Beta -fructofuranosidase and its gene, method of isolating beta -fructofuranosidase gene, system for producing beta -fructofuranosidase, and beta -fructofuranosidase variant

Abstract

A novel β-fructofuranosidase gene and a β-fructofuranosidase encoded by the gene, a process for isolating a β-fructofuranosidase gene using the novel β-fructofuranosidase gene, and a novel β-fructofuranosidase obtained by this isolation process are disclosed. A novel mold fungus having no β-fructofuranosidase activity suitable for the production of β-fructofuranosidase, and a system for producing a recombinant β-fructofuranosidase using the novel mold fungus as a host is disclosed. Further, a β-fructofuranosidase variant which selectively and efficiently produces a specific fructooligosaccharide such as 1-kestose from sucrose is disclosed.

What is claimed is:  
     
         1 . A DNA fragment comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID No. 1 or a homologue thereof.  
     
     
         2 . A DNA fragment according to  claim 1  comprising the nucleotide sequence of SEQ ID No. 2.  
     
     
         3 . A DNA encoding the amino acid sequence of SEQ ID No. 1 or a homologue thereof.  
     
     
         4 . A DNA according to  claim 3  comprising the nucleotide sequence of SEQ ID No. 2.  
     
     
         5 . A polypeptide comprising the amino acid sequence of SEQ ID No. 1 or a homologue thereof.  
     
     
         6 . A recombinant plasmid wherein a DNA according to  claim 3  or 4 is integrated into the plasmid vector.  
     
     
         7 . A host cell transformed by a recombinant plasmid according to  claim 6 .  
     
     
         8 . A process for producing a β-fructofuranosidase comprising: 
 cultivating a host cell according to  claim 7 , and  
 collecting the β-fructofuranosidase from the host and/or the culture thereof.  
 
     
     
         9 . A process for producing fructooligosaccharides comprising a step of bringing into contact with sucrose a host cell according to  claim 7  or β-fructofuranosidase obtained in  claim 8 .  
     
     
         10 . A process for isolating a β-fructofuranosidase gene by making use of the homology thereof to a nucleotide sequence comprising all or part of the nucleotide sequence of SEQ No. 2.  
     
     
         11 . A process according to  claim 10  comprising: 
 preparing a gene library which presumably contains a β-fructofuranosidase gene,  
 screening the gene library using a nucleotide sequence comprising all or part of the nucleotide sequence of SEQ ID No. 2 to select sequences which hybridize with the nucleotide sequence comprising all or part of the nucleotide sequence of SEQ ID No. 2 from the gene library, then isolating the selected sequences, and  
 isolating a β-fructofuranosidase gene from the sequences which have been selected and isolated from the gene library.  
 
     
     
         12 . A process according to  claim 11  wherein the gene library is a genomic DNA library or a cDNA library.  
     
     
         13 . A process according to  claim 10  comprising: 
 preparing a primer consisting of a nucleotide sequence which comprises all or part of the nucleotide sequence of SEQ ID No. 2,  
 carrying out PCR process on the primer using a sample which presumably contains a β-fructofuranosidase gene as a template, and  
 isolating a β-fructofuranosidase gene from the amplified PCR product.  
 
     
     
         14 . A process according to any one of  claims 11  to  13  wherein the gene library which presumably contains a β-fructofuranosidase gene or the sample which presumably contains a β-fructofuranosidase is derived from a Eumycetes species.  
     
     
         15 . A process according to  claim 14  wherein the Eumycetes species is an Aspergillus, Penicillium or Scopulariopsis species.  
     
     
         16 . A polypeptide comprising the amino acid sequence of SEQ ID No. 11 or a homologue thereof.  
     
     
         17 . A DNA encoding a polypeptide according to  claim 16 .  
     
     
         18 . A DNA according to  claim 17  comprising the nucleotide sequence of SEQ ID No. 12.  
     
     
         19 . A polypeptide comprising the amino acid sequence of SEQ ID No. 13 or a homologue thereof.  
     
     
         20 . A DNA encoding a polypeptide according to  claim 19 .  
     
     
         21 . A DNA according to  claim 20  comprising the nucleotide sequence of SEQ ID No. 14.  
     
     
         22 . An Aspergillus mold fungus without β-fructofuranosidase activity.  
     
     
         23 . A mold fungus according to  claim 22  which has been deprived of β-fructofuranosidase activity by deleting all or part of the β-fructofuranosidase gene on the chromosome DNA of the original Aspergillus mold fungus.  
     
     
         24 . A mold fungus according to  claim 23  which is  Aspergillus niger.    
     
     
         25 . A mold fungus according to  claim 24  which is  Aspergillus niger  NIA1602 (FERM BP-5853).  
     
     
         26 . A process for producing a β-fructofuranosidase comprising: 
 transforming a mold fungus according to any one of  claims 22  to  25  using a DNA construction comprising a DNA encoding a β-fructofuranosidase,  
 cultivating the transformant, and  
 collecting the β-fructofuranosidase from the transformant and/or the culture thereof.  
 
     
     
         27 . A β-fructofuranosidase variant having fructosyltransferase activity obtained by a mutation in the original β-fructofuranosidase thereof, 
 wherein the mutation comprises an insertion, substitution or deletion of one or more amino acids in, or an addition to either or both of the terminals of, the amino acid sequence of the original β-fructofuranosidase, and  
 the variant makes the composition of the fructooligosaccharide mixture produced from sucrose as a result of fructosyltransfer reaction by the variant β-fructofuranosidase different from the composition of the fructooligosaccharide mixture produced by the original β-fructofuranosidase.  
 
     
     
         28 . A β-fructofuranosidase variant according to  claim 27  which improves the selectivity and/or efficiency of 1-kestose in the fructooligosaccharide mixture.  
     
     
         29 . A β-fructofuranosidase variant according to  claim 27  or  28  wherein the original β-fructofuranosidase is derived from a Eumycetes species.  
     
     
         30 . A β-fructofuranosidase variant according to  claim 29  wherein the original β-fructofuranosidase is derived from an Aspergillus, Penicillium, Scopulariopsis, Aureobasidium or Fusarium species.  
     
     
         31 . A β-fructofuranosidase variant according to  claim 30  wherein the original β-fructofuranosidase is the β-fructofuranosidase consisting of the amino acid sequence of SEQ ID No. 1 or a homologue thereof.  
     
     
         32 . A β-fructofuranosidase variant according to  claim 31 , wherein one or more amino acid residues at the positions selected from the group consisting of positions 170, 300, 313 and 386 in the amino acid sequence of SEQ ID No. 1, or, for a homologue of the amino acid sequence of SEQ ID No. 1, or one or more amino acid residues at the positions selected from the group consisting of the positions equivalent to the positions 170, 300, 313 and 386, are substituted by other amino acids.  
     
     
         33 . A β-fructofuranosidase variant according to  claim 32 , wherein amino acid residue at position 170 in the amino acid sequence of SEQ ID No. 1 or the amino acid residue at the position equivalent to position 170 is substituted by an aromatic amino acid selected from the group consisting of tryptophan, phenylalanine and tyrosine.  
     
     
         34 . A β-fructofuranosidase variant according to  claim 32 , wherein amino acid residue at position 300 in the amino acid sequence of SEQ ID No. 1 or the amino acid residue at the position equivalent to position 300 is substituted by an amino acid selected from the group consisting of tryptophan, valine, glutamic acid and aspartic acid.  
     
     
         35 . A β-fructofuranosidase variant according to  claim 32 , wherein amino acid residue at position 313 in the amino acid sequence of SEQ ID No. 1 or the amino acid residue at the position equivalent to position 313 is substituted by a basic amino acid selected from the group consisting of lysine, arginine and histidine.  
     
     
         36 . A β-fructofuranosidase variant according to  claim 32 , wherein amino acid residue at position 386 in the amino acid sequence of SEQ ID No. 1 or the amino acid reside at the position equivalent to position 386 is substituted by a basic amino acid selected from the group consisting of lysine, argine and histidine.  
     
     
         37 . A β-fructofuranosidase variant according to  claim 32 , wherein amino acid residues at positions 170, 300 and 313 in the amino acid sequence of SEQ ID No. 1 or the amino acid residues at the positions equivalent to positions 170, 300 and 313 are substituted by tryptophan, tryptophan and lysine, respectively.  
     
     
         38 . A β-fructofuranosidase variant according to  claim 32 , wherein amino acid residues at the positions 170, 300 and 313 in the amino acid sequence of SEQ ID No. 1 or the amino acid residues at the positions equivalent to positions 170, 300 and 313 are substituted by tryptophan, valine and lysine, respectively.  
     
     
         39 . A DNA encoding a variant β-fructofuranosidase according to any one of  claims 27  to  38 .  
     
     
         40 . A vector expressing a variant β-fructofuranosidase which comprises a DNA according to  claim 39 .  
     
     
         41 . A host cell comprising an expression vector according to  claim 40 .  
     
     
         42 . A host cell according to  claim 41  wherein the host cell is a mold fungus according to any one of  claims 22  to  25 .  
     
     
         43 . A process for producing a variant β-fructofuranosidase according to any one of  claims 27  to  38  comprising: 
 transforming a host cell using a DNA according to  claim 39  or an expressing vector according to  claim 40 ,  
 cultivating the transformant, and  
 collecting the β-fructofuranosidase from the transformant and/or the culture thereof.  
 
     
     
         44 . A process for producing a variant β-fructofuranosidase according to  claim 43  wherein the host cell is a mold fungus according to any one of  claims 22  to  25 .  
     
     
         45 . A process for producing fructooligosaccharides comprising bringing into contact with sucrose a host cell according to  claim 41  or  42  or a variant β-fructofuranosidase according to any one of  claims 27  to  38 .  
     
     
         46 . A mold fungus according to any one of  claims 22  to  25  transformed by a DNA fragment or a DNA according to any one of  claims 1  to  4 .  
     
     
         47 . A process for producing a β-fructofuranosidase comprising: 
 cultivating a mold fungus according to  claim 46 , and  
 collecting the β-fructofuranosidase from the mold fungus and/or the culture thereof.  
 
     
     
         48 . A mold fungus according to any one of  claims 22  to  25  transformed by a DNA according to  claim 17  or  20 .  
     
     
         49 . A process for producing a β-fructofuranosidase comprising: 
 cultivating a mold fungus according to  claim 48 , and  
 collecting the β-fructofuranosidase from the mold fungus and/or the culture thereof.