# US 11,896,034 B2 · Polypeptides having lysozyme activity, polynucleotides encoding same and uses and compositions thereof

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- Number: US 11,896,034 B2
- Document ID: US11896034B2
- Status: Granted
- Kind: B2
- Application number: 17/212,019
- Filed: 2021-03-25
- Published: 2021-07-15
- Granted: 2024-02-13
- Earliest claimed: 2016-12-21
- Assignees: Novozymes A/S
- Applicants: Novozymes A/S
- Inventors: Mikkel Klausen; Kirk Matthew Schnorr; Lars Kobberoee Skov; Soeren Nymand-Grarup; Marianne Thorup Cohn; Peter Bjarke Olsen; Ming Li; Ye Liu
- Web page: https://patentgrep.com/patent/US11896034B2

## CPC

- A23K 20/147: Polymeric derivatives, e.g. peptides or proteins
- A23K 20/189: Enzymes
- 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 20/174: Vitamins
- A23K 20/22: Compounds of alkali metals
- A23K 20/24: Compounds of alkaline earth metals, e.g. magnesium
- A23K 20/30: {Oligoelements}
- A23K 50/30: for swines
- A23K 50/75: for poultry
- C12N 9/2462: {Lysozyme (3.2.1.17)}
- C12Y 302/01017: Lysozyme (3.2.1.17)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A23K: FODDER
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention relates animal feed or animal feed additives comprising one or more polypeptides having lysozyme activity. The invention also relates to polypeptides having lysozyme activity, polynucleotides encoding the polypeptides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

## Claims

### Claim 1

A granule comprising one or more Glycoside Hydrolase Family 25 (GH25) polypeptides having lysozyme activity, wherein the polypeptide has improved activity compared to the activity of the polypeptide of SEQ ID NO: 39 and wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 3;
(b) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 6;
(c) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 9;
(d) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 12;
(e) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 18;
(f) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 21;
(g) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 24;
(h) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 30;
(i) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 33;
(j) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 38;
(k) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 33 and SEQ ID NO: 38, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1-20 positions;
(l) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), or (k), and a N-terminal and/or C-terminal histidine tag (His-tag) and/or histidine-glutamine tag (HQ-tag);
(m) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), or (k) and a N-terminal and/or C-terminal extension of between 1 and 10 amino acids; and
(n) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), or (k) or having lysozyme activity and having at least 90% of the length of the mature polypeptide.

### Claim 2

An isolated polypeptide having lysozyme activity, selected from the group consisting of:
(a) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 3;
(b) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 6;
(c) a polypeptide having at least 91% sequence identity to the polypeptide of SEQ ID NO: 9;
(d) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 12;
(e) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 21;
(f) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 24;
(g) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 30;
(h) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 33;
(i) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 38;
(j) a variant of the polypeptide of SEQ ID NO: 3, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1-20 positions;
(k) a variant of the polypeptide of SEQ ID NO: 6, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1-20 positions;
(l) a variant of the polypeptide of SEQ ID NO: 9, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1-18 positions;
(m) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 30 and SEQ ID NO: 33, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any thereof in 1-20 positions;
(n) a variant of the polypeptide of SEQ ID NO: 38, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1-20 positions;
(o) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), or (n) and a N-terminal and/or C-terminal histidine tag (His-tag) and/or histidine-glutamine tag (HQ-tag);
(p) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), or (n) and a N-terminal and/or C-terminal extension of between 1 and 10 amino acids; and
(q) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), or (n) having lysozyme activity and having at least 90% of the length of the mature polypeptide.

### Claim 3 (depends on 2)

The polypeptide of claim 2, wherein the polypeptide comprises or consists of amino acids 1 to 207 of SEQ ID NO: 3, amino acids 1 to 216 of SEQ ID NO: 6, amino acids 1 to 204 of SEQ ID NO: 9, amino acids 1 to 203 of SEQ ID NO: 12, amino acids 1 to 208 of SEQ ID NO: 18, amino acids 1 to 207 of SEQ ID NO: 21, amino acids 1 to 207 of SEQ ID NO: 24, amino acids 1 to 217 of SEQ ID NO: 30, amino acids 1 to 207 of SEQ ID NO: 33 or amino acids 1 to 207 of SEQ ID NO: 38.

### Claim 4 (depends on 2)

A composition comprising the polypeptide of claim 2.

### Claim 5 (depends on 2)

An animal feed additive comprising the polypeptide of claim 2 and one or more components selected from the group consisting of:
one or more vitamins;
one or more minerals;
one or more amino acids;
one or more prebiotics;
one or more organic acids;
one or more other feed ingredients;
one or more additional enzymes;
one or more probiotics; and
one or more phytogenics.

### Claim 6 (depends on 2)

An animal feed comprising a polypeptide of claim 2 and plant based material.

### Claim 7

A method of hydrolysing peptidoglycan in a bacterial cell wall, comprising treating the bacterial cell wall with one or more Glycoside Hydrolase Family 25 (GH25) polypeptides having lysozyme activity, wherein the polypeptide has improved activity compared to the activity of the polypeptide of SEQ ID NO: 39 and wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide having at least 90% sequence identity to the polypeptide of SEQ ID NO: 3;
(b) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 6;
(c) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 9;
(d) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 12;
(e) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 18;
(f) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 21;
(g) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 24;
(h) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 30;
(i) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 33;
(j) a polypeptide having at least 85% sequence identity to the polypeptide of SEQ ID NO: 38;
(k) a variant of the polypeptide selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 33 and SEQ ID NO: 38, wherein the variant has lysozyme activity and comprises one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions or any combination thereof in 1-20 positions;
(l) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), or (k), and a N-terminal and/or C-terminal histidine tag (His-tag) and/or histidine-glutamine tag (HQ-tag);
(m) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), or (k) and a N-terminal and/or C-terminal extension of between 1 and 10 amino acids; and
(n) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), or (k) having lysozyme activity and having at least 90% of the length of the mature polypeptide.