US 8,877,478 B2 · Phytases, nucleic acids encoding them and methods for making and using them

Abstract

This invention relates to phytases, polynucleotides encoding them, uses of the polynucleotides and polypeptides of the invention, as well as the production and isolation of such polynucleotides and polypeptides. In particular, the invention provides polypeptides having phytase activity under high temperature conditions, and phytases that retain activity after exposure to high temperatures. The phytases of the invention can be thermotolerant and/or thermostable at low temperatures, in addition to higher temperatures. The phytases of the invention can be used in foodstuffs to improve the feeding value of phytate rich ingredients. The phytases of the invention can be formulated as foods or feeds or supplements for either to, e.g., aid in the digestion of phytate. The foods or feeds of the invention can be in the form of pellets, liquids, powders and the like. In one aspect, phytases of the invention are stabile against thermal denaturation during pelleting; and this decreases the cost of the phytase product while maintaining in vivo efficacy and detection of activity in feed.

Claims44

Dependent claims

An isolated, synthetic, or recombinant variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO:

2 and one single amino acid substitution to the amino acid sequence of SEQ ID NO:
2, wherein said one single amino acid substitution is selected from the group consisting of:
A47F, T136F, N159V, N159E, T163R, D164R, G179R, V233W, Q275V, R289A, and T349Y;
wherein said variant polypeptide has phytase activity.

1 more element

An isolated, synthetic, or recombinant variant polypeptide comprising the amino acid sequence selected from the group consisting of:

(a) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159V, G179R, C226D, V233W, Q275V, R289A, and T349Y to the amino acid sequence of SEQ ID NO:
2;
(b) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, N159V, T163R, D164R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(c) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, T136H, N159V, T163R, D164R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(d) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, T136H, N159V, T163R, D164R, E168R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(e) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97V, T136H, N159V, T163R, E168R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(f) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97V, T136H, N159V, T163R, D164R, E168R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(g) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, T136H, N159V, T163R, D164R, E168R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(h) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159V, T163R, D164R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(i) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159V, T163R, D164R, E168R, G179R, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(j) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159E, T163R, D164R, G179R, V233W, Q275V, R289A, T349Y and L363P to the amino acid sequence of SEQ ID NO:
2;
(k) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97V, T136H, N159V, T163R, D164R, G179R, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(l) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159V, T163R, D164R, E168R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(m) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, N159V, D164R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(n) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, N159V, T163R, D164R, E168R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(o) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97V, T136H, N159E, T163R, D164R, E168R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(p) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159V, T163R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(q) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97E, T136H, N159V, T163R, D164R, E168R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(r) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, T136H, N159E, T163R, D164R, G179R, C226D, V233W, Q275V, R289A and T349Y to the amino acid sequence of SEQ ID NO:
2;
(s) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, T136H, N159V, D164R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(t) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97V, T136H, N159V, D164R, G179R, C226D, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
(u) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, C97V, T136H, N159V, D164R, 0179R, V233W, Q275V and T349Y to the amino acid sequence of SEQ ID NO:
2;
and
(v) a variant polypeptide comprising an amino acid sequence that is at least 95% identical to the amino sequence as set forth in SEQ ID NO:
2 and the combination of amino acid substitutions of A47F, T136H, N159V, C226D, V233W and T349Y to the amino acid sequence of SEQ ID NO:
2;
wherein said variant polypeptide has phytase activity.

65 more elements

Dependencies are inferred from the claim text.

Phytases, nucleic acids encoding them and methods for making and using them

Life of this patent

Phytases, nucleic acids encoding them and methods for making and using them

Life of this patent

Abstract

Claims44