Roche Diabetes Care Inc

Inventors

• Roedel Wolfgang
• Horn Carina
• Steinke Nelli
• Bucci Nadine
• Meier Thomas
• Schmuck Rainer
• Nagel Rolf
• Heindl Dieter

Classifications

• C12Y101/01047

  Glucose 1-dehydrogenase (1.1.1.47) · CPC title

• C12N9/0006

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

• C12Y101/01002

  Alcohol dehydrogenase (NADP+) (1.1.1.2), i.e. aldehyde reductase · CPC title

• C12Y101/01001 Primary

  Alcohol dehydrogenase (1.1.1.1) · CPC title

• C12Y106/05002

  NAD(P)H dehydrogenase (quinone) (1.6.5.2) · CPC title