Compositions of enzymes stabilized with stable coenzymes

Abstract

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

The invention claimed is: 
     
         1 . A composition, comprising:
 an enzyme stabilized with a stabilized coenzyme, wherein the enzyme exhibits a decrease in enzyme activity of less than 50% based on an initial value of enzyme activity when stored for a period of time of at least 2 weeks in an environment that includes at least one of a temperature of at least 20° C. and a presence of light at a wavelength of ≧300 nm, and wherein the enzyme is selected from the group consisting of an alcohol dehydrogenase (EC I.1.1.2), a glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and a diaphorase (EC1.6.99.2).   
     
     
         2 . The composition of  claim 1 , wherein the decrease in enzyme activity is less than 30%. 
     
     
         3 . The composition of  claim 1 , wherein the decrease in enzyme activity is less than 20%, 
     
     
         4 . The composition of  claim 1 , wherein the period of time is at least four weeks. 
     
     
         5 . The composition of  claim 1 , wherein the period of time is at least eight weeks. 
     
     
         6 . The composition of  claim 1 , wherein the temperature is at least 25° C. 
     
     
         7 . The position of  claim 1 , wherein the temperature is at least 30° C. 
     
     
         8 . The composition of  claim 1 , wherein the enzyme further exhibits the decrease in enzyme activity of less than 50% when relative air humidity is at least 50%. 
     
     
         9 . The composition of  claim 1 , wherein the enzyme further exhibits the decrease in enzyme activity of less than 50% in the absence of desiccants. 
     
     
         10 . The composition of  claim 1 , wherein the enzyme further exhibits the decrease in enzyme activity of less than 50% in the absence of desiccants. 
     
     
         11 . The composition of  claim 1 , wherein the stabilized coenzyme is one of a stabilized nicotinamide adenine dinucleotide (NAD/NADH) compound, a stabilized nicotinamide adenine dinucleotide phosphate (NADP/NADPH) compound and a compound of formula (I): 
       
         
           
           
               
               
           
         
       
     
     
         12 . The composition of  claim 1 , wherein the stabilized coenzyme includes a compound of the 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 ; and 
         Z═a linear or cyclic organic residue; 
         provided that Z and the pyridine residue are not linked by a glycosidic bond; or a salt or optionally a reduced form thereof. 
       
     
     
         13 . The composition of  claim 1 , wherein Z is a saturated or unsaturated carbocyclic or heterocyclic 5-membered ring of the general formula (III): 
       
         
           
           
               
               
           
         
         in which 
         a single or double bond may be present between R 5′  and R 5″ , and 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 . 
       
     
     
         14 . The composition of  claim 1 , wherein the stabilized coenzyme includes a compound of the 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 ; and 
         Z═a linear or cyclic organic residue; 
         provided that Z and the pyridine residue are not linked by a glycosidic bond; or a salt or optionally a reduced form thereof. 
       
     
     
         15 . The composition of  claim 1 , wherein Z is a saturated or unsaturated carbocyclic or heterocyclic 5-membered ring of the general formula (III): 
       
         
           
           
               
               
           
         
         in which 
         a single or double bond may be present between R 5′  and R 5″ , and 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   2 , 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 . 
       
     
     
         16 . The composition of  claim 1 , wherein the stabilized coenzyme comprises carbaNAD. 
     
     
         17 . The composition of  claim 1 , 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″ , and 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 ; and 
         wherein the Z and the pyridine residue are not linked by a glycosidic bond; or a salt or optionally a reduced form thereof. 
       
     
     
         18 . A detection reagent for determining an analyte concentration or presence, the detection reagent comprising the composition of  claim 1 . 
     
     
         19 . A test element comprising one of the composition of  claim 1  and the detection reagent of  claim 18 .