2-O-sulfation enzyme mutant and 3-O-sulfation enzyme mutant, and method for using same

Patent metadata

Field	Value
Publication number	US-12139728-B2
Application number	US-202218056853-A
Country	US
Kind code	B2
Filing date	Nov 18, 2022
Priority date	Sep 5, 2017
Publication date	Nov 12, 2024
Grant date	Nov 12, 2024

Abstract

The present invention provides a 2-OST mutant exhibiting a high activity. Specifically, the present invention provides a 2-O-sulfation enzyme mutant, having a substitution of a leucine residue at position 321 with a basic amino acid residue in any one amino acid sequence of: (a) the amino acid sequence of SEQ ID NO: 2; (b) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 2; (c) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2; (d) the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; (e) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, or additions in the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; (f) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of amino acid residues at positions 69 to 356 in the amino acid sequence of SEQ ID NO: 2; and having a 2-O-sulfate transfer activity.

First claim

The invention claimed is:

1. A 3-O-sulfation enzyme mutant comprising an amino acid sequence selected from the group consisting of:
(a′) the amino acid sequence of SEQ ID NO: 8;
(b′) an amino acid sequence comprising one thirty amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 8;
(c′) an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8;
(d′) the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8;
(e′) an amino acid sequence comprising one to twenty amino acid substitutions, deletions, insertions, or additions in the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8; and
(f′) an amino acid sequence having 90% or more identity to the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8; and

wherein the 3-O-sulfation enzyme mutant has a substitution, relative to the amino acid sequence of SEQ ID NO: 8, selected from the group consisting of:
(i) a methionine residue at position 77 is substituted with a lysine residue;
(ii) a proline residue at position 125 is substituted with an alanine residue;
(iii) a valine residue at position 164 is substituted with an isoleucine residue;
(iv) an asparagine residue at position 167 is substituted with a histidine residue;
(v) a lysine residue at position 171 is substituted with a glutamine residues; and
(vi) a tyrosine residue at position 259 is substituted with a phenylalanine residue;

wherein the 3-O-sulfation enzyme mutant has a 3-O-sulfate transfer activity.

2. A method of producing a modified heparosan compound in which a hydroxyl group at 3-position of an α-D-glucosamine residue in a heparosan compound is sulfated, comprising contacting said heparosan compound with the 2-O-sulfation enzyme mutant of claim 1 to produce the modified heparosan compound comprising a sulfated hydroxyl group at the 3-position of an α-D-glucosamine residue in said heparosan.

3. The method according to claim 2 , wherein the modified heparosan compound is selected from the group consisting of N-sulfated 6-O-sulfated heparosan, N-sulfated 6-O-sulfated epimerized heparosan, N-sulfated 2-O-sulfated 6-O-sulfated heparosan, N-sulfated 2-O-sulfated 6-O-sulfated epimerized depolymerized heparosan, N-sulfated 6-O-sulfated depolymerized heparosan, N-sulfated 6-O-sulfated epimerized depolymerized heparosan, N-sulfated 2-O-sulfated 6-O-sulfated depolymerized heparosan, and N-sulfated 2-O-sulfated 6-O-sulfated epimerized depolymerized heparosan.

4. The method according to claim 2 , wherein the 3-O-sulfation enzyme mutant is produced by a transformed microorganism or an extract thereof.

5. The method according to claim 4 , wherein the transformed microorganism is a bacterium belonging to the genus Escherichia.

6. The method according to claim 5 , wherein the bacterium belonging to the genus Escherichia is Escherichia col.

7. A method of producing a heparan sulfate, comprising subjecting heparosan to a treatment comprising (1) N-deacetylation of α-D-glucosamine residue, (2) depolymerization, (3) N-sulfation of the α-D-glucosamine residue, (4) C5-epimerization of a hexuronic acid residue, (5) 2-O-sulfation of the hexuronic acid residue by contacting said heparosan with the 3-O-sulfation enzyme mutant of claim 1 , (6) 6-O-sulfation of the α-D-glucosamine residue, and (7) 3-O-sulfation of the α-D-glucosamine residue to produce said heparan sulfate.

8. The 3-O-sulfation enzyme mutant according to claim 1 , wherein the amino acid sequence is selected from the group consisting of:
(a′) the amino acid sequence of SEQ ID NO: 8;
(b′) an amino acid sequence comprising one to ten amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 8;
(c′) an amino acid sequence having 95% or more identity to the amino acid sequence of SEQ ID NO: 8;
(d′) the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8;
(e′) an amino acid sequence comprising one to ten amino acid substitutions, deletions, insertions, or additions in the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8; and
(f′) an amino acid sequence having 95% or more identity to the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8.

9. The 3-O-sulfation enzyme mutant according to claim 1 , wherein the amino acid sequence is selected from the group consisting of:
(a′) the amino acid sequence of SEQ ID NO: 8;
(b′) an amino acid sequence comprising one to five amino acid substitutions, deletions, insertions, or additions in the amino acid sequence of SEQ ID NO: 8;
(c′) an amino acid sequence having 98% or more identity to the amino acid sequence of SEQ ID NO: 8;
(d′) the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8;
(e′) an amino acid sequence comprising one to five amino acid substitutions, deletions, insertions, or additions in the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8; and
(f′) an amino acid sequence having 98% or more identity to the amino acid sequence consisting of amino acid residues at positions 48 to 311 in the amino acid sequence of SEQ ID NO: 8.

Assignees

• Ajinomoto Kk

Inventors

• Tsuji Chihiro
• Shimizu Tomoko
• Tagami Uno
• Mihara Yasuhiro
• Sugiki Masayuki
• Nakano Shogo
• MOTOYAMA Tomoharu
• Ito Sohei

Classifications

• C12Y208/02

  Sulfotransferases (2.8.2) · CPC title

• C12P19/26

  Preparation of nitrogen-containing carbohydrates · CPC title

• C08B37/0075 Primary

  Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N9/13 Primary

  transferring sulfur containing groups (2.8) · CPC title