Beta-hexosyl-transferases and uses thereof

Abstract

This invention relates generally to the discovery of novel recombinant forms of β-hexosyl-transferases (BHT) and uses thereof to produce galacto-oligosaccharides (GOS) or as food additives.

What is claimed is: 
     
       1. An isolated DNA encoding a recombinant β-hexosyl-transferase (rBHT) protein having the amino acid sequence set forth in SEQ ID NO: 12, 14, or 20. 
     
     
       2. The isolated DNA encoding the recombinant β-hexosyl-transferase (rBHT) protein of  claim 1  having the amino acid sequence set forth in SEQ ID NO: 12 or 14. 
     
     
       3. The isolated DNA encoding the recombinant β-hexosyl-transferase (rBHT) protein of  claim 1 , wherein the nucleotide sequence of the DNA has at least 97% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 11, 13, or 19. 
     
     
       4. The isolated DNA encoding the recombinant β-hexosyl-transferase (rBHT) protein of  claim 3 , wherein the nucleotide sequence of the DNA has at least 97% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 13 or 15. 
     
     
       5. A method for producing enzymatically active recombinant β-hexosyl-transferase (rBHT) protein in a cultured eukaryotic host cell which comprises transforming the eukaryotic host cell with a plasmid under the control of a suitable promotor wherein the plasmid contains an isolated DNA encoding a rBHT protein having the amino acid sequence set forth in SEQ ID NO: 12, 14, or 20 wherein the promoter is operably linked to the isolated DNA, and culturing the transformed cell under conditions such that the rBHT protein is produced, wherein the rBHT protein produced by the transformed cell has 3-hexosyl-transferase enzymatic activity. 
     
     
       6. The method of  claim 5 , wherein the enzymatically active rBHT protein has a specific activity of 8 U/mg at 20° C. 
     
     
       7. The method of  claim 5 , wherein the eukaryotic host cell is a yeast cell. 
     
     
       8. An isolated DNA A heterologous expression vector comprising a nucleotide sequence encoding a recombinant β-hexosyl-transferase (rBHT) protein, wherein the nucleotide sequence encoding the rBHT protein consists of the DNA has a sequence having at least 99.5% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 11, 13, or 19 28, and wherein the rBHT protein has β-hexosyl-transferase enzymatic activity. 
     
     
       9. The isolated DNA coding for the recombinant β-hexosyl-transferase (rBHT) protein of  claim 8 , wherein the nucleotide sequence of the DNA has at least 99.5% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 11 or 13. 
     
     
       10. The expression vector of claim 8, wherein the expression vector comprises a nucleic acid sequence encoding a signal peptide that is operably linked to the rBHT protein encoded by SEQ ID NO: 28 at the rBHT protein's N-terminus.  
     
     
       11. The expression vector of claim 10, wherein the signal peptide is selected from the group consisting of: a signal peptide from S. cerevisiae alpha mating factor (αMF), a signal peptide from interleukin-7 (IL-7), a signal peptide from interleukin-2 receptor (IL2RB), a signal peptide from interleukin-4 receptor (IL4RA), a signal peptide from type I interleukin-1 receptor (IL1R1), and a signal peptide from type II interleukin-1 receptor (IL1R2).  
     
     
       12. The expression vector of claim 8, wherein the expression vector comprises a nucleic acid sequence encoding an affinity tag that is operably linked to the rBHT protein encoded by SEQ ID NO: 28 at the rBHT protein's C-terminus.  
     
     
       13. The expression vector of claim 12, wherein the affinity tag is selected from the group consisting of: a polyhistidine (HIS) tag, a FLAG tag, a hemagluttanin (HA) tag, a maltose-binding protein (MBP) tag, a glutathione S transferase (GST) tag, a thioredoxin (Trx) tag, a polyHIS/NI or Co tag, a T7 tag, and a streptavidin tag.  
     
     
       14. The expression vector of claim 8, wherein the rBHT protein has at least 6-fold higher enzymatic activity compared to a corresponding full length rBHT protein.  
     
     
       15. A host cell comprising the expression vector of claim 8.   
     
     
       16. The host cell of claim 15, wherein the host cell is a eukaryotic cell.  
     
     
       17. The host cell of claim 16, wherein the host cell is a yeast cell.