Beta-hexosyl-transferases and uses thereof

Patent metadata

Field	Value
Publication number	US-9783789-B2
Application number	US-201314649819-A
Country	US
Kind code	B2
Filing date	Dec 9, 2013
Priority date	Dec 7, 2012
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

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

First claim

What is claimed is:

1. A method for producing enzymatically active recombinant β-hexosyl-transferase (rBHT) protein which comprises
transforming a Pichia pastoris 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: 4, 6, 8, 10, 12, 14, 16, 18 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 β-hexosyl-transferase enzymatic activity.

2. The method of claim 1 , wherein the rBHT protein comprises the amino acid sequence of SEQ ID NO: 12 or 14.

3. The method of claim 1 , wherein the nucleotide sequence of the isolated DNA has at least 97% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 or 19.

4. The method of claim 3 , wherein the nucleotide sequence of the isolated DNA has at least 97% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 11, 13, or 15.

5. The method of claim 1 , wherein the isolated DNA is linked to a DNA encoding a signal peptide.

6. The method of claim 5 , wherein the signal peptide is an S. cerevisiae α-factor signal peptide.

7. The method of claim 1 , wherein the suitable promotor is an alcohol oxidase promotor.

8. The method of claim 1 , wherein the enzymatically active rBHT protein has a specific activity of 8 U/mg at 20° C.

9. The method of claim 1 , wherein the nucleotide sequence of the isolated DNA has at least 99.5% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17 or 19.

10. The method of claim 9 , wherein the nucleotide sequence of the isolated DNA has at least 99.5% sequence identity with the nucleic acid sequence set forth in SEQ NO: 11, 13 or 15.

Assignees

• Univ North Carolina State

Inventors

• Bruno-Barcena Jose M
• Dagher Suzanne
• Azcarate-Peril Maria

Classifications

• A23G9/34

  characterised by carbohydrates used, e.g. polysaccharides (characterised by the dairy products used A23G9/40) · CPC title

• A23G9/40

  characterised by the dairy products used · CPC title

• A23L19/00

  Products from fruits or vegetables; Preparation or treatment thereof (marmalades, jams, jellies or the like A23L21/10; treating harvested fruit or vegetables in bulk A23N) · CPC title

• A23C9/1216

  Other enzymes · CPC title

• C12N9/1051 Primary

  Hexosyltransferases (2.4.1) · CPC title