Creatine amidinohydrolase, production thereof and use thereof

Abstract

A creatine amidinohydrolase having the following physicochemical properties: Action: catalyzing the following reaction; creatine+H 2 O→sarcosine+urea Optimum temperature: about 40-50° C. Optimum pH: pH about 8.0-9.0 Heat stability: not more than about 50° C. (pH 7.5, 30 min) Km value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: about 3.5-10.0 mM Molecule weight: about 43,000 (SDS-PAGE) Isoelectric point: about 3.5 4.5, a method for producing said enzyme, comprising culture of microorganism producing said enzyme, a method for the determination of creatine or creatinine in a sample using said enzyme, and a reagent therefor.

1. A creatine amidinohydrolase having the following physicochemical properties:
 Action: catalyzing the following reaction; 
 creatine+H 2 O→sarcosine+urea  
   Optimum temperature: about 40-50° C.    Optimum pH: pH about 8.0-9.0    K m  value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: 3.5-10.0 mM    Molecular weight: about 43,000 (SDS-PAGE)    Isoelectric point: about 3.5.    
     
     
       2. A creatine amidinohydrolase having the following physicochemical properties:
 Action: catalyzing the following reaction; 
 creatine+H 2 O→sarcosine+urea  
   Optimum temperature: about 40-50° C.    Optimum pH: about 8.0-9.0    K m  value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: 4.5±1.0 mM    Molecular weight: about 43,000 (SDS-PAGE)    Isoelectric point: about 3.5.    
     
     
       3. The creatine amidinohydrolase of  claim 2 , which is obtained from Escherichia coli JM109 (pCRH273M2) (FERM BP-5375). 
     
     
       4. A creatine amidinohydrolase having the following physiocochemical properties:
 Action: catalyzing the following reaction; 
 creatine+H 2 O→sarcosine+urea  
   Optimum temperature: about 40-50° C.    Optimum pH: pH about 8.0-9.0    K m  value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: 6.5±1.0 mM    Molecular weight: about 43,000 (SDS-PAGE)    Isoelectric point: about 3.5.    
     
     
       5. The creatine amidinohydrolase of  claim 4 , which is obtained from Escherichia coli JM109 (pCRH273M1) (FERM BP-5374). 
     
     
       6. A creatine amidinohydrolase having the following physicochemical properties:
 Action: catalyzing the following reaction; 
 creatine+H 2 O→sarcosine+urea  
   Optimum temperature: about  40 - 50 ° C.    Optimum pH: pH about  8 . 0 - 9 . 0     K m  value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: 9.0±1.0 mM    Molecular weight: about 43,000 (SDS-PAGE)    Isoelectric point: about 3.5.    
     
     
       7. The creatine amidinohydrolase of  claim 6 , which is obtained from Escherichia coli JM109 (pCRH273M3) (FERM BP-5376). 
     
     
       8. A method for producing the creatine amidinohydrolase of  claim 1 , comprising culturing a microorganism producing said creatine amidinohydrolase in a nutrient medium and recovering said creatine amidinohydrolase from the resulting culture. 
     
     
       9. The method of  claim 8 , wherein said microorganism is selected from the group consisting of Escherichia coli JM109 (pCRH273M1) (FERM BP-5374), Escherichia coli JM109 (pCRH273M2) (FERM BP-5375), and Escherichia coli JM109 (pCRH273M3) (FERM BP-5376). 
     
     
       10. A reagent for determination of creatine in a sample, comprising the creatine amidinohydrolase of  claim 1 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       11. The reagent of  claim 10 , in which the composition for the detection of hydrogen peroxide comprises an enzyme having a peroxidase activity, a chromophore, and a buffer. 
     
     
       12. The reagent of  claim 11 , in which the enzyme having the peroxidase activity is selected from the group consisting of peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase, and myeloperoxidase. 
     
     
       13. The reagent of  claim 11 , in which the chromophore comprises a hydrogen receptor and a coupler. 
     
     
       14. The reagent of  claim 13 , in which the hydrogen receptor is 4-aminoantipyrine or a 3-methyl-2-benzothiazoline-hydrazine derivative. 
     
     
       15. The reagent of  claim 13 , in which the coupler is an aniline derivative or a phenol derivative. 
     
     
       16. A method for determining creatine in a sample, which comprises measuring absorbance of the pigment produced by the reaction of the reagent of  claim 10  with the sample. 
     
     
       17. A reagent for determination of creatinine in sample, comprising a creatinine amidohydrolase, the creatine amidinohydrolase of  claim 1 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       18. The reagent of  claim 17 , in which the composition for the detection of hydrogen peroxide comprises and enzyme having a peroxidase activity, a chromophore, and a buffer. 
     
     
       19. The reagent of  claim 18 , in which the enzyme having the peroxidase activity is selected from the group consisting of peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase and myeloperoxidase. 
     
     
       20. The reagent of  claim 18 , in which the chromophore comprises a hydrogen receptor and a coupler. 
     
     
       21. The reagent of  claim 20 , in which the hydrogen receptor is 4-aminoantipyrine or a 3-methyl-2-benzothiazoline-hydrozine derivative. 
     
     
       22. The reagent of  claim 20 , in which the coupler is an aniline derivative or a phenol derivative. 
     
     
       23. A method for determining creatinine in a sample, which comprises measuring absorbance of the pigment produced by the reaction of the reagent of  claim 17  with the sample. 
     
     
       24. A creatine amidinohydrolase ( i )  encoded by a nucleic acid sequence obtained by mutating  ( a )  the nucleic acid sequence of SEQ ID NO: 2  or  ( b )  a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1  and  ( ii )  having the following physicochemical properties:      Action: catalyzing the following reaction:      creatine+H   2   O→sarcosine+urea          Km values for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  3 . 5 - 10 . 0  mM        Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7 . 5   )      Optimum pH: pH about  8 . 0 - 9 . 0   ( at a temperature of about  37 ° C. )      Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point of  4 . 5 .      
     
     
       25. A creatine amidinohydrolase ( i )  encoded by a nucleic acid sequence obtained by mutating  ( a )  the nucleic acid sequence of SEQ ID NO: 2  or  ( b )  a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1  and  ( ii )  having the following physicochemical properties:      Action: catalyzing the following reaction:      creatine+H   2   O→sarcosine+urea          Km values for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  4 . 5 ± 1 . 0  mM        Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7 . 5   )      Optimum pH: pH about  8 . 0 - 9 . 0   ( at a temperature of about  37 ° C. )      Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       26. A creatine amidinohydrolase ( i )  encoded by a nucleic acid sequence obtained by mutating  ( a )  the nucleic acid sequence of SEQ ID NO: 2  or  ( b )  a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1  and  ( ii )  having a following physicochemical properties:      Action: catalyzing the following reaction:      creatine+H   2   O→sarcosine+urea          Km values for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  6 . 5 ± 1 . 0  mM        Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7.5)      Optimum pH: pH about  8 . 0 - 9 . 0   ( at a temperature of about  37 ° C. )      Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       27. A creatine amidinohydrolase ( i )  encoded by a nucleic acid sequence obtained by mutating  ( a )  the nucleic acid sequence of SEQ ID NO: 2  or  ( b )  a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1  and  ( ii )  having the following physiocochemical properties:      Action: catalyzing the following reaction:      creatine+H   2   O→sarcosine+urea          Km values for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  9 . 0 ± 1 . 0  mM        Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7 . 5   )      Optimum pH: about  8 . 0 - 9 . 0   ( at a temperature of about  37 ° C. )      Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       28. A method for producing the creatine amidinohydrolase of  claim 24 , comprising culturing a microorganism producing said creatine amidinohydrolase in a nutrient medium and recovering said creatine amidinohydrolase from the resulting culture. 
     
     
       29. A reagent for determination of creatine in a sample, comprising the creatine amidinohydrolase of  claim 24 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       30. A method for determining a creatine in a sample, which comprises measuring absorbance of a pigment produced by the reaction of the reagent of  claim 29  with the sample. 
     
     
       31. A reagent for determination of creatinine in a sample, comprising a creatinine amidohydrolase, the creatine amidinohydrolase of  claim 24 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       32. A method for determining creatinine in a sample, which comprises measuring- absorbance of a pigment produced by the reaction of the reagent of    claim 31    with the sample.    
     
     
       33. A method for producing the creatine amidinohydrolase of  claim 25 , comprising culturing a microorganism producing said creatine amidinohydrolase in a nutrient medium and recovering said creatine amidinohydrolase from the resulting culture. 
     
     
       34. A reagent for determination of creatine in a sample, comprising the creatine amidinohydrolase of  claim 25 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       35. A method for determining creatine in a sample, which comprises measuring- absorbance of a pigment produced by the reaction of the reagent of    claim 34    with the sample.    
     
     
       36. A reagent for determination of creatinine in a sample, comprising a creatinine amidohydrolase, the creatine amidinohydrolase of  claim 25 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       37. A method for determining creatinine in a sample, which comprises measuring- absorbance of a pigment produced by the reaction of the reagent of    claim 36    with the sample.    
     
     
       38. A creatine amidinohydrolase having the following physicochemical properties:
   Action: catalyzing the following reaction;      creatine+H   2   O→sarcosine+urea          Optimum temperature: about  40 - 50 ° C. ( at pH of about  7 . 5   )      Optimum pH: pH about  8 . 0 - 9 . 0   ( at a temperature of about  37 ° C. )      K   m    value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  3 . 5 - 10 . 0  mM        Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       39. A creatine amidinohydrolase having the following physicochemical properties:
   Action: catalyzing the following reaction;      creatine+H   2   O→sarcosine+urea          Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7 . 5   )      Optimum pH: about  8 . 0 - 9 . 0   ( at temperature of about  37 ° C. )      K   m    value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  4 . 5 ± 1 . 0  mM        Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       40. The creatine amidinohydrolase of  claim 39 , which is obtained from Escherichia coli JM 109  ( pCRH 273 M 2   )( FERM BP -   5375   ). 
     
     
       41. A creatine amidinohydrolase having the following physiocochemical properties:
   Action: catalyzing the following reaction;      creatine+H   2   O→sarcosine+urea          Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7 . 5   )      Optimum pH: pH about  8.0-9.0 ( at a temperature of about  37 ° C. )      K   m    value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  6 . 5 ± 1 . 0  mM        Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       42. The creatine amidinohydrolase of  claim 41 , which is obtained from Escherichia coli JM 109  ( pCRH 273 M 1   )( FERM BP -   5374   ). 
     
     
       43. A creatine amidinohydrolase having the following physicochemical properties:
   Action: catalyzing the following reaction;      creatine+H   2   O→sarcosine+urea          Optimum temperature: about  40 - 50 ° C.  ( at pH of about  7 . 5   )      Optimum pH: pH about  8 . 0 - 9 . 0   ( at a temperature of about  37 ° C. )      K   m    value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase:  9 . 0 ± 1 . 0  mM        Molecular weight: about  43 , 000   ( SDS - PAGE )      Isoelectric point: about  4 . 5 .      
     
     
       44. The creatine amidinohydrolase of  claim 43 , which is obtained from Escherichia coli JM 109  ( pCRH 273 M 3   )( FERM BP -   5376   ). 
     
     
       45. A method for producing the creatine amidinohydrolase of  claim 38 , comprising culturing a microorganism producing said creatine amidinohydrolase in a nutrient medium and recovering said creatine amidinohydrolase from the resulting culture. 
     
     
       46. The method of  claim 45 , wherein said microorganism is selected from the group consisting of Escherichia coli JM 109  ( pCRH 273 M 1   )( FERM BP -   5374   ),  Escherichia coli JM 109   ( pCRH 273 M 2   )( FERM BP -   5375   ),  and Escherichia coli JM 109   ( pCRH 273 M 3   )( FERM BP -   5376   ). 
     
     
       47. A reagent for determination of creatine in a sample, comprising the creatine amidinohydrolase of  claim 38 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       48. The reagent of  claim 47 , in which the composition for the detection of hydrogen peroxide comprises an enzyme having a peroxidase activity, a chromophore, and a buffer. 
     
     
       49. The reagent of  claim 48 , in which the enzyme having the peroxidase activity is selected from the group consisting of peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase, and myeloperoxidase. 
     
     
       50. The reagent of  claim 48 , in which the chromophore comprises a hydrogen receptor and a coupler. 
     
     
       51. The reagent of  claim 50 , in which the hydrogen receptor is  4 - aminoantipyrine or a  3   - methyl -   2   - benzothiazoline - hydrazine derivative.   
     
     
       52. The reagent of  claim 50 , in which the coupler is an aniline derivative or a phenol derivative. 
     
     
       53. A method for determining creatine in a sample, which comprises measuring absorbance of the pigment produced by the reaction of the reagent of  claim 47  with the sample. 
     
     
       54. A reagent for determination of creatinine in sample, comprising a creatinine amidohydrolase, the creatine amidinohydrolase of  claim 15 , a sarcosine oxidase, and a composition for the detection of hydrogen peroxide. 
     
     
       55. The reagent of  claim 54 , in which the composition for the detection of hydrogen peroxide comprises and enzyme having a peroxidase activity, a chromophore, and a buffer. 
     
     
       56. The reagent of  claim 55 , in which the enzyme having the peroxidase activity is selected from the group consisting of peroxidase, haloperoxidase, bromoperoxidase, lactoperoxidase, and myeloperoxidase. 
     
     
       57. The reagent of  claim 55 , in which the chromophore comprises a hydrogen receptor and a coupler. 
     
     
       58. The reagent of  claim 57 , in which the hydrogen receptor is  4 - aminoantipyrine or a  3   - methyl -   2   - benzothiazoline - hydrozine derivative.   
     
     
       59. The reagent of  claim 57 , in which the coupler is an aniline derivative or a phenol derivative.  
     
     
       60. A method for determining creatinine in a sample, which comprises measuring absorbance of the pigment produced by the reaction of the reagent of  claim 54  with the sample.