# US 8,772,011 B2 · Protease variants

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- Number: US 8,772,011 B2
- Document ID: US8772011B2
- Status: Granted
- Kind: B2
- Application number: 13/738,490
- Filed: 2013-01-10
- Published: 2013-07-25
- Granted: 2014-07-08
- Earliest claimed: 2003-10-10
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Leonardo De Maria; Carsten Andersen; Lars Lehmann Hylling Christensen; Soren Flensted Lassen; Peter Rahbek Ostergaard
- Web page: https://patentgrep.com/patent/US8772011B2

## CPC

- C12N 9/48: acting on peptide bonds (3.4)
- A23K 20/189: Enzymes
- C12N 9/52: derived from bacteria {or Archaea}
- C12N 9/58: derived from fungi
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- C12N 2310/111: spanning the whole gene, or a large part of it
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23K: FODDER
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

The invention relates to a novel 3D structure encoding a Nocardiopsis protease, as well as to variants of parent protease homologous to Nocardiopsis proteases, preferably of improved thermostability and/or with an amended temperature activity profile. The invention also relates to DNA sequences encoding such variants, their production in a recombinant host cell, as well as methods of using the variants, in particular within the field of animal feed and detergents. The invention furthermore relates to methods of generating and preparing protease variants of amended properties.

## Claims

### Claim 1

A variant of a parent protease, comprising a substitution selected from the group consisting of:
6C; 7P; 8C; 9C; 10E,D; 12E,D; 13E,D,P; 16C; 17C; 18C; 23P; 24K,P,R; 25K,P,R; 27P; 32C; 33C; 37C; 39R,K; 42E,D; 43W; 46D,E; 47D,E; 52C; 55C; 56R,K; 58E,D; 62C; 63C; 67C; 69D,E; 70D,E; 71C; 72K,R; 76C; 81D,E,P; 82D,E,P; 84D,E; 85C; 89C,D,E; 92P,R; 93P; 94C,P; 95E,D; 96E,D,P; 97R,K; 98P; 99R,K; 103C; 105C,P; 106C; 111R,K; 113E,D; 118R,K; 120E,D; 122K; 124R,K; 125P; 127K; 129E,D; 130E,D; 134C; 135P; 136P; 139C; 140E,D; 141C; 144C; 145C; 146C; 147W; 148C; 149C; 150E,D; 151P,E,D; 155C; 156C; 160D,E; 161D,E; 162K,R; 163C; 166D,E; 170C; 172C; 173C; 174P; 175P; 176P; 180R,K; 181R,K; 184P; 187P; and 188R,K
wherein
(a) the variant has protease activity;
(b) each position corresponds to a position of amino acids 1 to 188 of SEQ ID NO: 2; and
(c) the variant has a sequence identity to amino acids 1 to 188 of SEQ ID NO: 2 of at least 90%.

### Claim 2 (depends on 1)

The variant of claim 1, which has a sequence identity to amino acids 1 to 188 of SEQ ID NO: 2 of at least 95%.

### Claim 3 (depends on 1)

The variant of claim 1, which comprises a substitution selected from the group consisting of 6C; 7P; 8C; 9C; 10E,D; 12E,D; 13E,D,P; 16C; 17C; 18C; 23P; 24K,P,R; 25K,P,R; 27P; 32C; 33C; 37C; 39R,K; and 42E,D.

### Claim 4 (depends on 1)

The variant of claim 1, which comprises a substitution selected from the group consisting of 43W; 46D,E; 47D,E; 52C; 55C; 56R,K; 58E,D; 62C; 63C; 67C; 69D,E; 70D,E; 710; 72K,R; 76C; 81D,E,P; 82D,E,P; 84D,E; and 85C.

### Claim 5 (depends on 1)

The variant of claim 1, which comprises 89C,D,E; 92P,R; 93P; 94C,P; 95E,D; 96E,D,P; 97R,K; 98P; 99R,K; 103C; 105C,P; 106C; 111R,K; 113E,D; 118R,K; 120E,D; 122K; 124R,K; and 125P.

### Claim 6 (depends on 1)

The variant of claim 1, which comprises a substitution selected from the group consisting of 127K; 129E,D; 130E,D; 134C; 135P; 136P; 139C; 140E,D; 141C; 144C; 145C; 146C; 147W; 148C; and 149C.

### Claim 7 (depends on 1)

The variant of claim 1, which comprises a substitution selected from the group consisting of 150E,D; 151P,E,D; 155C; 156C; 160D,E; 161D,E; 162K,R; 163C; 166D,E; 170C; 172C; 173C; 174P; 175P; 176P; 180R,K; 181R,K; 184P; 187P; and 188R,K.

### Claim 8 (depends on 1)

An animal feed additive comprising at least one variant of claim 1, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 9 (depends on 2)

An animal feed additive comprising at least one variant of claim 2, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 10 (depends on 3)

An animal feed additive comprising at least one variant of claim 3, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 11 (depends on 4)

An animal feed additive comprising at least one variant of claim 4, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 12 (depends on 5)

An animal feed additive comprising at least one variant of claim 5, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 13 (depends on 6)

An animal feed additive comprising at least one variant of claim 6, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 14 (depends on 7)

An animal feed additive comprising at least one variant of claim 7, and
(a) at least one fat soluble vitamin;
(b) at least one water soluble vitamin; and/or
(c) at least one trace mineral.

### Claim 15 (depends on 1)

An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising a variant of claim 1.

### Claim 16 (depends on 2)

An animal feed composition having a crude protein content of 50 to 800 g/kg and comprising a variant of claim 2.

### Claim 17 (depends on 1)

A method for improving the nutritional value of an animal feed, comprising adding a variant of claim 1 to the feed.

### Claim 18 (depends on 2)

A method for improving the nutritional value of an animal feed, comprising adding a variant of claim 2 to the feed.

### Claim 19 (depends on 1)

A method for the treatment of proteins, comprising adding a variant of claim 1 to at least one protein or protein source.

### Claim 20 (depends on 2)

A method for the treatment of proteins, comprising adding a variant of claim 2 to at least one protein or protein source.