Novel beta-galactoside-alpha2,3-sialyltransferase, a gene encoding thereof, and a method for producing thereof

Abstract

The present invention provides a novel β-galactoside-α2,3-sialyltransferase and a nucleic acid encoding the sialyltransferase. The present invention also provides a microorganism producing the sialyltransferase, as well as a method for producing the sialyltransferase using such a microorganism. The present invention further provides a vector carrying a nucleic acid encoding the sialyltransferase, and a host cell transformed with the vector, as well as a method for producing a recombinant β-galactoside-α2,3-sialyltransferase. The present invention further provides a method for preparing a sialylsugar chain, which uses the sialyltransferase of the present invention.

1 . An isolated protein comprising an amino acid sequence selected from SEQ ID NO: 31 or amino acid residues 23-402 of
 SEQ ID NO: 31.   
     
     
         2 . An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which comprises an amino acid sequence comprising conservative substitution of one or more amino acids in, and sharing an amino homology of at least 90% or more with, an amino acid sequence selected from SEQ ID NO: 31, or amino acid residues 23-402 of SEQ ID NO: 31. 
     
     
         3 . An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which comprises an amino acid sequence sharing an amino acid homology of at least 90% or more with an amino acid sequence selected from SEQ ID NO: 31, or amino acid residues 23-402 of SEQ ID NO: 31. 
     
     
         4 . An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which is encoded by a nucleic acid comprising a nucleotide sequence selected from SEQ ID NO: 30, or nucleotides 67-1209 of SEQ ID NO: 30. 
     
     
         5 . An isolated protein having β-galactoside-α2,3-sialyltransferase activity, which is encoded by a nucleic acid comprising a nucleotide sequence hybridizable under conditions including hybridization at 65° C. in 0.5M sodium phosphate pH7.2, 1 mM EDTA, 7% SDS, 1% BSA, and washing at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 5% SDS, 0.5% BSA and at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 1% SDS, with the full length complementary stand of a nucleotide sequence of SEQ ID NO: 30. 
     
     
         6 . The isolated protein according to  claim 3 , which is obtained from a microorganism which belongs to Vibrionaceae. 
     
     
         7 . An isolated nucleic acid encoding a protein comprising an amino acid sequence selected from SEQ ID NO: 31 or amino acid residues 23-402 of SEQ ID NO: 31. 
     
     
         8 . An isolated nucleic acid encoding a protein having β-galactoside-α2,3-sialyltransferase activity, wherein the protein comprises an amino acid sequence comprising conservative substitution of one or more amino acids in, and sharing an amino acid homology of at least 90% or more with, an amino acid sequence selected from SEQ ID NO: 31 or amino acid residues 23-402 of SEQ ID NO: 31. 
     
     
         9 . An isolated nucleic acid encoding a protein having β-galactoside-α2,3-sialyltransferase activity, wherein the protein comprises an amino acid sequence sharing a homology of at least 90% with an amino acid sequence selected from SEQ ID NO: 31 or amino acid residues 23-402 of SEQ ID NO: 31. 
     
     
         10 . An isolated nucleic acid comprising a nucleotide sequence selected from SEQ ID NO: 30 or nucleotides 67-1209 of SEQ ID NO: 30. 
     
     
         11 . An isolated nucleic acid encoding a protein having β-galactoside-α2,3-sialyltransferase activity, wherein the nucleic acid comprises a nucleotide sequence hybridizable under conditions including hybridization at 65° C. in 0.5M sodium phosphate pH7.2, 1 mM EDTA, 7% SDS, 1% BSA, and washing at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 5% SDS, 0.5% BSA and at 65° C. in 40 mM sodium phosphate buffer pH 7.2, 1 mM EDTA, 1% SDS, with the full length complementary stand of a nucleotide sequence of SEQ ID NO: 30. 
     
     
         12 . A recombinant vector comprising the nucleic acid according to  claim 9 . 
     
     
         13 . An isolated host cell transformed with the recombinant vector according to  claim 12 . 
     
     
         14 . An isolated microorganism of the Vibrionaceae, which expresses the protein according to  claim 1 . 
     
     
         15 . The microorganism according to  claim 14 , which is  Vibrio  sp. strain JT-FAJ-16 (Accession No. NITE BP-98). 
     
     
         16 . A method for producing a recombinant protein having β-galactoside-α2,3-sialyltransferase activity, which comprises the following steps:
 1) transforming and insolating a host cell with a recombinant vector comprising the nucleic acid according to  claim 7 ; 
 2) culturing the transformed host cell; and 
 3) isolating the protein having β-galactoside-α2,3-sialyltransferase activity from the cultured host cell or the culture supernatant. 
 
     
     
         17 . A method for preparing a sialylated sugar chain, which comprises:
 (i) preparing a solution containing the protein according to  claim 1 , a glycosyl donor substrate and a glycosyl acceptor substrate;   (ii) causing sialic acid transfer reaction in the solution; and   (iii) obtaining the generated sialylated sugar chain from the reaction solution.   
     
     
         18 . The isolated protein of  claim 1 , which comprises SEQ ID NO: 31. 
     
     
         19 . The method of  claim 16 , wherein said nucleic acid encodes a protein comprising SEQ ID NO: 31.