Novel Taste-Modifying Polypeptide Nas, Dna Thereof and Use Thereof

Abstract

It is an object of the invention to find a substance with a better taste-modifying function and determine the structure of the taste-modifying substance, as well as to elucidate the structure thereof at a gene level and determine the primary structure of the substance and obtain the gene encoding the substance. Additionally, it is an object of the invention to provide a novel taste-modifying composition characteristically containing the taste-modifying substance. Specifically, the invention provides the polypeptide shown below in (A) or (B), a protein dimer neoculin comprising the polypeptide NAS and the polypeptide NBS and having a taste-modifying activity: (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with the polypeptide NBS.

1 . A polypeptide NAS shown below in (A) or (B):
 (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         2 . A polypeptide NAS according to  claim 1 , which is glycosylated with an N-linked sugar chain comprising mannose/N-acetylglucosamine/fucose/xylose at a ratio of 3/2/1/1. 
     
     
         3 . DNA of a gene encoding a polypeptide NAS shown below in (A) or (B):
 (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         4 . DNA of a gene according to  claim 3 , which is a DNA shown below in (A) or (B):
 (A) DNA containing the nucleotide sequence comprising the nucleotides 70 to 408 in the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing;   (B) DNA hybridizing with the DNA of a nucleotide sequence comprising the nucleotides 70 to 408 in the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing or with the DNA of a nucleotide sequence capable of functioning as a probe prepared from at least a part of the nucleotide sequence under stringent conditions and encoding a polypeptide capable of forming the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         5 . A polypeptide PNAS shown below in (A) or (B):
 (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.3 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.3 in the sequence listing, which polypeptide grows to the mature polypeptide NAS via processing so as to be able to form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         6 . A polypeptide PNAS according to  claim 5 , which is glycosylated with an N-linked sugar chain comprising mannose/N-acetylglucosamine/fucose/xylose at a ratio of 3/2/1/1. 
     
     
         7 . DNA of a gene encoding a polypeptide PNAS shown below in (A) or (B):
 (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.3 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.3 in the sequence listing, which polypeptide grows to the mature polypeptide NAS via processing so as to be able to form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         8 . DNA of a gene according to  claim 7 , which is a DNA shown below in (A) or (B):
 (A) DNA containing a nucleotide sequence comprising the nucleotides 4 to 477 in the nucleotide sequence shown in SEQ ID NO. 1 in the sequence listing;   (B) DNA hybridizing with the DNA of the nucleotide sequence comprising the nucleotides 4 to 477 in the nucleotide sequence shown in SEQ ID NO.1 in the sequence listing or with the DNA of a nucleotide sequence capable of functioning as a probe prepared from at least a part of the nucleotide sequence under stringent conditions and encoding a polypeptide growing to the mature polypeptide NAS via processing so as to be able to form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         9 . A dimeric protein neoculin comprising a polypeptide NAS according to  claim 1  and a polypeptide NBS shown below in (a) or (b) and having a taste-modifying activity:
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         10 . A taste-modifying composition containing the dimeric protein neoculin according to  claim 9  as the active ingredient. 
     
     
         11 . A recombinant vector carrying a nucleotide sequence constituting the DNA according to  claim 3 . 
     
     
         12 . A recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         13 . A recombinant vector according to  claim 11 , which is a vector functioning in eukaryotic organisms. 
     
     
         14 . A recombinant vector according to  claim 12 , which is a vector functioning in eukaryotic organisms. 
     
     
         15 . A recombinant vector according to  claim 11 , which is a vector functioning in filamentous fungi. 
     
     
         16 . A recombinant vector according to  claim 12 , which is a vector functioning in filamentous fungi. 
     
     
         17 . A recombinant vector according to  claim 11 , which is a vector functioning in koji molds. 
     
     
         18 . A recombinant vector according to  claim 12 , which is a vector functioning in koji molds. 
     
     
         19 . A transformant carrying a first recombinant vector according to  claim 11  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         20 . A method for producing a dimeric protein neoculin comprising:
 (i) a polypeptide NAS shown below in (A) or (B):   (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which Polypeptide can be a subunit constituting curculin; and   (ii) a polypeptide NBS shown below in (a) or (b), and having a taste-modifying activity:   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,   including a step of culturing a transformant according to  claim 19 .   
     
     
         21 . A dimeric protein neoculin comprising a polypeptide NAS according to  claim 2  and a polypeptide NBS shown below in (a) or (b) and having a taste-modifying activity:
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         22 . A recombinant vector carrying a nucleotide sequence constituting the DNA according to  claim 4 . 
     
     
         23 . A recombinant vector carrying a nucleotide sequence constituting the DNA according to  claim 7 . 
     
     
         24 . A recombinant vector carrying a nucleotide sequence constituting the DNA according to  claim 8 . 
     
     
         25 . A transformant carrying a first recombinant vector according to  claim 11  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,
 wherein the first and second recombinant vectors are vectors functioning in eukaryotic organisms. 
   
     
     
         26 . A method for producing a dimeric protein neoculin comprising:
 (i) a polypeptide NAS shown below in (A) or (B):   (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin; according to  claim 25  and   (ii) a polypeptide NBS shown below in (a) or (b), and having a taste-modifying activity:   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,   including a step of culturing a transformant according to  claim 25 .   
     
     
         27 . A transformant carrying a first recombinant vector according to  claim 11  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,
 wherein the first and second recombinant vectors are vectors functioning in filamentous fungi. 
   
     
     
         28 . A method for producing a dimeric protein neoculin comprising:
 (i) a polypeptide NAS shown below in (A) or (B):   (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin; and   (ii) a polypeptide NBS shown below in (a) or (b), and having a taste-modifying activity:   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,   including a step of culturing a transformant according to  claim 27 .   
     
     
         29 . A transformant carrying a first recombinant vector according to  claim 11  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,
 wherein the first and second recombinant vectors are vectors functioning in koji molds. 
   
     
     
         30 . A method for producing a dimeric protein neoculin comprising:
 (i) a polypeptide NAS shown below in (A) or (B):   (A) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.2 in the sequence listing;   (B) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.2 in the sequence listing, which polypeptide can form the neoculin dimer having a taste-modifying activity together with a polypeptide NBS shown below in (a) or (b):   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin; and   (ii) a polypeptide NBS shown below in (a) or (b), and having a taste-modifying activity:   (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin,   including a step of culturing a transformant according to  claim 29 .   
     
     
         31 . A transformant carrying first recombinant vector according to  claim 22  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         32 . A transformant carrying a first recombinant vector according to  claim 23  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.   
     
     
         33 . A transformant carrying a first recombinant vector according to  claim 24  and a second recombinant vector carrying a nucleotide sequence constituting the DNA of the gene encoding a polypeptide NBS shown below in (a) or (b):
 (a) a polypeptide comprising an amino acid sequence shown in SEQ ID NO.6 in the sequence listing;   (b) a polypeptide comprising an amino acid sequence with the substitution, deletion, insertion, addition or inversion of one or several amino acids in the amino acid sequence shown in SEQ ID NO.6 in the sequence listing, which polypeptide can be a subunit constituting curculin.