Novel fructosyl peptide oxidase

Abstract

The present invention has an object of providing a novel fructosyl peptide oxidase having superior physicochemical properties such as stability that is useful as an enzyme for clinical diagnosis, and an object of providing a method for producing the fructosyl peptide oxidase. A novel fructosyl peptide oxidase having physicochemical properties useful as an enzyme for clinical diagnosis, and a method for producing a novel fructosyl peptide oxidase are provided herein, the method comprising: culturing a microorganism capable of producing the oxidase in a medium; and collecting the oxidase from the culture. Furthermore, a fructosyl peptide oxidase gene coding for a novel fructosyl peptide oxidase, recombinant DNA wherein the gene is inserted into vector DNA, and a method for producing a novel fructosyl peptide oxidase are provided herein, the method comprising: culturing, in a medium, a transformant or a transductant including the gene; and collecting the novel fructosyl peptide oxidase from the culture.

1 . A fructosyl peptide oxidase which acts on fructosyl valyl histidine in the presence of oxygen and catalyzes a reaction that produces α-ketoaldehyde, valyl histidine and hydrogen peroxide. 
     
     
         2 . The fructosyl peptide oxidase of  claim 1 , wherein an activity of 80% or higher remains after a heat treatment at 45° C. for 10 minutes. 
     
     
         3 . The fructosyl peptide oxidase of  claim 1  which has a molecular weight of about 52,000 Da (SDS-PAGE). 
     
     
         4 . The fructosyl peptide oxidase of  claim 1  which:
 (a) acts on fructosyl valyl histidine in the presence of oxygen and catalyzes a reaction that produces a-ketoaldehyde, valyl histidine and hydrogen peroxide;   (b) exhibits optimal activity within the range of pH 6.0-8.0;   (c) is active within the temperature range of 20-45° C.;   (d) retains at least 80% of its activity following a heat treatment at 45° C. for 10 minutes;   (e) is stable within the range of pH 6.0-9.0; and   (f) has a molecular weight of about 52,000 Da (SDS-PAGE).   
     
     
         5 . A method for producing a fructosyl peptide oxidase comprising:
 culturing a filamentous fungus that produces the fructosyl peptide oxidase of  claim 1  in a medium; and   recovering the fructosyl peptide oxidase.   
     
     
         6 . The method of  claim 5 , wherein the filamentous fungus is selected from a group consisting of  Achaetomiella, Achaetomium, Thielavia, Chaetomium, Gelasinospora, Microascus, Coniochaeta  and  Eupenicillium.    
     
     
         7 . The method of  claim 6 , wherein the filamentous fungus is  Achaetomiella virescens  ATCC 32393 or  Chaetomium  is  Chaetomium  sp. NISL 9335 (FERM BP-7799). 
     
     
         8 . The fructosyl peptide oxidase of  claim 1 , which has less activity on ε-fructosyl lysine than it does on fructosyl valyl histidine or fructosyl glycine. 
     
     
         9 . The fructosyl peptide oxidase of  claim 8 , which retains an activity of 80% or higher after a heat treatment at 45° C. for 10 minutes. 
     
     
         10 . The fructosyl peptide oxidase of  claim 8  which:
 (a) exhibits an optimal activity within the range of pH 6.0-8.0;   (b) is active within a temperature range of 20-40° C.;   (c) retains an activity of 80% or higher following a heat treatment at 45° C. for 10 minutes; and   (d) is stable within a range of pH 6.0-9.0.   
     
     
         11 . A method for producing a fructosyl peptide oxidase comprising:
 culturing a filamentous fungus that produces the fructosyl peptide oxidase of  claim 8  in a medium; and   recovering the fructosyl peptide oxidase.   
     
     
         12 . The method of  claim 11 , wherein the filamentous fungus belongs to  Eupenicillium  or  Coniochaeta.    
     
     
         13 . The method of  claim 12 , wherein the filamentous fungus is selected from the group consisting of  Eupenicillium terrenum  ATCC 18547 , Eupenicillium senticosum  IFO 9158 , Eupenicillium idahoense  IFO 9510 , Eupenicillium euglaucum  TO 31729, and  Coniochaeta  sp. NISL 9330 (FERM BP-7798). 
     
     
         14 . A protein having a fructosyl peptide oxidase activity selected from the group consisting of:
 (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 1;   (b) a protein comprising an amino acid sequence having deletion, substitution and/or addition of one to several amino acids relative to the amino acid sequence represented by SEQ ID NO: 1, and having a fructosyl peptide oxidase activity; and   (c) a protein comprising an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 1, and having a fructosyl peptide oxidase activity.   
     
     
         15 . A nucleic acid selected from the group consisting of:
 (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 1;   (b) a protein comprising an amino acid sequence having deletion, substitution and/or addition of one to several amino acids relative to the amino acid sequence represented by SEQ ID NO: 1, and having a fructosyl peptide oxidase activity; and   (c) a protein comprising an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 1, and having a fructosyl peptide oxidase activity.   
     
     
         16 . A nucleic acid selected from the group consisting of:
 (a) DNA comprising a nucleotide sequence represented by SEQ ID NO: 2;   (b) DNA which hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to a full-length or 15 or more consecutive bases of the DNA comprising the nucleotide sequence represented by SEQ ID NO: 2; and   (c) DNA which has at least 80% homology with a full-length or 15 or more consecutive bases of the DNA comprising the nucleotide sequence represented by SEQ ID NO: 2.   
     
     
         17 . A DNA vector comprising the nucleic acid of  claim 15 . 
     
     
         18 . A host cell transformed or transduced with the DNA vector of  claim 17 . 
     
     
         19 . A method for producing a fructosyl peptide oxidase, comprising:
 culturing host cell of  claim 18  in a medium; and   recovering the fructosyl peptide oxidase.   
     
     
         20 . A protein having a fructosyl peptide oxidase activity selected from the group consisting of:
 (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 3;   (b) a protein comprising an amino acid sequence having deletion, substitution and/or addition of one to several amino acids relative to the amino acid sequence represented by SEQ ID NO: 3, and having a fructosyl peptide oxidase activity; and   (c) a protein comprising an amino acid sequence having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 3, and having a fructosyl peptide oxidase activity.   
     
     
         21 . A nucleic acid which encodes:
 (a) a protein comprising an amino acid sequence represented by SEQ ID NO: 3;   (b) a protein comprising an amino acid sequence having deletion, substitution and/or addition of one to several amino acids relative to the amino acid sequence represented by SEQ ID NO: 3; or   (c) a protein having at least 80% homology with the amino acid sequence represented by SEQ ID NO: 3.   
     
     
         22 . A nucleic acid comprising:
 (a) DNA comprising a nucleotide sequence represented by SEQ ID NO: 4;   (b) DNA which hybridizes under stringent conditions with DNA comprising a nucleotide sequence complementary to a full-length or 15 or more consecutive bases of the DNA comprising the nucleotide sequence represented by SEQ ID NO: 4or   (c) DNA which has at least 80% homology with a full-length or 15 or more consecutive bases of the DNA comprising the nucleotide sequence represented by SEQ ID NO: 4.   
     
     
         23 . A DNA vector comprising the nucleic acid of  claim 21 . 
     
     
         24 . A host cell that has been transformed or transduced with the recombinant DNA vector of  claim 23 . 
     
     
         25 . A method for producing a fructosyl peptide oxidase comprising:
 culturing the host cell of  claim 24  in a medium; and   recovering a fructosyl peptide oxidase encoded by said recombinant DNA vector.   
     
     
         26 . An isolated or purified polypeptide selected from the group consisting of:
 (x) a polypeptide comprising the entire amino acid sequence of SEQ ID NO: 1;   (y) a polypeptide comprising SEQ ID NO: 1, but having the deletion, substitution and/or addition of one to twenty amino acids relative to the amino acid sequence of SEQ ID NO: 1; and   (z) a polypeptide comprising an amino acid sequence having at least 80% homology with the amino acid sequence of SEQ ID NO: 1.