US 12,139,728 B2
2-O-sulfation enzyme mutant and 3-O-sulfation enzyme mutant, and method for using same
Chihiro Tsuji, Kanagawa (JP); Tomoko Shimizu, Kanagawa (JP); Uno Tagami, Kanagawa (JP); Yasuhiro Mihara, Kanagawa (JP); Masayuki Sugiki, Kanagawa (JP); Shogo Nakano, Shizuoka (JP); Tomoharu Motoyama, Shizuoka (JP); and Sohei Ito, Shizuoka (JP)
Assigned to AJINOMOTO CO., INC., Tokyo (JP)
Filed by AJINOMOTO CO., INC., Tokyo (JP)
Filed on Nov. 18, 2022, as Appl. No. 18/056,853.
Application 18/056,853 is a division of application No. 16/795,991, filed on Feb. 20, 2020, granted, now 11,851,688.
Application 16/795,991 is a continuation of application No. PCT/JP2018/033897, filed on Sep. 5, 2018.
Claims priority of application No. 2017-170637 (JP), filed on Sep. 5, 2017.
Prior Publication US 2023/0105158 A1, Apr. 6, 2023
Int. Cl. C12N 9/10 (2006.01); C08B 37/00 (2006.01); C12P 19/26 (2006.01)
CPC C12N 9/13 (2013.01) [C08B 37/0075 (2013.01); C12P 19/26 (2013.01); C12Y 208/02 (2013.01)] 9 Claims
 
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.