# US 8,540,984 B2 · Phytases with improved thermal stability

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- Number: US 8,540,984 B2
- Document ID: US8540984B2
- Status: Granted
- Kind: B2
- Application number: 12/375,432
- Filed: 2007-08-03
- Published: 2010-03-18
- Granted: 2013-09-24
- Earliest claimed: 2006-08-03
- Assignees: Cornell Research Foundation, Inc.
- Applicants: Xingen Lei
- Inventors: Xingen Lei
- Web page: https://patentgrep.com/patent/US8540984B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides variant phytase enzymes having increased thermal stability relative to their counterpart parent enzymes. The modifications to the enzymes include both single substitutions and various combinations of substitutions that provide improved stability and activity. The invention further provides nucleic acids encoding the variant phytase enzymes, host cells and vectors containing and expressing them, as well as feed compositions useful for providing improved nutrition, particularly with respect to the bioavailability of dietary phosphate, calcium, iron and zinc, among others.

## Claims

### Claim 1

An isolated phytase comprising an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:1 over a region of at least 100 amino acid residues and containing at least one substitution of at least one amino acid residue selected from the group consisting of residues 46, 65, 103, 112, 144, 209, 227, and 344 of SEQ ID NO:1.

### Claim 2 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution is a multiple substitution comprising K65E/K97M/S209G, or a conservative substitution thereof.

### Claim 3 (depends on 1)

An animal feed composition comprising the isolated phytase of claim 1.

### Claim 4 (depends on 3)

A foodstuff comprising an animal feed composition of claim 3.

### Claim 5

An isolated E. coli phytase polypeptide of SEQ ID NO: 1 carrying a modification of at least one of residues 46, 65, 103, 112, 144, 209, 227, and 344, wherein said phytase has increased thermostability relative to a phytase of SEQ ID NO: 1.

### Claim 6 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises at least one of K46E, K65E, G103S, D112N, D144N, K209G, V227A, and G344D.

### Claim 7 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residues 65, 97, and 209.

### Claim 8 (depends on 7)

The isolated phytase polypeptide of claim 7, wherein said modification comprises K65E/K97M/S209G.

### Claim 9 (depends on 5)

An animal feed composition comprising a phytase of claim 5.

### Claim 10

An isolated phytase polypeptide, wherein said phytase polypeptide comprises an amino acid sequence having at least 96 percent sequence identity to SEQ ID NO:1 over a region of at least 100 amino acid residues and containing substitutions at at least two amino acid residues corresponding to residues selected from the group consisting of residues 46, 65, 103, 112, 144, 209, 227, and 344 of SEQ ID NO:1, which phytase polypeptide has increased thermostability relative to a phytase of SEQ ID NO: 1.

### Claim 11 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitutions at residues corresponding to residues 144 and 227 of SEQ ID NO: 1.

### Claim 12 (depends on 10)

An animal feed composition comprising an isolated phytase polypeptide of claim 10.

### Claim 13 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 46.

### Claim 14 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 65.

### Claim 15 (depends on 14)

The isolated phytase of claim 14, wherein said at least one substitution comprises K65E, or a conservative substitution thereof.

### Claim 16 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 103.

### Claim 17 (depends on 16)

The isolated phytase of claim 16, wherein said at least one substitution comprises G103S, or a conservative substitution thereof.

### Claim 18 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 112.

### Claim 19 (depends on 18)

The isolated phytase of claim 18, wherein said at least one substitution comprises D112N, or a conservative substitution thereof.

### Claim 20 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 144.

### Claim 21 (depends on 20)

The isolated phytase of claim 20, wherein said at least one substitution comprises D144N, or a conservative substitution thereof.

### Claim 22 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 209.

### Claim 23 (depends on 22)

The isolated phytase of claim 22, wherein said at least one substitution comprises K209G, or a conservative substitution thereof.

### Claim 24 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 227.

### Claim 25 (depends on 24)

The isolated phytase of claim 24, wherein said at least one substitution comprises V227A, or a conservative substitution thereof.

### Claim 26 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution comprises substitution of amino acid residue 344.

### Claim 27 (depends on 26)

The isolated phytase of claim 26, wherein said at least one substitution comprises G344D, or a conservative substitution thereof.

### Claim 28 (depends on 1)

The isolated phytase of claim 1, wherein said at least one substitution is a multiple substitution comprising K65E/K97M/G103S/G344D, or a conservative substitution thereof.

### Claim 29 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 46.

### Claim 30 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 65.

### Claim 31 (depends on 30)

The isolated phytase polypeptide of claim 30, wherein said modification comprises K65E.

### Claim 32 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 103.

### Claim 33 (depends on 32)

The isolated phytase polypeptide of claim 32, wherein said modification comprises G103S.

### Claim 34 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 112.

### Claim 35 (depends on 34)

The isolated phytase polypeptide of claim 34, wherein said modification comprises D112N.

### Claim 36 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 144.

### Claim 37 (depends on 36)

The isolated phytase polypeptide of claim 36, wherein said modification comprises D144N.

### Claim 38 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 209.

### Claim 39 (depends on 38)

The isolated phytase polypeptide of claim 38, wherein said modification comprises K209G.

### Claim 40 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 227.

### Claim 41 (depends on 40)

The isolated phytase polypeptide of claim 40, wherein said modification comprises V227A.

### Claim 42 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises modification of amino acid residue 344.

### Claim 43 (depends on 42)

The isolated phytase polypeptide of claim 42, wherein said modification comprises G344D.

### Claim 44 (depends on 5)

The isolated phytase polypeptide of claim 5, wherein said modification comprises K65E/K97M/G103S/G344D.

### Claim 45 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 46 of SEQ ID NO:1.

### Claim 46 (depends on 45)

The isolated phytase polypeptide of claim 45, wherein the substitution at the residue corresponding to residue 46 of SEQ ID NO:1 is K46E, or a conservative substitution thereof.

### Claim 47 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 65 of SEQ ID NO:1.

### Claim 48 (depends on 47)

The isolated phytase polypeptide of claim 47, wherein the substitution at the residue corresponding to residue 65 of SEQ ID NO:1 is K65E, or a conservative substitution thereof.

### Claim 49 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 103 of SEQ ID NO:1.

### Claim 50 (depends on 49)

The isolated phytase polypeptide of claim 49, wherein the substitution at the residue corresponding to residue 103 of SEQ ID NO:1 is G103S, or a conservative substitution thereof.

### Claim 51 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 112 of SEQ ID NO:1.

### Claim 52 (depends on 51)

The isolated phytase polypeptide of claim 51, wherein the substitution at the residue corresponding to residue 112 of SEQ ID NO:1 is D112N, or a conservative substitution thereof.

### Claim 53 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 144 of SEQ ID NO:1.

### Claim 54 (depends on 53)

The isolated phytase polypeptide of claim 53, wherein the substitution at the residue corresponding to residue 144 of SEQ ID NO:1 is D144N, or a conservative substitution thereof.

### Claim 55 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 209 of SEQ ID NO:1.

### Claim 56 (depends on 55)

The isolated phytase polypeptide of claim 55, wherein the substitution at the residue corresponding to residue 209 of SEQ ID NO:1 is K209G, or a conservative substitution thereof.

### Claim 57 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 227 of SEQ ID NO:1.

### Claim 58 (depends on 57)

The isolated phytase polypeptide of claim 57, wherein the substitution at the residue corresponding to residue 227 of SEQ ID NO:1 is V227A, or a conservative substitution thereof.

### Claim 59 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide comprises substitution at the residue corresponding to residue 344 of SEQ ID NO:1.

### Claim 60 (depends on 59)

The isolated phytase polypeptide of claim 59, wherein substitution at the residue corresponding to residue 344 of SEQ ID NO:1 is G344D, or a conservative substitution thereof.

### Claim 61 (depends on 10)

The isolated phytase polypeptide of claim 10, wherein the phytase polypeptide further comprises substitution at the residue corresponding to residue 97 of SEQ ID NO:1.

### Claim 62 (depends on 61)

The isolated phytase polypeptide of claim 61, wherein the phytase polypeptide comprises substitutions K65E, K97M, and S209G at residues corresponding, respectively, to residues 65, 97, and 209 of SEQ ID NO: 1.

### Claim 63 (depends on 61)

The isolated phytase polypeptide of claim 61, wherein the phytase polypeptide comprises substitutions K65E, K97M, G103S, and G344D at residues corresponding, respectively, to residues 65, 97, 103, and 344 of SEQ ID NO: 1.

### Claim 64 (depends on 13)

The isolated phytase of claim 13, wherein said at least one substitution comprises K46E, or a conservative substitution thereof.

### Claim 65 (depends on 29)

The isolated phytase polypeptide of claim 29, wherein said modification comprises K46E.