Inactivation Method

Abstract

The present invention relates to the field of thermostable enzymes, methods for their inactivation, and related uses, kits and reagents. In particular, the invention relates to a method for reversible inactivation of thermophilic enzymes by chemical modification under aqueous or non-aqueous conditions.

1 . A method for inactivating a thermostable enzyme, comprising reacting the enzyme with a dicarboxylic acid imide. 
     
     
         2 . A method according to  claim 1 , wherein the dicarboxylic acid imide comprises a compound of formula I: 
       
         
           
           
               
               
           
         
         wherein: 
         X is hydrogen or an electron withdrawing group; 
         Y is a linker group. 
       
     
     
         3 . A method according to  claim 2 , wherein:
 Y is alkylene, alkenylene, alkynylene, a bivalent carbocyclic ring, or a bivalent heterocyclic ring,   wherein alkylene, alkenylene, alkynylene, the carbocyclic ring, or the heterocyclic ring:   a) optionally comprises one or more heteroatoms independently selected from N, O and S;   b) is optionally modified with one or more substituents independently selected from the following groups: alkyl, alkenyl, alkynyl, alkoxy, alkoxyalkyl, alkoxycarbonyl, alkoxycarbonylalkyl, acyl, acyloxy, acyloxyalkyl, amino, alkylamino, carboxy, cyano, thiocyano, halogen, hydroxy, nitro, acylamino, aminocarbonyl, carboxyamino;   c) is optionally modified with one or more further substituents, attached either (i) to the same carbon atom or heteroatom, or (ii) to different carbon atoms or heteroatoms, wherein the further substituents, together with the carbon atoms or heteroatoms to which they are attached, form a carbocyclic ring, or a heterocyclic ring containing one or more heteroatoms independently selected from N, O and S.   
     
     
         4 . A method according to  claim 3 , wherein Y represents a radical of formula Ia or IIb: 
       
         
           
           
               
               
           
         
         wherein: 
         R 1 , R 2 , R 3  and R 4  are each independently hydrogen or alkyl; 
         or R 1  and R 2  together with the carbon atoms to which they are attached form a cycloalkylene, cycloalkenylene, cycloalkynylene or arylene group; 
         and each a is a bond linking the radical to a carbon atom of a carbonyl group shown in formula I. 
       
     
     
         5 .- 36 . (canceled) 
     
     
         37 . An inactivated thermostable enzyme obtainable by a method as defined in  claim 1 . 
     
     
         38 . A dicarboxylic acid imide-derivatised thermostable enzyme. 
     
     
         39 . An enzyme according to  claim 38 , wherein the enzyme is derivatised at one or more amino groups in the enzyme. 
     
     
         40 . An enzyme as defined in  claim 39 , wherein the derivatised enzyme comprises a moiety of formula IIIa or IIIb: 
       
         
           
           
               
               
           
         
         wherein X and Y are as defined in any of  claims 2  to  5 ; and 
         β is a bond linking the moiety to the enzyme. 
       
     
     
         41 .- 58 . (canceled) 
     
     
         59 . A method for amplifying a nucleic acid sequence, comprising:
 a) contacting a dicarboxylic acid imide-derivatised nucleic acid polymerase with the nucleic acid sequence at a first temperature in a reaction mixture, wherein the derivatised nucleic acid polymerase is at least partially inactivated at the first temperature;   b) heating the reaction mixture to a second temperature higher than the first temperature, thereby reactivating the nucleic acid polymerase;   c) amplifying the nucleic acid sequence in the reaction mixture by means of the reactivated nucleic acid polymerase.   
     
     
         60 . A method according to  claim 59 , further comprising a step of adding nucleic acid primers to the reaction mixture at the first temperature. 
     
     
         61 . A method according to  claim 59 , wherein the pH of the reaction mixture is 8.3 to 8.7. 
     
     
         62 . A method according to  claim 59 , wherein the concentration of Mg 2+  ions in the reaction mixture is 1.5 to 5.0 mM. 
     
     
         63 .- 72 . (canceled) 
     
     
         73 . A kit comprising a dicarboxylic acid imide-derivatised thermostable nucleic acid polymerase and one or more components for performing a nucleic acid amplification reaction. 
     
     
         74 . A kit according to  claim 73 , wherein a component of the kit comprises a deoxynucleoside triphosphate. 
     
     
         75 . A kit according to  claim 74 , wherein a component comprises a mixture of deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate and deoxycytidine triphosphate. 
     
     
         76 .- 81 . (canceled) 
     
     
         82 . A reaction mixture comprising a dicarboxylic acid imide-derivatised thermostable nucleic acid polymerase and one or more components for performing a nucleic acid amplification reaction. 
     
     
         83 . (canceled) 
     
     
         84 . A compound of formula I: 
       
         
           
           
               
               
           
         
         wherein: 
         X is halogen, hydroxyl, alkoxycarbonyl, alkoxy, acyl or acyloxy; Y represents a radical of formula Ia or IIb: 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 1 , R 2 , R 3  and R 4  are each independently hydrogen or alkyl; 
         or R 1  and R 2  together with the carbon atoms to which they are attached form a cycloalkylene, cycloalkenylene, cycloalkynylene or arylene group; 
         and each α is a bond linking the radical to a carbon atom of a carbonyl group shown in formula I. 
       
     
     
         85 . A compound according to  claim 84 , wherein X is C 1-6 alkoxycarbonyl. 
     
     
         86 . A compound according to  claim 84 , wherein:
 (i) Y represents a radical of formula IIa and R 1  to R 4  are each independently C 1-4 alkyl or hydrogen, or (ii) Y represents a radical of formula IIb, and R 1  and R 2  are each independently hydrogen or C 1-4 alkyl, or R 1  and R 2  together with the carbon atoms to which they are attached form a 5-7 membered cycloalkylene, cycloalkenylene or arylene group.   
     
     
         87 . A compound according to  claim 86 , which is N-ethoxycarbonyl-2,3-dimethylmaleimide, N-iso-butoxycarbonyl-2,3-dimethylmaleimide, N-ethoxycarbonyl-3,4,5,6-tetrahydrophthalimide, N-ethoxycarbonyl-1,2,3,6-tetrahydrophthalimide, N-ethoxycarbonyl-maleimide or N-ethoxycarbonyl-succinimide. 
     
     
         88 . (canceled)