Method for preparing mogroside having no β-1,6-glucoside bond

Patent metadata

Field	Value
Publication number	US-10626430-B2
Application number	US-201615544316-A
Country	US
Kind code	B2
Filing date	Jan 19, 2016
Priority date	Jan 20, 2015
Publication date	Apr 21, 2020
Grant date	Apr 21, 2020

Abstract

The present invention provides a method for preparing a mogroside having no β-1,6-glucoside bond comprising the step of reacting glycosidase ASBGL2, AOBGL2, AOBGL1, ASBGL1, or a variant thereof with a mogroside having at least one β-1,6-glucoside bond, thereby cleaving said β-1,6-glucoside bond.

First claim

The invention claimed is:

1. A method for preparing a mogroside having no β-1,6-glucoside bond comprising reacting a protein selected from the group consisting of proteins (a) to (c) shown below with a mogroside having at least one β-1,6-glucoside bond, thereby cleaving said β-1,6-glucoside bond:
(a) a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8;
(b) a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, wherein 1 to 42 amino acids have been deleted, substituted, inserted, and/or added, and having an activity to cleave a β-1,6-glucoside bond of a mogroside; and
(c) a protein having an amino acid sequence having 95% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, and having an activity to cleave a β-1,6-glucoside bond of a mogroside.

2. The method according to claim 1 , wherein the protein selected from the group consisting of proteins (a) to (c) further includes a secretory signal peptide.

3. The method according to claim 1 , wherein the mogroside having at least one β-1,6-glucoside bond further has at least one β-1,2-glucoside bond.

4. The method according to claim 3 , wherein the mogroside having at least one β-1,6-glucoside bond and at least one β-1,2-glucoside bond is selected from mogroside V, siamenoside I, mogroside IV, and mogroside IIIA 1 .

5. The method according to claim 1 , wherein the mogroside having no β-1,6-glucoside bond is selected from mogroside IIIE and mogroside IIA.

6. A method for producing a mogroside having no β-1,6-glucoside bond comprising culturing a non-human transformant obtained by introducing a polynucleotide selected from the group consisting of polynucleotides (a) to (d) shown below into a host producing a mogroside having at least one β-1,6-glucoside bond:
(a) a polynucleotide having a nucleotide sequence selected from the group consisting of positions 61 to 2601 of SEQ ID NO: 1, positions 61 to 2707 of SEQ ID NO: 2, positions 61 to 2601 of SEQ ID NO: 5 and positions 61 to 2708 of SEQ ID NO: 6;
(b) a polynucleotide encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8;
(c) a polynucleotide encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, wherein 1 to 42 amino acids have been deleted, substituted, inserted, and/or added, and having an activity to cleave a β-1,6-glucoside bond of a mogroside; and
(d) a polynucleotide encoding a protein having an amino acid sequence having 95% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, and having an activity to cleave a β-1,6-glucoside bond of a mogroside.

7. The method according to claim 6 , wherein the polynucleotide selected from the group consisting of polynucleotides (a) to (d) further includes a polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide.

8. The method according to claim 7 , wherein the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide is a polynucleotide consisting of a nucleotide sequence set forth in any of positions 1 to 60 of SEQ ID NO: 1, positions 1 to 60 of SEQ ID NO: 5, positions 1 to 57 of SEQ ID NO: 9, positions 1 to 57 of SEQ ID NO: 13, SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47.

9. The method according to claim 8 , wherein the polynucleotide containing the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide consists of a nucleotide sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NOS: 17 to 19.

10. The method according to claim 6 , wherein the polynucleotide is inserted into an expression vector.

11. The method according to claim 6 , wherein the transformant is transformed yeast or a transformed plant.

12. A method for preparing a mogroside having no β-1,6-glucoside bond comprising contacting an enzyme agent derived from a non-human transformed cell obtained by introducing, into a host cell, a polynucleotide selected from the group consisting of polynucleotides (a) to (d) shown below, with a mogroside having at least one β-1,6-glucoside bond, thereby cleaving said β-1,6-glucoside bond:
(a) a polynucleotide having a nucleotide sequence selected from the group consisting of positions 61 to 2601 of SEQ ID NO: 1, positions 61 to 2707 of SEQ ID NO: 2, positions 61 to 2601 of SEQ ID NO: 5, and positions 61 to 2708 of SEQ ID NO: 6;
(b) a polynucleotide encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8;
(c) a polynucleotide encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, wherein 1 to 42 amino acids have been deleted, substituted, inserted, and/or added, and having an activity to cleave a β-1,6-glucoside bond of a mogroside; and
(d) a polynucleotide encoding a protein having an amino acid sequence having 95% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, and having an activity to cleave a β-1,6-glucoside bond of a mogroside.

13. The method according to claim 12 , wherein the polynucleotide selected from the group consisting of polynucleotides (a) to (d) further includes a polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide.

14. The method according to claim 13 , wherein the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide is a polynucleotide consisting of a nucleotide sequence set forth in any of positions 1 to 60 of SEQ ID NO: 1, positions 1 to 60 of SEQ ID NO: 5, positions 1 to 57 of SEQ ID NO: 9, positions 1 to 57 of SEQ ID NO: 13, SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 47.

15. The method according to claim 14 , wherein the polynucleotide containing the polynucleotide consisting of a nucleotide sequence encoding a secretory signal peptide consists of a nucleotide sequence set forth in any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NOS: 17 to 19.

16. The method according to claim 12 , wherein the polynucleotide is inserted into an expression vector.

17. The method according to claim 12 , wherein the transformed cell is a transformed bacterium or transformed yeast.

18. The method according claim 12 , wherein the mogroside having at least one β-1,6-glucoside bond further has at least one β-1,2-glucoside bond.

19. The method according to claim 18 , wherein the mogroside having at least one β-1,6-glucoside bond and at least one β-1,2-glucoside bond is selected from mogroside V, siamenoside I, mogroside IV, and mogroside IIIA 1 .

20. The method according to claim 12 , wherein the mogroside having no β-1,6-glucoside bond is selected from mogroside IIIE and mogroside IIA.

21. The method according to claim 1 , wherein the protein (b) is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 8, wherein 1 to 14 amino acids have been deleted, substituted, inserted, and/or added, and having an activity to cleave a β-1,6-glucoside bond of a mogroside.

Assignees

• Suntory Holdings Ltd

Inventors

• Ochiai Misa
• Ono Eiichiro

Classifications

• C12P19/44 Primary

  Preparation of O-glycosides, e.g. glucosides {(polysaccharides and not substituted disaccharides C12P19/04, C12P19/12)} · CPC title

• C12N9/24

  acting on glycosyl compounds (3.2) · CPC title

• C12N15/09

  Recombinant DNA-technology · CPC title

• C07K2319/02

  containing a signal sequence · CPC title

• A61P3/10

  for hyperglycaemia, e.g. antidiabetics · CPC title