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 50° C. (pH 7.5, 30 min) Km value for creatine in a coupling assay using a sarcosine oxidase and a peroxidase: about 3.5-10.00 mM Molecular weight: about 43,000 (SDS-PAGE) Isoelectric point: 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: 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 Escherchia coli JM109 (pCRH273M2) (FERM BP-5375). 
     
     
       4. 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: 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 Escherchia 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 Escherchia 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 reaction 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 the 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 a 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 an 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-hydrazine 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 the absorbance of the pigment produced by the reaction of the reagent of  claim 17  with the sample. 
     
     
       24. A method of preparing a creatine amidinohydrolase comprising:
 ( i )  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  to provide mutant nucleic acid sequences,      ( ii )  determining Km values for creatine of proteins encoded by the mutant nucleic acid sequences in a coupling assay using a sarcosine oxidase and a peroxidase,      ( iii )  selecting and isolating a desired mutant nucleic acid sequence that encodes a creatine amidinohydrolase 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,    
   Molecular weight: about  43 , 000   ( SDS - PAGE )  
   Isoelectric point: about  4 . 5     
     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. )    ( iv )  expressing the desired mutant nucleic acid sequence in a host to produce creatine amidinohydrolase, and      ( v )  harvesting the produced creatine amidinohydrolase.      
     
     
       25. The method of  claim 24 , wherein the sarcosine oxidase is originated from the genus Arthrobacter, Corynebacterium, Alcaligenes, Pseudomonas, Micrococcus, or Bacillus.  
     
     
       26. A method of preparing a creatine amidinohydrolase comprising:
 ( i )  selecting  ( a )  a nucleic acid sequence of SEQ ID NO: 2  or  ( b )  a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1  to provide a source nucleic acid sequence,      ( ii )  mutating the source nucleic acid sequence to provide mutant nucleic acid sequences that encode mutant creatine amidinohydrolases,      ( iii )  selecting a mutant nucleic acid sequence that encodes a creatine amidinohydrolase which has a reduced Km value as compared to the Km value of creatine amidinohydrolase encoded by the source nucleic acid sequence by:  
 ( A )  determining a first activity of creatine amidinohydrolase encoded by the source nucleic acid sequence with a first concentration of creatine and a second activity of creatine amidinohydrolase encoded by the source nucleic acid sequence with a second concentration of creatine, wherein the second concentration of creatine is less than the first concentration of creatine,    
 ( B )  determining a first activity of the mutant creatine amidinohydrolase with the first concentration of creatine and a second activity of the mutant creatine amidinohydrolase with the second concentration of creatine, wherein the second concentration of creatine is less than the first concentration of creatine,    
 ( C )  calculating a ratio of the second activity of the creatine amidinohydrolase encoded by the source nucleic acid sequence divided by the first activity of the creatine amidinohydrolase encoded by the source nucleic acid sequence,    
 ( D )  calculating a ratio of the second activity of the mutant creatine amidinohydrolase divided by the first activity of the mutant creatine amidinohydrolase,    
 ( E )  comparing the ratio calculated in step  ( iii )( C )  to the ratio calculated in step  ( iii )( D ) , wherein a mutant creatine amidinohydrolase that has a reduced Km value as compared to the Km value of creatine amidinohydrolase encoded by the source nucleic acid sequence has a greater ratio than the ratio for creatine amidinohydrolase encoded by the source nucleic acid,    
   ( iv )  selecting and isolating a desired mutant nucleic acid sequence that encodes a creatine amidinohydrolase 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,    
   Molecular weight: about  43 , 000   ( SDS - PAGE )  
   Isoelectric point: about  4 . 5     
   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. )  
   ( v )  expressing the desired mutant nucleic acid sequence in a host to produce creatine amidinohydrolase; and      ( vi )  harvesting the produced creatine amidinohydrolase.