# US 7,608,444 B2 · Use of acid stable protease in animal feed

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- Number: US 7,608,444 B2
- Document ID: US7608444B2
- Status: Granted
- Kind: B2
- Application number: 11/074,491
- Filed: 2005-03-08
- Published: 2005-07-07
- Granted: 2009-10-27
- Earliest claimed: 2000-02-08
- Assignees: Dsm IP Assets B.V.
- Inventors: Peter Rahbek Oestergaard; Carsten Sjoeholm; Anna-Marie Kluenter
- Web page: https://patentgrep.com/patent/US7608444B2

## CPC

- C12N 9/52: derived from bacteria {or Archaea}
- A23J 3/16: from soybean
- A23J 3/346: {of vegetable proteins}
- A23K 10/30: from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms (obtained by microbiological or biochemical processes, e.g. using yeasts or enzymes, A23K10/10)
- A23K 20/189: Enzymes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A23J: PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23K: FODDER

## Abstract

The present invention relates to acid-stable proteases of the subtilisin family, their use in animal feed, feed-additives and feed compositions containing such proteases, and methods for the treatment of vegetable proteins using such proteases.

## Claims

### Claim 1

An animal feed composition comprising (a) at least one acid-stable subtilisin and (b) a vegetable protein or protein source, wherein the composition has a crude protein content of 50-800 g/kg, wherein the acid stability of the subtilisin means that the activity of the subtilisin in purified form, in a dilution corresponding to A280=1.0, is at least 40% of the reference activity of the subtilisin, wherein the activity of the subtilisin is measured after two hours incubation at a temperature of 37° C. in a buffer of 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100 mM CABS, 1 mM CaCl2, 150 M KCl, and 0.01% Triton X-100 (pH 3.5), and wherein the reference activity is measured after two hours incubation at a temperature of 5° C. in the same buffer but adjusted to pH 9.0, wherein the activity and reference activity are measured after these incubations, at 25° C. in Suc AAPF pNA (pH 9.0).

### Claim 2 (depends on 1)

The animal feed composition of claim 1, wherein the activity of the pure subtilisin is at least 45% of the reference activity of the subtilisin.

### Claim 3 (depends on 2)

The animal feed composition of claim 2, wherein the activity of the subtilisin is at least 50% of the reference activity.

### Claim 4 (depends on 3)

The animal feed composition of claim 3, wherein the activity of the subtilisin is at least 60% of the reference activity.

### Claim 5 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin has a pH optimum in the range of 6.0-11.0.

### Claim 6 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin is an Acremonium chrysogenum, ATCC 48272 subtilisin.

### Claim 7 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin is an Acremonium kiliense, ATCC 20338 subtilisin.

### Claim 8 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin is a Bacillus sp., NCIMB 40484 subtilisin.

### Claim 9 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin is a Bacillus alcalophilus, NCIMB 10438 subtilisin.

### Claim 10 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin is a Fusarium oxysporum, IFO 4471 subtilisin.

### Claim 11 (depends on 1)

The animal feed composition of claim 1, wherein the subtilisin is a Paecilomyces lilacinus, CBS 102449 subtilisin.

### Claim 12 (depends on 1)

The animal feed composition of claim 1, wherein the amount of the purified subtilisin corresponds to 0.01-200 mg subtilisin protein per kg feed.

### Claim 13 (depends on 1)

The animal feed composition of claim 1, which further comprises at least one enzyme selected from the group consisting of galactanase, beta-glucanase, phytase, and xylanase.

### Claim 14 (depends on 1)

The animal feed composition of claim 1, which further comprises one or more fat soluble vitamins and/or water soluble vitamins, and (c) one or more trace minerals.