Polypeptides having Lysozyme Activity, Polynucleotides Encoding Same and Uses and Compositions Thereof

• US 20200131492A1
• Filed: 05/11/2018
• Published: 04/30/2020
• Est. Priority Date: 05/12/2017
• Status: Active Grant

• Current Assignee: Novozymes A/S (Novozymes)
• Original Assignee: Novozymes A/S (Novozymes)
• Inventors: Liu, Ye, Li, Ming, Schnorr, Kirk Matthew, Olsen, Peter Bjarke
• Granted Patent: US 11,859,170 B2
• Time in Patent Office: Days
• CPC Class Codes: 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
• CPC Class Codes: A23K 20/189 Enzymes
• CPC Class Codes: A23K 40/10 by agglomeration; by granulation, e.g. making powders
• CPC Class Codes: A23K 40/30 by encapsulating; by coating
• CPC Class Codes: A23K 50/30 for swines
• CPC Class Codes: A23K 50/75 for poultry
• CPC Class Codes: A23K 50/80 for aquatic animals, e.g. fish, crustaceans or molluscs
• CPC Class Codes: C12N 9/2462 Lysozyme (3.2.1.17)
• CPC Class Codes: C12Y 302/01017 Lysozyme (3.2.1.17)

Abstract

Animal feed or animal feed additives comprising one or more polypeptides having lysozyme activity. 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

1-20. -20. (canceled)

21. A composition comprising at least 0.01 mg of LYS polypeptide per kilogram of composition, wherein the polypeptide (a) has lysozyme activity and (b) comprises one or more LAD catalytic domains;

wherein the LAD catalytic domain gives a domT score of at least 180 when queried using a Profile Hidden Markov Model (HMM) prepared using SEQ ID NOs;

46 to 187 and hmmbuild software program.

22. The composition of claim 21, wherein the polypeptide further comprises one or more lysozyme enhancing domains, wherein the lysozyme enhancing domain gives a domT score of at least 100 when queried using a Profile Hidden Markov Model prepared using SEQ ID NOs:

188 to 316 and hmmbuild software program, and wherein the query is carried out using the hmmscan software program.

23. The composition of claim 21, wherein the polypeptide is selected from the group consisting of:

(a) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

3;

(b) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

6;

(c) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

9;

(d) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

12;

(e) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

15;

(f) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

18;

(g) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

21;

(h) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

24;

(i) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

27;

(j) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

30;

(k) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

33;

(l) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

36;

(m) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

39;

(n) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

42;

(o) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

45;

(p) 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;

15, SEQ ID NO;

18, SEQ ID NO;

21, SEQ ID NO;

24, SEQ ID NO;

27, SEQ ID NO;

30, SEQ ID NO;

33, SEQ ID NO;

36, SEQ ID NO;

39, SEQ ID NO;

42 and SEQ ID NO;

45, 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 positions;

(q) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal His-tag and/or HQ-tag;

(r) a polypeptide comprising the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) and a N-terminal and/or C-terminal extension of up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; and

(s) a fragment of the polypeptide of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (l), (m), (n), (o) or (p) having lysozyme activity and having at least 90% of the length of the mature polypeptide.

24. The composition of claim 21, wherein the LYS polypeptide comprises one or more motifs selected from the group consisting of

25. The composition of claim 21, wherein the polypeptide is obtained or obtainable from the taxonomic phylum Ascomycota, preferably the taxonomic subphylum Pezizomycotina.

26. A granule comprising the composition of claim 21.

27. The granule of claim 26, wherein the granule is coated.

28. An animal feed additive comprising the composition of claim 21.

29. An animal feed comprising plant-based material and the composition of claim 21.

30. A liquid formulation comprising the composition of claim 21, wherein the LYS polypeptide is dosed between 0.01% to 25% w/w of the liquid formulation.

31. The liquid formulation of claim 30, which further comprises 20% to 80% w/w of polyol.

32. A method of improving one or more performance parameters of an animal comprising administering to the animal the composition of claim 21.

33. A method of preparing an animal feed, comprising mixing the composition of claim 21 with plant-based material.

34. An isolated polypeptide having lysozyme activity, selected from the group consisting of:

(a) a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO;

3;

(b) a polypeptide having at least 94% sequence identity to the polypeptide of SEQ ID NO;

6;

(c) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

9;

(d) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

12;

(e) a polypeptide having at least 87% sequence identity to the polypeptide of SEQ ID NO;

15;

(f) a polypeptide having at least 81% sequence identity to the polypeptide of SEQ ID NO;

18;

(g) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

21;

(h) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

24;

(i) a polypeptide having at least 87% sequence identity to the polypeptide of SEQ ID NO;

27;

(j) a polypeptide having at least 96.2% sequence identity to the polypeptide of SEQ ID NO;

30;

(k) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

33;

(l) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

36;

(m) a polypeptide having at least 81% sequence identity to the polypeptide of SEQ ID NO;

39;

(n) a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO;

42;

(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 His-tag and/or 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 up to 10 amino acids, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 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.

35. The polypeptide of claim 34, wherein the polypeptide comprises or consists of amino acids 1 to 316 of SEQ ID NO:

2, amino acids 1 to 316 of SEQ ID NO;

3, amino acids 1 to 322 of SEQ ID NO;

4, amino acids 1 to 318 of SEQ ID NO;

6, amino acids 1 to 318 of SEQ ID NO;

7, amino acids 1 to 326 of SEQ ID NO;

8, amino acids 1 to 316 of SEQ ID NO;

10, amino acids 1 to 316 of SEQ ID NO;

11, amino acids 1 to 324 of SEQ ID NO;

12, amino acids 1 to 316 of SEQ ID NO;

14, amino acids 1 to 316 of SEQ ID NO;

15, amino acids 1 to 324 of SEQ ID NO;

16, amino acids 1 to 316 of SEQ ID NO;

18, amino acids 1 to 316 of SEQ ID NO;

19, amino acids 1 to 324 of SEQ ID NO;

20, amino acids 1 to 316 of SEQ ID NO;

22, amino acids 1 to 316 of SEQ ID NO;

23, amino acids 1 to 324 of SEQ ID NO;

24, amino acids 1 to 516 of SEQ ID NO;

26, amino acids 1 to 516 of SEQ ID NO;

27, amino acids 1 to 524 of SEQ ID NO;

28, amino acids 1 to 317 of SEQ ID NO;

30, amino acids 1 to 317 of SEQ ID NO;

31, amino acids 1 to 325 of SEQ ID NO;

32, amino acids 1 to 316 of SEQ ID NO;

34, amino acids 1 to 316 of SEQ ID NO;

35, amino acids 1 to 324 of SEQ ID NO;

36, amino acids 1 to 316 of SEQ ID NO;

38, amino acids 1 to 316 of SEQ ID NO;

39 or amino acids 1 to 324 of SEQ ID NO;

40.

36. A polynucleotide encoding the polypeptide of claim 34.

37. A nucleic acid construct or expression vector comprising the polynucleotide of claim 36 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

38. A recombinant host cell comprising the polynucleotide of claim 36 operably linked to one or more control sequences that direct the production of the polypeptide.

39. A method of producing a polypeptide having lysozyme activity, comprising:

(a) cultivating the recombinant host cell of claim 38 under conditions conducive for production of the polypeptide; and

(b) recovering the polypeptide.