US 12,258,592 B2
Phytase mutant
Aixi Bai, Jiangsu (CN); and Feng Li, Jiangsu (CN)
Assigned to NANJING BESTZYME BIO-ENGINEERING CO., LTD., Nanjing (CN)
Appl. No. 17/059,696
Filed by Nanjing Bestzyme Bio-engineering Co., Ltd., Jiangsu (CN)
PCT Filed May 30, 2019, PCT No. PCT/CN2019/089212
§ 371(c)(1), (2) Date Nov. 30, 2020,
PCT Pub. No. WO2019/228441, PCT Pub. Date Dec. 5, 2019.
Claims priority of application No. 201810540167.5 (CN), filed on May 30, 2018.
Prior Publication US 2021/0207112 A1, Jul. 8, 2021
Int. Cl. C12N 9/16 (2006.01)
CPC C12N 9/16 (2013.01) [C12Y 301/03008 (2013.01)] 15 Claims
 
1. A thermostable phytase variant having phytase activity that has at least 95% sequence identity to a polypeptide selected from the group consisting of the polypeptides of SEQ ID NO: 1, 2, 3, 79, and 99, wherein the phytase variant comprises a cysteine or a homocysteine residue at positions corresponding to positions 346 and 393 of the polypeptide of SEQ ID NO: 1, such that a disulfide bond is formed between the two residues, and optionally, the phytase variant further comprises one or more combinations of residues selected from (A) to (O), such that a disulfide bond is formed between the two residues of each combination:
(A) a cysteine or a homocysteine residue at positions corresponding to positions 34 and 174 of the polypeptide of SEQ ID NO: 1;
(B) a cysteine or a homocysteine residue at positions corresponding to positions 56 and 103 of the polypeptide of SEQ ID NO: 1;
(C) a cysteine or a homocysteine residue at positions corresponding to positions 57 and 366 of the polypeptide of SEQ ID NO: 1;
(D) a cysteine or a homocysteine residue at positions corresponding to positions 61 and 366 of the polypeptide of SEQ ID NO: 1;
(E) a cysteine or a homocysteine residue at positions corresponding to positions 82 and 296 of the polypeptide of SEQ ID NO: 1;
(F) a cysteine or a homocysteine residue at positions corresponding to positions 128 and 203 of the polypeptide of SEQ ID NO: 1;
(G) a cysteine or a homocysteine residue at positions corresponding to positions 140 and 262 of the polypeptide of SEQ ID NO: 1;
(H) a cysteine or a homocysteine residue at positions corresponding to positions 156 and 191 of the polypeptide of SEQ ID NO: 1;
(I) a cysteine or a homocysteine residue at positions corresponding to positions 165 and 245 of the polypeptide of SEQ ID NO: 1;
(J) a cysteine or a homocysteine residue at positions corresponding to positions 191 and 210 of the polypeptide of SEQ ID NO: 1;
(K) a cysteine or a homocysteine residue at positions corresponding to positions 196 and 211 of the polypeptide of SEQ ID NO: 1;
(L) a cysteine or a homocysteine residue at positions corresponding to positions 264 and 312 of the polypeptide of SEQ ID NO: 1;
(M) a cysteine or a homocysteine residue at positions corresponding to positions 315 and 380 of the polypeptide of SEQ ID NO: 1;
(N) a cysteine or a homocysteine residue at positions corresponding to positions 322 and 356 of the polypeptide of SEQ ID NO: 1; and
(O) a cysteine or a homocysteine residue at positions corresponding to positions 349 and 390 of the polypeptide of SEQ ID NO: 1;
wherein the thermostable phytase variant does not comprise the combinations of residues of (C) and (D) at the same time; or does not comprise the combinations of residues (H) and (J) at the same time.