Dispersin variants and their use in animal feed

Abstract

The present invention relates to animal feed additives comprising dispersin variants that are gastric stable, to the gastric stable polypeptides and to the polynucleotides expressing the variants.

Description

[0001] DISPERSIN VARIANTS AND THEIR USE IN ANIMAL FEED

[0002] Reference to a Sequence Listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] Field of the Invention

[0005] The present invention relates to dispersin variants for use in animal feed as well as, polynucleotides encoding the variants and methods of producing the variants.

[0006] Background of the Invention

[0007] Polypeptides having hexosaminidase activity include dispersins such as Dispersin B (DspB) which are p-N-acetylglucosamininidases belonging to the Glycoside Hydrolase 20 family (GH20). Dispersin B is produced by the periodontal pathogen, Aggregatibacter actinomycetemcomitans, a Gram-negative oral bacterium. Dispersin B is a p-hexosaminidase that specifically hydrolyzes p-1 ,6-glycosidic linkages of acetylglucosamine polymers e.g. found in biofilm. Dispersin B contains three highly conserved acidic residues: an aspartic acid at residue 183 (D183), a glutamic acid at residue 184 (E184), and a glutamic acid at residue 332 (E332). Biofilm have been found attached to various surfaces including medical devices such as implants. W004061117 A2 (Kane Biotech INC) describe use of compositions comprising DspB for reducing biofilm caused by poly-N-acetylglucosamine-producing bacteria and Kane et al. describes the use of compositions for reduction of biofilm on medical devises and for wound care.

[0008] WO 2017 / 186943 discloses the parent wild-type GH45 dispersin of SEQ ID NO: 1. WO 2020 / 207944 dislcoses variants thereof. SEQ ID NO: 1 is not suitable for use in animal feed due to its lack of gastric stability. The present invention provides dispersin (GH45) variants with improved properties compared to its parent enzymes for use as an animal feed additive. The dispersin (GH45) variants with improved properties in animal feed are intended to help maintain the general health of an animal by improving the intestinal health of the animal or maintaining / preserving intestinal paracellular permeability or gut integrity in an animal.

[0009] Current methodologies face considerable limitations when addressing biofilm-associated problems. One major issue is the inherent difficulty in producing enzymes that remain stable in the gastric environment, which is crucial for human oral use. Additionally, the escalating problem of antibiotic resistance has rendered many conventional treatments ineffective, necessitating exorbitantly high concentrations of antibiotics to achieve desired outcomes. This not only increases treatment costs but also exacerbates the risk of adverse side effects.

[0010] Biofilms contribute to a multitude of complications by providing a reservoir of cells within a sessile community, which while beneficial to pathogen survival, can be detrimental to the external environment. In the context of the raising of livestock, bacterial colonization of undesired or unfavorable bacterial species in a population can have overall negative consequences on the health of livestock without necessarily rendering them ill but nonetheless impacting their growth.

[0011] It is therefore an objective of the present disclosure to overcome the above limitations at least in part. This objective includes providing new strategies to mitigate the impact of antibacterial resistance while effectively prevent or treat infections. The invention aims to harness the potential of microorganisms that secrete enzymes capable of degrading extracellular polymeric substances (EPS).

[0012] Summary of the Invention

[0013] Without being bound by theory, PNAG (poly-p-1 ,6-linked N-acetyl-D-glucosamine) is believed to protect pathogenic bacteria from macrophage-mediated killing. The activity of dispersin in the gut may degrade PNAG, thereby exposing bacterial cells to immune recognition and facilitating their clearance by immune cells. This mechanism may contribute to reducing pathogenic load in the gastrointestinal environment. The inventors have demonstrated that dispersins hydrolyze synthetic PNAG using MALDI-TOF-MS analysis.

[0014] An aspect of the invention is directed to an animal feed additive comprising a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 .

[0015] A further aspect of the invention is directed to an animal feed additive comprising a polypeptide having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 and comprising mutations selected from the group consisting of i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111 R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P

[0016] N260Q H272V S279D Y281 P K308Q K309E K312Q ix. Q1G Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aG; xii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324al; xiii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q L324*; xiv. *1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; and xv. Q1* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q.

[0017] The dispersin (GH45) variants with improved properties in animal feed are intended to help maintain the general health of an animal by improving the intestinal health of the animal. A further aspect of the invention is directed to use of a dispersin variant polypeptide having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 or the animal feed additive of the invention, to stabilize the healthy microflora of animals, in particular livestock.

[0018] An aspect of the invention is directed to variants of SEQ ID NO:1 having a. at least 80% sequence identity to SEQ ID NO: 2, such as at least 85% sequence identity to SEQ ID NO:2, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:2; b. at least 80% sequence identity to SEQ ID NO: 3, such as at least 85% sequence identity to SEQ ID NO:3, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:3; c. at least 80% sequence identity to SEQ ID NO: 4, such as at least 85% sequence identity to SEQ ID NO:4, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:4; d. at least 80% sequence identity to SEQ ID NO: 5, such as at least 85% sequence identity to SEQ ID NO:5, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:5; e. at least 80% sequence identity to SEQ ID NO: 6, such as at least 85% sequence identity to SEQ ID NO:6, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:6; f. at least 80% sequence identity to SEQ ID NO: 7, such as at least 85% sequence identity to SEQ ID NO:7, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:7; g. at least 80% sequence identity to SEQ ID NO: 8, such as at least 85% sequence identity to SEQ ID NO:8, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:8; h. at least 80% sequence identity to SEQ ID NO: 9, such as at least 85% sequence identity to SEQ ID NO:9, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:9; i. at least 80% sequence identity to SEQ ID NO: 10, such as at least 85% sequence identity to SEQ ID NO: 10, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO: 10; j. at least 80% sequence identity to SEQ ID NO: 11 , such as at least 85% sequence identity to SEQ ID NO:11 , such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO:11 ; k. at least 80% sequence identity to SEQ ID NO: 12, such as at least 85% sequence identity to SEQ ID NO: 12, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO: 12; l. at least 80% sequence identity to SEQ ID NO: 13, such as at least 85% sequence identity to SEQ ID NO: 13, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO: 13; m. at least 80% sequence identity to SEQ ID NO: 14, such as at least 85% sequence identity to SEQ ID NO: 14, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO: 14; n. at least 80% sequence identity to SEQ ID NO: 15, such as at least 85% sequence identity to SEQ ID NO: 15, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO: 15; and o. at least 80% sequence identity to SEQ ID NO: 16, such as at least 85% sequence identity to SEQ ID NO: 16, such as at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or 100% sequence identity to SEQ ID NO: 16.

[0019] The dispersin (GH45) variants with improved properties in animal feed are intended to help maintain the general health of an animal by improving the intestinal health of the animal. Another aspect of the invention is use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving intestinal health of an animal or maintaining / preserving intestinal paracellular permeability or gut integrity in an animal.

[0020] A further aspect is a method of increasing the ratio of lactic acid bacteria to Enterococcus species ratio of an animal comprising feeding the animal a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or the animal feed additive of the invention. Another aspect is a method of increasing the relative and / or absolute level of beneficial gut microbes in an animal, wherein beneficial gut microbes are selected from the group Lactic acid bacteria, Bifidiobacteria and Corynebacterium sp.

[0021] A further aspect of the invention is use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing the relative and / or the absolute level of Enterococcus cecorum in the intestine and / or the relative and / or the absolute level of Escherichia colijn the intestine.

[0022] Another aspect of the invention is directed to a polypeptide of the invention for use in the treatment or prevention of Escherichia coli, Clostridioides difficile or Helicobacter pylori infections or other PNAG producing infections in an animal in need thereof. Clostridioides difficile and Helicobacter pylori are bacteria known to cause severe gastrointestinal infections. PNAG producing infections are to be understood as PNAG producing microorganisms or bacteria causing infections. In yet another embodiment, Salmonella typhimurium infections are treated or prevented. One advantage of this arrangement is the potential for the polypeptide to inhibit or eradicate these bacterial infections, thereby reducing morbidity and mortality associated with such infections.

[0023] Another aspect of the invention is directed to a method for increasing the relative and / or absolute level of beneficial gut microbes in a human or animal, wherein beneficial gut microbes are selected from the group Lactic acid bacteria, Bifidobacteria, and Corynebacterium sp. Beneficial gut microbes refer to microorganisms in the gastrointestinal tract that contribute positively to health, aiding in digestion, immune function, and protection against pathogens. One advantage of this arrangement is that the polypeptide can selectively promote the growth of these beneficial bacteria, leading to a healthier and more balanced gut microbiome. This, in turn, can support various aspects of health, including improved digestion, enhanced immune response, and reduced risk of gastrointestinal diseases.

[0024] Another aspect is a method of improving the performance of an animal comprising feeding the animal a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or the animal feed additive of the invention, said performance selected from the group consisting of a. a reduced feed conversion ratio (FCR), and b. an increased average body weight gain.

[0025] A further aspect of the invention is directed to a method of reducing the intestinal levels of microbial species that generates exopolysaccharides containing (3-1 ,6-linked poly-N- acetylglucosamine (polyGIcNAc) comprising feeding the animal a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for or the animal feed additive of the invention.

[0026] Another aspect of the invention is directed to a dispersin variant having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 comprising a substitution at positions 3, 15, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309 and 312 and further comprising a substitution, an insertion or a deletion at a position selected from the group consisting of 1 , 49, 111 , 124, 148, 171 , 249, and 324.

[0027] The variants of the invention have hexosaminidase activity, which involves the enzymatic cleavage of terminal N-acetylhexosamines from glycoproteins and glycolipids. One advantage of this arrangement is its potential to degrade biofilm components effectively. This enzymatic action can disrupt the extracellular polymeric substances (EPS) matrix within biofilms, thereby reducing the protective barrier that bacteria use to shield themselves from external stresses, including antibiotic treatments. By breaking down the biofilm matrix, the hexosaminidase activity facilitates enhanced penetration of antimicrobial agents, leading to more effective eradication of bacterial communities.

[0028] One of the key components of EPS in many biofilms is poly-p-1 ,6-linked N-acetyl-D- glucosamine (PNAG). By hydrolyzing these polysaccharides into smaller, more accessible mono- and oligosaccharides, dispersin makes these sugars available to beneficial gut bacteria such as lactic acid bacteria, including Bifidobacteria and Lactobacillus.

[0029] Mono- and oligosaccharides serve as prebiotics, which are non-digestible food ingredients that promote the growth and activity of beneficial microorganisms. In the gut, lactic acid bacteria like Bifidobacteria utilize these prebiotic sugars for their metabolism, leading to the production of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. SCFAs play a crucial role in maintaining gut health by reducing inflammation, enhancing the integrity of the gut barrier, and modulating the immune system.

[0030] By providing readily available prebiotic nutrients to beneficial bacteria, dispersin indirectly supports their proliferation. The increased population of Bifidobacteria, Lactobacillus and other lactic acid bacteria helps outcompete pathogenic microorganisms, reducing the risk of infections and inflammation. Additionally, the metabolic activities of these beneficial bacteria produce antiinflammatory compounds that further contribute to reducing inflammation in the gut.

[0031] Thus, dispersin helps reduce inflammation by breaking down complex polysaccharides into simpler sugars that act as prebiotics for beneficial lactic acid bacteria. This process not only supports the growth of a healthy gut microbiome but also aids in the production of metabolites that have anti-inflammatory properties, promoting overall gut health and reducing the risk of inflammatory diseases. Another aspect of the invention is directed to the use of polypeptide of the invention to reduce the likelihood of development of antibiotic resistance. The use of dispersins allow at least in part for the continued utilization of antibiotics susceptible to resistance.

[0032] An aspect is of the invention is directed to a polypeptide according to the invention for use in the prevention of a disease, particularly that said polypeptide prevents gut diseases by promoting the growth of Bifidobacterium and / or Lactobacillus in the gut.

[0033] Another aspect of the invention is directed to a polypeptide according to the invention for reducing pathogenic bacterial loads in feces. This, in turn, contributes to controlling disease spread in livestock, including in a flock, a brood, a herd, a litter, a sounder, a drift, a drove, or on a farm.

[0034] Brief Description of the Figures

[0035] Figure 1 is an alignment of the polypeptides of SEQ ID NOs: 1 to 16.

[0036] Figure 2 illustates the results for E.coli shedding, as measured by log CFU count, of Challenged, Unchallenged, Challenged with dispersin addition and Unchallenged with dispersin addition, at Day 3, Day 7. Day 14 and Day 21. Analysis of microbial counts revealed that by day 14, dispersin having SEQ ID NO:5 had a significant effect on ETEC shedding, resulting in a 2- log reduction compared to the challenged control group

[0037] Figure 3 shows the fecal score of Challenged, Unchallenged, Challenged with dispersin addition and Unchallenged with dispersin addition, from Day 0 to Day 14. A score of 0 is normal feces consistency. A score of 1 illustrates soft feces. A scope of 2 illustrates diarrhea, albeit mild disarrhea. A score above 2 illustrates mild to moderate to severe diarrhea. The figure illustrates that the presence of dispersin (C and D) keeps fecal scores at or below 0.5 for most days. The figures illustrates the use of dispersin prevents diarrhea.

[0038] Sequence Listing

[0039] SEQ ID NO: 1 is the amino acid sequence of the mature polypeptide dispersin 45, a wild-type dispersin obtained from Terribacillus saccharophilus and described in WO 2017 / 186943

[0040] SEQ ID NO:2 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0041] SEQ ID NO:3 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0042] SEQ ID NO:4 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0043] SEQ ID NO:5 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1 SEQ ID NO:6 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1

[0044] SEQ ID NO:7 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0045] SEQ ID NO:8 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0046] SEQ ID NO:9 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0047] SEQ ID NQ:10 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0048] SEQ ID NO:11 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0049] SEQ ID NO:12 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0050] SEQ ID NO:13 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0051] SEQ ID NO:14 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0052] SEQ ID NO:15 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0053] SEQ ID NO:16 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0054] SEQ ID NO: 17 is the amino acid sequence of the mature polypeptide of wild-type Dispersin B, also know as Dispersin 27 from Aggregatibacter actinomycetemcomitans, wherein MNYIKKIILSLFLLGLFSVLNC is the signal peptide (SEQ ID NO.34).

[0055] SEQ ID NO: 18 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0056] 1.

[0057] SEQ ID NO: 19 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0058] 2.

[0059] SEQ ID NO: 20 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0060] 3. SEQ ID NO: 21 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0061] 4.

[0062] SEQ ID NO: 22 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0063] 5.

[0064] SEQ ID NO: 23 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0065] 6.

[0066] SEQ ID NO: 24 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0067] 7.

[0068] SEQ ID NO: 25 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0069] 8.

[0070] SEQ ID NO: 26 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0071] 9.

[0072] SEQ ID NO: 27 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0073] 10.

[0074] SEQ ID NO: 28 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0075] 11.

[0076] SEQ ID NO: 29 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0077] 12.

[0078] SEQ ID NO: 30 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0079] 13.

[0080] SEQ ID NO: 31 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0081] 14.

[0082] SEQ ID NO: 32 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0083] 15.

[0084] SEQ ID NO: 33 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO:

[0085] 16.

[0086] SEQ ID NO:34 is the signal peptide of the mature polypeptide of wild-type Dispersin B, also know as Dispersin 27 from Aggregatibacter actinomycetemcomitans (SEQ ID NO: 17). DETAILED DESCRIPTION OF THE INVENTION

[0087] Definitions

[0088] In accordance with this detailed description, the following definitions apply. Note that the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0089] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0090] The term “dispersin’’ and the abbreviation “Dsp” means a polypeptide having hexosaminidase activity, EC 3.2.1.- that catalyzes the hydrolysis of p-1 ,6-glycosidic linkages of N-acetyl-glucosamine polymers (poly-N-acetylglucosamine) found e.g. in biofilm.

[0091] The term “dispersin variant” or “variant” means a polypeptide which comprises an alteration at one or more (e.g., several) positions compared to the parent or reference polypeptide, in this instance of a parent or reference dispersin. The alteration may be a substitution, insertion or deletion. A substitution means replacement of the amino acid occupying a position with a different amino acid, a deletion means removal of an amino acid occupying a position and an insertion means adding amino acids e.g. 1 to 10 amino acids, preferably 1-3 amino acids adjacent to an amino acid occupying a position. The term “dispersin variant” means a polypeptide having hexosaminidase, preferably beta-1 ,6 N-acetylglucosaminidase activity or is active to poly-beta- 1 ,6-N-actylglucosamin (PNAG) and which comprise an alteration, i.e., a substitution, insertion, and / or deletion at one or more (or one or several) positions compared to the parent dispersin e.g. compared to SEQ ID NO: 1. The term “dispersin activity” means a polypeptide having hexosaminidase, preferably beta-1 ,6 N-acetylglucosaminidase activity or is active to poly-beta- 1 ,6-N-actylglucosamin (PNAG). The variants e.g. dispersin variants of the present invention preferably have at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the dispersin activity of the polypeptide shown in SEQ ID NO: 1.

[0092] The term “hexosaminidases” means a polypeptide having hexosaminidase activity (hexosaminidases), and includes EC 3.2.1. e.g. that catalyzes the hydrolysis of of N-acetyl-D- hexosamine or N-acetyl-glucosamine polymers found e.g. in biofilm. The term includes dispersins and includes polypeptides having N-acetylglucosaminidase activity and p-N- acetylglucosamininidase activity. The term “polypeptide having hexosaminidase activity” may be used interchangeably with the term hexosaminidases and similar the term “polypeptide having p- N-acetylglucosaminidase activity” may be used interchangeably with the term p-N- acetylglucosamininidases. For the purposes of the present invention, hexosaminidase activity is determined according to the procedure described in Example 4.

[0093] In one embodiment, the polypeptides of the present invention have at least 80%, at least 90%, at least 95%, or at least 100% of the hexosaminidase activity of the mature polypeptide of SEQ ID NO 2. In one embodiment, the polypeptides of the present invention have at least 80%, at least 90%, at least 95%, or at least 100% of the hexosaminidase activity of the mature polypeptide of SEQ ID NO 4. In one embodiment, the polypeptides of the present invention have at least 80%, at least 90%, at least 95%, or at least 100% of the hexosaminidase activity of the mature polypeptide of SEQ ID NO 6. In one embodiment, the polypeptides of the present invention have at least 80%, at least 90%, at least 95%, or at least 100% of the hexosaminidase activity of the mature polypeptide of SEQ ID NO 13. In one embodiment, the polypeptides of the present invention have at least 80%, at least 90%, at least 95%, or at least 100% of the hexosaminidase activity of the mature polypeptide of SEQ ID NO 15.

[0094] The term “animal” refers to production animals. Non-ruminant animals include monogastric animals, e.g. pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), and young calves

[0095] Animals further include fish and crustaceans. Examples of fish include but are not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish. Examples of crustaceans include but are not limited to shrimps and prawns.

[0096] The term “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for an animal typically comprises concentrates as well as vitamins, minerals, amino acids and / or other feed ingredients (such as in a premix) as well as plant or animal sources of protein.

[0097] The term “composition” refers to a composition comprising a carrier and at least one enzyme of the present invention. The compositions described herein may be mixed with an animal feed and referred to as a “mash feed.”

[0098] The terms “effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the enzyme(s) sufficient to improve the digestion or yield of an animal. The actual effective dosage in absolute numbers depends on factors including: the state of health of the animal in question, other ingredients present. The “effective amount”, “effective concentration”, or “effective dosage” of the enzyme(s) may be determined by routine assays known to those skilled in the art.

[0099] The terms “pellet" and / or "pelleting" refer to solid rounded, spherical and / or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the terms extrusion or extruding are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.

[0100] The term “poultry” means domesticated birds kept by humans for the eggs they produce and / or their meat and / or their feathers. Poultry includes broilers and layers. Poultry include members of the superorder Galloanserae (fowl), especially the order Galliformes (which includes chickens, Guineafowls, quails and turkeys) and the family Anatidae, in order Anseriformes, commonly known as "waterfowl" and including domestic ducks and domestic geese. Poultry also includes other birds that are killed for their meat, such as the young of pigeons. Examples of poultry include chickens (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.

[0101] The term "stable" is a term that is known in the art, and in a preferred aspect, stable is intended to mean the ability of the microorganism to remain in a spore form until it is administered to an animal to improve the health of the animal.

[0102] The term “swine” or “pigs” means domesticated pigs kept by humans for food, such as their meat. Swine includes members of the genus Sus, such as Sus scrofa domesticus or Sus domesticus and include piglets, growing pigs, and sows.

[0103] The term “vegetable protein” refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.

[0104] The term “catalytic domain” means the region of an enzyme containing the catalytic machinery of the enzyme.

[0105] The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

[0106] The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0107] The term “control sequences” means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e. , from the same gene) or foreign (i.e. , from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

[0108] The term “expression” includes any step involved in the production of a variant including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0109] An "expression vector" refers to a linear or circular DNA construct comprising a DNA sequence encoding a variant, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0110] A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a variant has been introduced. Exemplary host strains are microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells.

[0111] The term “isolated” means a variant, nucleic acid, cell, or other specified material or component that is separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide, nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted variant expressed in a host cell.

[0112] The term “mature polypeptide” means a polypeptide in its mature form following N-terminal processing and / or C-terminal processing (e.g., removal of signal peptide).

[0113] The term “mutant” means a polynucleotide encoding a variant.

[0114] The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0115] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a variant. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0116] The term “parent” or “parent dispersin’’ means a dispersin to which an alteration is made to produce the enzyme variants of the present invention. The term purified means a nucleic acid, variant or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified variant or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, usually at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The term "enriched" refers to a compound, variant, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0117] In one aspect, the term "purified" as used herein refers to the variant or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the variant being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the variant is separated from some of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0118] Accordingly, the variant may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The variant may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host organism for recombinantly produced variant. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated.

[0119] It is, therefore, preferred that the substantially pure variant is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material present in the preparation. The variant of the present invention is preferably in a substantially pure form ( / .e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the variant by well-known recombinant methods or by classical purification methods.

[0120] The term "recombinant" is used in its conventional meaning to refer to the manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, variant or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant” is synonymous with “genetically modified” and “transgenic”.

[0121] The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide.

[0122] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0123] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0124] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0125] For purposes of the present invention, the sequence identity between two polynucleotide sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0126] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0127] The term “variant” means a polypeptide having hexoaminidase activity comprising a substitution, an insertion (including extension), and / or a deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0128] The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally-occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild- type sequence).

[0129] Conventions for Designation of Variants

[0130] For purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is a Disp45 (Dispersin 45) is used to determine the corresponding amino acid positions in another dispersin. The amino acid sequence of another dispersin is aligned with the polypeptide disclosed in SEQ ID NO: 1 , and based on the alignment, the amino acid position number corresponding to any amino acid residue in the polypeptide disclosed in SEQ ID NO: 1 is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0131] In describing the variants of the present invention, the nomenclature described below is adapted for ease of reference. The accepted IIIPAC single letter or three letter amino acid abbreviation is employed.

[0132] Substitutions. For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid. Accordingly, the substitution of threonine at position 226 with alanine is designated as T226A . Multiple mutations are separated by addition marks (“+”) or by commas, e.g., “G205R, S411 F” or “G205R + S411 F”, representing substitutions at positions 205 and 411 of glycine (G) with arginine (R) and serine (S) with phenylalanine (F), respectively. Because the amino acid residue at a given position varies from parent to parent, the amino acid to be substituted may be indicated with X, e.g., X226A.

[0133] Deletions. For an amino acid deletion, the following nomenclature is used: Original amino acid, position, *. Accordingly, the deletion of the amino acid at position 195 is designated as “X195*”. Multiple deletions are separated by addition marks (“+”) or by commas, e.g., “X195* + X411*” or “X195*, X411*”.

[0134] Insertions. For an amino acid insertion, the following nomenclature is used: Original amino acid, position, original amino acid, inserted amino acid. Accordingly, the insertion of lysine after the amino acid at position 195 is designated “X195XK”. An insertion of multiple amino acids is designated [Original amino acid, position, original amino acid, inserted amino acid #1 , inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after the amino acid at position 195 is indicated as “X195XKA”.

[0135] In such cases, the inserted amino acid residue(s) are numbered by the addition of lower case letters to the position number of the amino acid residue preceding the inserted amino acid residue(s). In the above example, the sequence would thus be:

[0136] Alternatively, an insertion of an amino acid residue such as lysine after the amino acid at position 195 may be indicated by “195aK”, and the insertion of two or more additional amino acid residues such as lysine and alanine after the amino acid at position 195 may be indicated by “195aK, 195bA”.

[0137] Nomenclature

[0138] For purposes of the present invention, the nomenclature [IV] or [l / V] means that the amino acid at this position may be isoleucine (lie, I) or valine (Vai, V). Likewise, the nomenclature [LVI] and [L / V / l] means that the amino acid at this position may be a leucine (Leu, L), valine (Vai, V) or isoleucine (lie, I), and so forth for other combinations as described herein. Unless otherwise limited further, the amino acid X is defined such that it may be any of the 20 natural amino acids. The term “comprising a substitution” is in the present context meant comprising a substitution compared to the starting dispersin or the parent. The thus, dispersin have a replacement of the amino acid in e.g. position 3 with another amino acid Gastric stable dispersins

[0139] An aspect of the present invention relates to dispersin variants, comprising a substitution, an insertion or a deletion at five or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1 , wherein the variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1 and wherein the variant has hexoaminidase activity.

[0140] In another embodiment, the variant has at most 30% sequence differences compared to the parent, e.g., the polypeptide of SEQ ID NO: 1 , wherein the variant has hexoaminidase activity. The variants may further comprise an extension of one or more amino acids at the N-terminal and / or C-terminal ends. Alternatively, the variants may further comprise a truncation of one or more amino acids at the N-terminal and / or C-terminal ends.

[0141] In an embodiment, the variant has a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 1 .

[0142] In one embodiment, the number of alterations in the variants of the present invention is 5 to 50, such as 5 to 40, such as at least 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 or 40 alterations. Most preferably the variant comprises at least 15 alterations, including a substitution, an insertion or a deletion compared to SEQ ID NO: 1 and has an improved pH stability, such as at least 16, at least 17 or at least 18 alterations, such as 15 to 50 alterations, such as 16 to 50 alterations, such as 17 to 50 alterations, such as 18 to 50 alterations, such as 19 to 50 alterations such as 20 to 50 alterations.

[0143] In one embodiment, the dispersin variant comprises an alteration at five or more positions selected from the list consisting of positions 1 , 2, 3, 12, 15, 18, 22, 23, 24, 25, 30, 49, 56, 57, 59, 62, 63, 68, 72, 74, 77, 82, 90, 99, 100, 106, 114, 123, 124, 125, 135, 138, 148, 163,

[0144] 167, 170, 171 , 173, 174, 175, 178, 179, 181 , 185, 186, 187, 188, 189, 199, 203, 204, 205, 207,

[0145] 210, 215, 221 , 225, 227, 232, 235, 244, 249, 252, 256, 260, 262, 263, 264, 265, 267, 270, 272,

[0146] 273, 274, 276, 278, 279, 280, 281 , 282, 283, 284, 288, 290, 291 , 296, 303, 304, 305, 306, 308,

[0147] 309, 312, 314, 315, 319, 321 , 322, 323, and 324 wherein each position corresponds to the position of the polypeptide of SEQ ID NO: 1 , wherein the variant has at least 70% sequence identity to SEQ ID NO: 1 , and wherein the variant has dispersin activity. The variant has improved stability compared to SEQ ID NO 1.

[0148] In another embodiment, a variant comprises a substitution, an insertion or a deletion at five or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324, wherein the variant has at least 70% sequence identity to SEQ ID NO: 1 , and wherein the variant has dispersin activity. The variant has improved stability compared to SEQ ID NO 1. In another embodiment, a variant comprises a substitution, an insertion or a deletion at six positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at seven positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another aspect, a variant comprises a substitution, an insertion or a deletion at seven positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at eight positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at nine positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at ten positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at eleven positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at twelve positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at thirteen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at fourteen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at fifteen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at sixteen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at seventeen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at eigteen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at nineteen positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at twenty positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at twenty-one positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at twenty-two positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at twenty-three positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at twenty-four positions corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In another embodiment, a variant comprises a substitution, an insertion or a deletion at each position corresponding to any of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 279, 281 , 308, 309, 312, and 324. In each of these embodiments, the variant has at least 70% sequence identity to SEQ I D NO: 1 , and the variant has dispersin activity.

[0149] In a preferred embodiment, a variant comprises a substitution at positions 3, 15, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309 and 312 and further comprises a substitution, an insertion or a deletion at a position selected from the group consisting of 1 , 49, 111 , 124, 148, 171 , 249, and 324. In a further preferred embodiment,

[0150] In an embodiment, the dispersin variant comprises at least fifteen alterations selected from the group consisting of: i. a substitution, an insertion or a deletion at position 1 selected from the group consisting of Q1G, Q1*, 1aG, ii. a substitution, an insertion or a deletion at position 3 selected from the group consisting of Q3F, Q3I, Q3L, Q3M, Q3P, Q3V, Q3Y, Q3T, iii. a substitution, an insertion or a deletion at position 15 selected from the group consisting of H15F, H15Y, iv. a substitution, an insertion or a deletion at position 49 selected from the group consisting of A49W, A49Y, v. a substitution, an insertion or a deletion at position 59 selected from the group consisting of N59A, N59C, N59D, N59E, N59F, N59M, N59R, N59V, N59W, vi. a substitution, an insertion or a deletion at position 111 selected from the group consisting of, D111A, D111E, D111M, D111 N, D111Q, D111 R, D111V, D111W, vii. a substitution, an insertion or a deletion at position 124 selected from the group consisting of Y124C, Y124I, Y124K, Y124L, Y124M, Y124Q, Y124R, Y124T, Y124V, Y124W, viii. a substitution, an insertion or a deletion at position 148 selected from the group consisting of K148A, K148D, K148L, K148V, ix. a substitution, an insertion or a deletion at position 163 x. a substitution, an insertion or a deletion at position 171 selected from the group consisting of D171A, D171C, D171 E, D171 K, D171 L, D171M, D171Q, D171 R, D171V, D171W, D171Y, xi. a substitution, an insertion or a deletion at position 186 selected from the group consisting of, S186D, S186E, S186H, S186I, S186K, S186L, S186M, S186N, S186Q, S186R, S186V, S186W, xii. a substitution, an insertion or a deletion at position 225 xiii. a substitution, an insertion or a deletion at position 227 selected from the group consisting of, N227A, N227Q, N227R, N227S, N227T, N227K xiv. a substitution, an insertion or a deletion at position 232 selected from the group consisting of E232D, E232V, xv. a substitution, an insertion or a deletion at position 235 selected from the group consisting of G235W, G235A, G235E, G235F, G235H, G235I, G235L, G235M, G235N, G235P, G235S, G235V, xvi. a substitution, an insertion or a deletion at position 249 selected from the group consisting of, L249H, L249K, L249Q, L249R, L249W, L249Y, xvii. a substitution, an insertion or a deletion at position 252 selected from the group consisting of N252P, N252C, xviii. a substitution, an insertion or a deletion at position 260 selected from the group consisting of N260*, N260A, N260C, N260E, N260I, N260K, N260L, N260M, N260Q, N260R, N260V, N260W, N260Y xix. a substitution, an insertion or a deletion at position 272 selected from the group consisting of H272D, H272I, H272M, H272P, H272V, H272W, xx. a substitution, an insertion or a deletion at position 279 selected from the group consisting of S279C, S279D, S279E, S279G, S279N xxi. a substitution, an insertion or a deletion at position 281 selected from the group consisting of Y281*, Y281A, Y281C, Y281H, Y281K, Y281N, Y281 P, Y281 R, xxii. a substitution, an insertion or a deletion at position 308 selected from the group consisting of K308A, K308D, K308G, K308I, K308L, K308Q, K308S, K308T, K308V, K308Y, xxiii. a substitution, an insertion or a deletion at position 309 selected from the group consisting of K309A, K309C, K309D, K309H, K309L, K309M, K309N, K309Q, K309S, K309T, K309I, xxiv. a substitution, an insertion or a deletion at position 312 selected from the group consisting of K312A, K312L, K312M, K312N, K312Q, K312S, K312W, and xxv. a substitution, an insertion or a deletion at position 324, preferably selected from the group consisting of L324*, *324aA, *324aG, *324al. wherein the variant has dispersin activity and wherein each position corresponds to the position of the polypeptide of SEQ I D NO: 1.

[0151] In an embodiment, the dispersin variant comprises the alterations from the group consisting of: i. a substitution, an insertion or a deletion at position 3 selected from the group consisting of Q3F, Q3I, Q3L, Q3M, Q3P, Q3V, Q3Y, Q3T, ii. a substitution, an insertion or a deletion at position 15 selected from the group consisting of H15F, H15Y, iii. a substitution, an insertion or a deletion at position 59 selected from the group consisting of N59A, N59C, N59D, N59E, N59F, N59M, N59R, N59V, N59W, iv. a substitution, an insertion or a deletion at position 148 selected from the group consisting of K148A, K148D, K148L, K148V, v. a substitution, an insertion or a deletion at position 163 vi. a substitution, an insertion or a deletion at position 186 selected from the group consisting of, S186D, S186E, S186H, S186I, S186K, S186L, S186M, S186N, S186Q, S186R, S186V, S186W, vii. a substitution, an insertion or a deletion at position 225 viii. a substitution, an insertion or a deletion at position 227 selected from the group consisting of, N227A, N227Q, N227R, N227S, N227T, N227K ix. a substitution, an insertion or a deletion at position 232 selected from the group consisting of E232D, E232V, x. a substitution, an insertion or a deletion at position 235 selected from the group consisting of G235W, G235A, G235E, G235F, G235H, G235I, G235L, G235M, G235N, G235P, G235S, G235V, xi. a substitution, an insertion or a deletion at position 252 selected from the group consisting of N252P, N252C, xii. a substitution, an insertion or a deletion at position 260 selected from the group consisting of N260*, N260A, N260C, N260E, N260I, N260K, N260L, N260M, N260Q, N260R, N260V, N260W, N260Y xiii. a substitution, an insertion or a deletion at position 272 selected from the group consisting of H272D, H272I, H272M, H272P, H272V, H272W, xiv. a substitution, an insertion or a deletion at position 279 selected from the group consisting of S279C, S279D, S279E, S279G, S279N xv. a substitution, an insertion or a deletion at position 281 selected from the group consisting of Y281*, Y281A, Y281C, Y281H, Y281K, Y281N, Y281 P, Y281 R, xvi. a substitution, an insertion or a deletion at position 308 selected from the group consisting of K308A, K308D, K308G, K308I, K308L, K308Q, K308S, K308T, K308V, K308Y, xvii. a substitution, an insertion or a deletion at position 309 selected from the group consisting of K309A, K309C, K309D, K309H, K309L, K309M, K309N, K309Q, K309S, K309T, K309I, xviii. a substitution, an insertion or a deletion at position 312 selected from the group consisting of K312A, K312L, K312M, K312N, K312Q, K312S, K312W, xix. a substitution, an insertion or a deletion at position 324, preferably selected from the group consisting of L324*, *324aA, *324aG, *324al; and and further comprises one or more alterations selected from the group consisting of i. a substitution, an insertion or a deletion at position 1 selected from the group consisting of Q1G, Q1*, 1aG, ii. a substitution, an insertion or a deletion at position 49 selected from the group consisting of A49W, A49Y, iii. a substitution, an insertion or a deletion at position 111 selected from the group consisting of, D111A, D111E, D111M, D111 N, D111Q, D111 R, D111V, D111W, iv. a substitution, an insertion or a deletion at position 124 selected from the group consisting of Y124C, Y124I, Y124K, Y124L, Y124M, Y124Q, Y124R, Y124T, Y124V, Y124W, v. a substitution, an insertion or a deletion at position 148 selected from the group consisting of K148A, K148D, K148L, K148V, vi. a substitution, an insertion or a deletion at position 171 selected from the group consisting of D171A, D171C, D171 E, D171 K, D171 L, D171M, D171Q, D171 R, D171V, D171W, D171Y, vii. a substitution, an insertion or a deletion at position 249 selected from the group consisting of, L249H, L249K, L249Q, L249R, L249W, L249Y, and viii. a substitution, an insertion or a deletion at position 324, preferably selected from the group consisting of L324*, *324aA, *324aG, *324al; and wherein the variant has dispersin activity and wherein each position corresponds to the position of the polypeptide of SEQ I D NO: 1.

[0152] In an embodiment, the dispersin variant comprises the alterations from the group consisting of: i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q ix. Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aG; xii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324al; xiii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q L324*; xiv. *1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; and xv. Q1* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q In a typical embodiment, the variant has improved pH activity compared to the parent enzyme. In a typical embodiment, the variant has improved pH stability compared to the parent enzyme. In an embodiment, the variant has improved specific activity compared to the parent enzyme. In an embodiment, the variant has improved catalytic efficiency compared to the parent enzyme. The polypeptide may be a fusion polypeptide comprising a variant of the invention. The parent may be a fusion polypeptide or cleavable fusion polypeptide. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post- translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0153] A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen- Wilson etal., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35- 48.

[0154] In an aspect, the variant is isolated. In another aspect, the variant is purified.

[0155] Animal Feed and Animal Feed Additives

[0156] An aspect of the invention of an animal feed additive comprising a gastric stable dispersin described herein.

[0157] An aspect of the invention of an animal feed additive comprising a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1. The animal feed additive suitably has at least 75% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1 .

[0158] In one embodiment, the animal feed additive comprises an embodiment of the dispersin variant of the invention.

[0159] In one embodiment, the polypeptide of the feed additive is typically a dispersin variant compriseing a substitution at positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324. The polypeptide of the feed additive is preferably a dispersin variant comprising a substitution at positions 3, 15, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309 and 312 and further comprising a substitution, an insertion or a deletion at a position selected from the group consisting of 1 , 49, 111 , 124, 148, 171 , 249, and 324.

[0160] In one embodiment, the polypeptide of the feed additive is a dispersin variant comprising at least fifteen alterations selected from the group consisting of: i. a substitution, an insertion or a deletion at position 1 selected from the group consisting of Q1G, Q1*, 1aG, ii. a substitution, an insertion or a deletion at position 3 selected from the group consisting of Q3F, Q3I, Q3L, Q3M, Q3P, Q3V, Q3Y, Q3T, iii. a substitution, an insertion or a deletion at position 15 selected from the group consisting of H15F, H15Y, iv. a substitution, an insertion or a deletion at position 49 selected from the group consisting of A49W, A49Y, v. a substitution, an insertion or a deletion at position 59 selected from the group consisting of N59A, N59C, N59D, N59E, N59F, N59M, N59R, N59V, N59W, vi. a substitution, an insertion or a deletion at position 111 selected from the group consisting of, D111A, D111 E, D111 M, D111 N, D111Q, D111 R, D111V, D111W, vii. a substitution, an insertion or a deletion at position 124 selected from the group consisting of Y124C, Y124I, Y124K, Y124L, Y124M, Y124Q, Y124R, Y124T, Y124V, Y124W, viii. a substitution, an insertion or a deletion at position 148 selected from the group consisting of K148A, K148D, K148L, K148V, ix. a substitution, an insertion or a deletion at position 163 x. a substitution, an insertion or a deletion at position 171 selected from the group consisting of D171A, D171C, D171 E, D171 K, D171 L, D171 M, D171Q, D171 R, D171V, D171W, D171Y, xi. a substitution, an insertion or a deletion at position 186 selected from the group consisting of, S186D, S186E, S186H, S186I, S186K, S186L, S186M, S186N, S186Q, S186R, S186V, S186W, xii. a substitution, an insertion or a deletion at position 225 xiii. a substitution, an insertion or a deletion at position 227 selected from the group consisting of, N227A, N227Q, N227R, N227S, N227T, N227K xiv. a substitution, an insertion or a deletion at position 232 selected from the group consisting of E232D, E232V, xv. a substitution, an insertion or a deletion at position 235 selected from the group consisting of G235W, G235A, G235E, G235F, G235H, G235I, G235L, G235M, G235N, G235P, G235S, G235V, xvi. a substitution, an insertion or a deletion at position 249 selected from the group consisting of, L249H, L249K, L249Q, L249R, L249W, L249Y, xvii. a substitution, an insertion or a deletion at position 252 selected from the group consisting of N252P, N252C, xviii. a substitution, an insertion or a deletion at position 260 selected from the group consisting of N260*, N260A, N260C, N260E, N260I, N260K, N260L, N260M, N260Q, N260R, N260V, N260W, N260Y xix. a substitution, an insertion or a deletion at position 272 selected from the group consisting of H272D, H272I, H272M, H272P, H272V, H272W, xx. a substitution, an insertion or a deletion at position 279 selected from the group consisting of S279C, S279D, S279E, S279G, S279N xxi. a substitution, an insertion or a deletion at position 281 selected from the group consisting of Y281*, Y281A, Y281C, Y281 H, Y281 K, Y281 N, Y281 P, Y281 R, xxii. a substitution, an insertion or a deletion at position 308 selected from the group consisting of K308A, K308D, K308G, K308I, K308L, K308Q, K308S, K308T, K308V, K308Y, xxiii. a substitution, an insertion or a deletion at position 309 selected from the group consisting of K309A, K309C, K309D, K309H, K309L, K309M, K309N, K309Q, K309S, K309T, K309I, xxiv. a substitution, an insertion or a deletion at position 312 selected from the group consisting of K312A, K312L, K312M, K312N, K312Q, K312S, K312W, and xxv. a substitution, an insertion or a deletion at position 324, preferably selected from the group consisting of L324*, *324aA, *324aG, *324al. wherein the variant has dispersin activity and wherein each position corresponds to the position of the polypeptide of SEQ I D NO: 1.

[0161] In one embodiment, the polypeptide of the feed additive is a dispersin variant comprising the alterations from the group consisting of: i. a substitution, an insertion or a deletion at position 3 selected from the group consisting of Q3F, Q3I, Q3L, Q3M, Q3P, Q3V, Q3Y, Q3T, ii. a substitution, an insertion or a deletion at position 15 selected from the group consisting of H15F, H15Y, iii. a substitution, an insertion or a deletion at position 59 selected from the group consisting of N59A, N59C, N59D, N59E, N59F, N59M, N59R, N59V, N59W, iv. a substitution, an insertion or a deletion at position 148 selected from the group consisting of K148A, K148D, K148L, K148V, v. a substitution, an insertion or a deletion at position 163 vi. a substitution, an insertion or a deletion at position 186 selected from the group consisting of, S186D, S186E, S186H, S186I, S186K, S186L, S186M, S186N, S186Q, S186R, S186V, S186W, vii. a substitution, an insertion or a deletion at position 225 viii. a substitution, an insertion or a deletion at position 227 selected from the group consisting of, N227A, N227Q, N227R, N227S, N227T, N227K ix. a substitution, an insertion or a deletion at position 232 selected from the group consisting of E232D, E232V, x. a substitution, an insertion or a deletion at position 235 selected from the group consisting of G235W, G235A, G235E, G235F, G235H, G235I, G235L, G235M, G235N, G235P, G235S, G235V, xi. a substitution, an insertion or a deletion at position 252 selected from the group consisting of N252P, N252C, xii. a substitution, an insertion or a deletion at position 260 selected from the group consisting of N260*, N260A, N260C, N260E, N260I, N260K, N260L, N260M, N260Q, N260R, N260V, N260W, N260Y xiii. a substitution, an insertion or a deletion at position 272 selected from the group consisting of H272D, H272I, H272M, H272P, H272V, H272W, xiv. a substitution, an insertion or a deletion at position 279 selected from the group consisting of S279C, S279D, S279E, S279G, S279N xv. a substitution, an insertion or a deletion at position 281 selected from the group consisting of Y281*, Y281A, Y281C, Y281H, Y281K, Y281N, Y281 P, Y281 R, xvi. a substitution, an insertion or a deletion at position 308 selected from the group consisting of K308A, K308D, K308G, K308I, K308L, K308Q, K308S, K308T, K308V, K308Y, xvii. a substitution, an insertion or a deletion at position 309 selected from the group consisting of K309A, K309C, K309D, K309H, K309L, K309M, K309N, K309Q, K309S, K309T, K309I, xviii. a substitution, an insertion or a deletion at position 312 selected from the group consisting of K312A, K312L, K312M, K312N, K312Q, K312S, K312W, xix. a substitution, an insertion or a deletion at position 324, preferably selected from the group consisting of L324*, *324aA, *324aG, *324al; and and further comprises one or more alterations selected from the group consisting of i. a substitution, an insertion or a deletion at position 1 selected from the group consisting of Q1G, Q1*, 1aG, ii. a substitution, an insertion or a deletion at position 49 selected from the group consisting of A49W, A49Y, iii. a substitution, an insertion or a deletion at position 111 selected from the group consisting of, D111A, D111 E, D111 M, D111 N, D111Q, D111 R, D111V, D111W, iv. a substitution, an insertion or a deletion at position 124 selected from the group consisting of Y124C, Y124I, Y124K, Y124L, Y124M, Y124Q, Y124R, Y124T, Y124V, Y124W, v. a substitution, an insertion or a deletion at position 148 selected from the group consisting of K148A, K148D, K148L, K148V, vi. a substitution, an insertion or a deletion at position 171 selected from the group consisting of D171A, D171C, D171 E, D171 K, D171 L, D171 M, D171Q, D171 R, D171V, D171W, D171Y, vii. a substitution, an insertion or a deletion at position 249 selected from the group consisting of, L249H, L249K, L249Q, L249R, L249W, L249Y, and viii. a substitution, an insertion or a deletion at position 324, preferably selected from the group consisting of L324*, *324aA, *324aG, *324al; and wherein the variant has dispersin activity and wherein each position corresponds to the position of the polypeptide of SEQ I D NO: 1.

[0162] In one embodiment, the animal feed additive comprising a polypeptide having at least 70% sequence identity to SEQ ID NO: 1 and comprising mutations selected from the group consisting of i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111 R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q ix. Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aG; xii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324al; xiii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q L324*; xiv. *1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; and xv. Q1* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q

[0163] Typically, the polypeptide has a residual activity at pH 3 of at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, or at least 99%, wherein the residual activity is measured after 30 minutes at a temperature of 40 °C according to Residual Activity Assay as described in Example 4. The animal feed additive suitably has a residual activity as measured at pH 3 and upon pepsin exposure, preferably at pH 3 and at a pepsin concentration of 2775 U / rnL wherein the residual activity is measured after 30 minutes at a temperature of 40 °C according to Residual Activity Assay as described in Example 4

[0164] In an embodiment of the invention, the animal feed additive of the invention comprises a polypeptide selected from the group consisting of a. a polypeptide having at least 80% sequence identity to SEQ ID NO: 2; b. a polypeptide having at least 80% sequence identity to SEQ ID NO: 3; c. a polypeptide having at least 80% sequence identity to SEQ ID NO: 4; d. a polypeptide having at least 80% sequence identity to SEQ ID NO: 5; e. a polypeptide having at least 80% sequence identity to SEQ ID NO: 6; f. a polypeptide having at least 80% sequence identity to SEQ ID NO: 7; g. a polypeptide having at least 80% sequence identity to SEQ ID NO: 8; h. a polypeptide having at least 80% sequence identity to SEQ ID NO: 9; i. a polypeptide having at least 80% sequence identity to SEQ ID NO: 10; j. a polypeptide having at least 80% sequence identity to SEQ ID NO: 11 ; k. a polypeptide having at least 80% sequence identity to SEQ ID NO: 12; l. a polypeptide having at least 80% sequence identity to SEQ ID NO: 13; m. a polypeptide having at least 80% sequence identity to SEQ ID NO: 14; n. a polypeptide having at least 80% sequence identity to SEQ ID NO: 15; and o. a polypeptide having at least 80% sequence identity to SEQ ID NO: 16.

[0165] In one embodiment, the animal feed additive further comprises an ingredient selected from the group consisting of a. at least one fat soluble vitamin, and / or b. at least one water soluble vitamin, and / or c. at least one trace mineral.

[0166] The animal feed additive typically is in solid form, such as in granule form.

[0167] The present invention also relates to animal feed compositions and animal feed additives comprising one or more hexoaminidases. In an embodiment, the animal feed or animal feed additive comprises a formulating agent and one or more hexoaminidases of the invention. In a suitable embodiment, the formulating agent comprises one or more of the following compounds: glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin and cellulose.

[0168] The enzyme of the invention may be formulated as a liquid or a solid or a semi-solid formulation. For a liquid formulation, the formulating agent may comprise a polyol (such as e.g. glycerol, ethylene glycol or propylene glycol), a salt (such as e.g. sodium chloride, sodium benzoate, potassium sorbate) or a sugar or sugar derivative (such as e.g. dextrin, glucose, sucrose, and sorbitol). Thus, in one embodiment, the composition is a liquid composition comprising the polypeptide of the invention and one or more formulating agents selected from the list consisting of glycerol, ethylene glycol, 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose, and sorbitol.

[0169] For a solid formulation, the formulation may be for example as a granule, spray dried powder or agglomerate. The formulating agent may comprise a salt (organic or inorganic zinc, sodium, potassium or calcium salts such as e.g. such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol).

[0170] In an embodiment, the solid composition is in granulated form. The granule may have a matrix structure where the components are mixed homogeneously. However, the granule typically comprises a core particle and one or more coatings, which typically are salt and / or wax coatings. The core particle can either be a homogeneous blend of dispersin variant of the invention optionally combined with one or more additional enzymes and optionally together with one or more salts or an inert particle with the dispersin variant of the invention optionally combined with one or more additional enzymes applied onto it.

[0171] In an embodiment, the material of the core particles are selected from the group consisting of inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starch or a sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), sugar or sugar derivative (such as e.g. sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starch, flour, cellulose and minerals.

[0172] The salt coating is typically at least 1 pm thick and can either be one particular salt or a mixture of salts, such as Na2SC>4, K2SO4, MgSCU and / or sodium citrate. Other examples are those described in e.g. WO 2008 / 017659, WO 2006 / 034710, WO 1997 / 05245, WO 1998 / 54980, WO 1998 / 55599, WO 2000 / 70034 or polymer coating such as described in WO 2001 / 00042.

[0173] In another embodiment, the composition is a solid composition comprising the dispersin variant of the invention and one or more formulating agents selected from the list consisting of sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate. In a preferred embodiment, the solid composition is in granulated form. In an embodiment, the solid composition is in granulated form and comprises a core particle, an enzyme layer comprising the dispersin variant of the invention and a salt coating.

[0174] In a further embodiment, the formulating agent is selected from one or more of the following compounds: glycerol, ethylene glycol, 1 , 2-propylene glycol or 1 , 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch and cellulose. In a preferred embodiment, the formulating agent is selected from one or more of the following compounds: 1 , 2-propylene glycol, 1 , 3-propylene glycol, sodium sulfate, dextrin, cellulose, sodium thiosulfate and calcium carbonate.

[0175] Animal feed compositions or diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01 / 58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore, such fish diets usually have a crude fat content of 200-310 g / kg.

[0176] An animal feed composition according to the invention has a crude protein content of 50-800 g / kg, and furthermore comprises at least one dispersin variant as defined herein.

[0177] Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolisable energy of 10-30 MJ / kg; and / or a content of calcium of 0.1-200 g / kg; and / or a content of available phosphorus of 0.1-200 g / kg; and / or a content of methionine of 0.1-100 g / kg; and / or a content of methionine plus cysteine of 0.1-150 g / kg; and / or a content of lysine of 0.5-50 g / kg.

[0178] In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and / or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01 / 58275 (R. 2-5).

[0179] Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e. Crude protein (g / kg)= N (g / kg) x 6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).

[0180] Metabolisable energy can be calculated on the basis of the NRC publication Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D.C., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

[0181] The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

[0182] In a particular embodiment, the animal feed composition of the invention contains at least one vegetable protein as defined above.

[0183] The animal feed composition of the invention may also contain animal protein, such as Meat and Bone Meal, Feather meal, and / or Fish Meal, typically in an amount of 0-25%. The animal feed composition of the invention may also comprise Dried Distillers Grains with Solubles (DDGS), typically in amounts of 0-30%.

[0184] In still further particular embodiments, the animal feed composition of the invention contains 0- 80% maize; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% Barley; and / or 0-30% oats; and / or 0-40% soybean meal; and / or 0-25% fish meal; and / or 0-25% meat and bone meal; and / or 0-20% whey.

[0185] The animal feed may comprise vegetable proteins. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% (w / w). Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example, materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal, rapeseed meal, and combinations thereof.

[0186] In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rapeseed, and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

[0187] Animal diets can e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, for mash feed a solid or liquid enzyme formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) dispersin variant preparation may also be added before or during the feed ingredient step. Typically a liquid dispersin variant preparation comprises the dispersin variant of the invention optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The enzyme may also be incorporated in a feed additive or premix.

[0188] Alternatively, the dispersin variant can be prepared by freezing a mixture of liquid enzyme solution with a bulking agent such as ground soybean meal, and then lyophilizing the mixture.

[0189] In an embodiment, the animal feed or animal feed additive comprises one or more additional enzymes. In an embodiment, the animal feed comprises one or more microbes. In an embodiment, the animal feed comprises one or more vitamins. In an embodiment, the animal feed comprises one or more minerals. In an embodiment, the animal feed comprises one or more amino acids. In an embodiment, the animal feed comprises one or more other feed ingredients.

[0190] In another embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more additional enzymes. In an embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more microbes. In an embodiment, the animal feed comprises the polypeptide of the invention, one or more formulating agents and one or more vitamins. In an embodiment, the animal feed or animal feed additive comprises one or more minerals. In an embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more amino acids. In an embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more other feed ingredients.

[0191] In a further embodiment, the animal feed or animal feed additive comprises the polypeptide of the invention, one or more formulating agents and one or more components selected from the list consisting of: one or more additional enzymes; one or more microbes; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

[0192] The final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme protein per kg diet, preferably between 0.05-100 mg / kg diet, more preferably 0.1-50 mg, even more preferably 0.2-20 mg enzyme protein per kg animal diet.

[0193] It is at present contemplated that the enzyme is administered in one or more of the following amounts (dosage ranges): 0.01-200; 0.05-100; 0.1-50; 0.2-20; 0.1-1 ; 0.2-2; 0.5-5; or 1-10; - all these ranges being in mg hexoaminidase protein per kg feed (ppm).

[0194] For determining mg of hexoaminidase per kg feed, the hexoaminidase is purified from the feed composition, and the specific activity of the purified hexoaminidase is determined using a relevant assay (see under dispersin activity). The hexoaminidase activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg hexoaminidase protein per kg feed is calculated.

[0195] In a particular embodiment, the animal feed additive of the invention is intended for being included (or prescribed as having to be included) in animal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This is so in particular for premixes.

[0196] The same principles apply for determining mg hexoaminidaseprotein in feed additives. Of course, if a sample is available of the hexoaminidase used for preparing the feed additive or the feed, the specific activity is determined from this sample (no need to purify the hexoaminidase from the feed composition or the additive).

[0197] The composition of the invention may further comprise one or more amino acids. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

[0198] In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and / or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and / or one or more macro minerals.

[0199] Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed.

[0200] Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3.

[0201] Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate.

[0202] Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc.

[0203] Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.

[0204] The nutritional requirements of these components (exemplified with poultry and piglets / pigs) are listed in Table A of WO 01 / 58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated.

[0205] In the alternative, the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01 / 58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide an in-feed-concentration within the range indicated in column four, or column five, or column six of Table A.

[0206] In a still further embodiment, the animal feed additive of the invention comprises at least one of the below vitamins, preferably to provide an in-feed-concentration within the ranges specified in the below Table 1 (for piglet diets, and broiler diets, respectively).

[0207] Table 1: Typical vitamin recommendations

[0208] The composition of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds / flavourings, polyunsaturated fatty acids (PLIFAs); reactive oxygen generating species, anti-microbial peptides and anti-fungal polypeptides. Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein. Examples of aroma compounds / flavourings are creosol, anethol, deca-, undeca-and / or dodecalactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin. Examples of antimicrobial peptides (AMP’s) are CAP18, Leucocin A, Tritrpticin, Protegrin-1 , Thanatin, Defensin, Lactoferrin, Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000), Plectasins, and Statins, including the compounds and polypeptides disclosed in WO 03 / 044049 and WO 03 / 048148, as well as variants or fragments of the above that retain antimicrobial activity. Examples of antifungal polypeptides (AFP’s) are the Aspergillus giganteus, and Aspergillus niger peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94 / 01459 and WO 02 / 090384. Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate.

[0209] The composition of the invention may further comprise at least one amino acid. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

[0210] The present invention also relates to liquid compositions comprising a variant of the invention. The composition may comprise an enzyme stabilizer (examples of which include polyols such as propylene glycol or glycerol, sugar or sugar alcohol, lactic acid, reversible protease inhibitor, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid). In some embodiments, filler(s) or carrier matenal(s) are included to increase the volume of such compositions. Suitable filler or carrier materials include, but are not limited to, various salts of sulfate, carbonate and silicate as well as talc, clay and the like. Suitable filler or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

[0211] In an aspect, the liquid formulation comprises 20-80% w / w of polyol. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative.

[0212] In another embodiment, the invention relates to liquid formulations comprising:

[0213] (A) 0.001-25% w / w of a variant of the present invention;

[0214] (B) 20-80% w / w of polyol;

[0215] (C) optionally 0.001-2% w / w preservative; and

[0216] (D) water.

[0217] In another embodiment, the invention relates to liquid formulations comprising:

[0218] (A) 0.001-25% w / w of a variant of the present invention;

[0219] (B) 0.001-2% w / w preservative;

[0220] (C) optionally 20-80% w / w of polyol; and

[0221] (D) water.

[0222] In another embodiment, the liquid formulation comprises one or more formulating agents, such as a formulating agent selected from the group consisting of polyol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetate and phosphate, preferably selected from the group consisting of sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin and calcium carbonate. In one embodiment, the polyols is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600, more preferably selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG) or any combination thereof.

[0223] In another embodiment, the liquid formulation comprises 20-80% polyol ( / .e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol. In one embodiment, the liquid formulation comprises 20-80% polyol, e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1 ,2-propylene glycol or 1 ,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight below about 600 and polypropylene glycol (PPG) having an average molecular weight below about 600. In one embodiment, the liquid formulation comprises 20-80% polyol ( / .e., total amount of polyol), e.g., 25-75% polyol, 30-70% polyol, 35-65% polyol, or 40-60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol and propylene glycol (MPG).

[0224] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% w / w preservative, e.g., 0.05-1 % w / w preservative or 0.1 -0.5% w / w preservative. In one embodiment, the liquid formulation comprises 0.001-2% w / w preservative ( / .e., total amount of preservative), e.g., 0.02- 1.5% w / w preservative, 0.05-1% w / w preservative, or 0.1-0.5% w / w preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof.

[0225] Use in Animal Feed

[0226] A dispersin variant of the invention may also be used in animal feed. In an embodiment, the present invention provides a method for preparing an animal feed composition comprising adding one or more hexoaminidases of the present invention to one or more animal feed ingredients.

[0227] One aspect of the present invention relates to a polypeptide having hexosaminidase activity. A polypeptide may be understood as a polymer consisting of a chain of amino acids linked by peptide bonds, which can perform a variety of functions in biological organisms, including enzymatic activity. Hexosaminidase activity refers to the enzymatic function of cleaving hexose sugars from larger molecules, a process that is significant in the degradation of complex carbohydrates like glycosaminoglycans.

[0228] It may be provided that the polypeptide has hexosaminidase activity. Hexosaminidase activity involves the enzymatic cleavage of terminal N-acetylhexosamines from glycoproteins and glycolipids. One advantage of this arrangement is its potential to degrade biofilm components effectively. This enzymatic action can disrupt the extracellular polymeric substances (EPS) matrix within biofilms, thereby reducing the protective barrier that bacteria use to shield themselves from external stresses, including antibiotic treatments. By breaking down the biofilm matrix, the hexosaminidase activity facilitates enhanced penetration of antimicrobial agents, leading to more effective eradication of bacterial communities.

[0229] One of the key components of EPS in many biofilms is poly-p-1 ,6-linked N-acetyl-D- glucosamine (PNAG). By hydrolyzing these polysaccharides into smaller, more accessible mono- and oligosaccharides, dispersin makes these sugars available to beneficial gut bacteria such as lactic acid bacteria, including Bifidobacteria.

[0230] Mono- and oligosaccharides serve as prebiotics, which are non-digestible food ingredients that promote the growth and activity of beneficial microorganisms. In the gut, lactic acid bacteria like Bifidobacteria utilize these prebiotic sugars for their metabolism, leading to the production of short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate. SCFAs play a crucial role in maintaining gut health by reducing inflammation, enhancing the integrity of the gut barrier, and modulating the immune system.

[0231] By providing readily available prebiotic nutrients to beneficial bacteria, dispersin indirectly supports their proliferation. The increased population of Bifidobacteria and other lactic acid bacteria helps outcompete pathogenic microorganisms, reducing the risk of infections and inflammation. Additionally, the metabolic activities of these beneficial bacteria produce antiinflammatory compounds that further contribute to reducing inflammation in the gut. Accordingly, as aspect of the invention is directed to the use of the polypeptides of the invention to prevent or reduce inflammation in the gut in livestock animals.

[0232] Thus, dispersin helps reduce inflammation by breaking down complex polysaccharides into simpler sugars that act as prebiotics for beneficial lactic acid bacteria. This process not only supports the growth of a healthy gut microbiome but also aids in the production of metabolites that have anti-inflammatory properties, promoting overall gut health and reducing the risk of inflammatory diseases. Accordingly, as aspect of the invention is directed to the use of the polypeptides of the invention to prevent or reduce inflammation in livestock animals.

[0233] It may further be provided that the polypeptide has at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1. Sequence identity refers to the degree of similarity between two sequences of amino acids, expressed as a percentage, with a higher percentage indicating greater similarity. One advantage of this arrangement is the preservation of functional integrity while allowing for variations that can enhance stability, specificity, or activity under different environmental conditions. This range of sequence identity ensures that the polypeptide retains its hexosaminidase activity while potentially offering improved characteristics such as increased resistance to proteolytic degradation or enhanced activity in varying pH levels. Such qualities are particularly advantageous for applications requiring gastric stability for oral administration, thus addressing one of the major limitations in current enzyme-based treatments for biofilm-related infections.

[0234] Another aspect of the invention is directed to the use of polypeptide of the invention to reduce the likelihood of development of antibiotic resistance. The use of dispersins allow at least in part for the continued utilization of antibiotics susceptible to resistance.

[0235] By incorporating these specific features, the polypeptides described in the present invention offer a promising approach to overcoming the challenges associated with biofilm degradation and infection prevention. The ability to maintain hexosaminidase activity while allowing for sequence variability provides a robust framework for developing effective treatments that can adapt to various clinical and industrial needs.

[0236] It may be provided that said polypeptide is for use in the prevention or reduction of inflammatory and metabolic diseases in a subject or patient in need thereof. Inflammatory and metabolic diseases are disorders characterized by abnormal inflammatory responses and metabolic imbalances, respectively. One advantage of this arrangement is the polypeptide's ability to modulate inflammatory pathways and metabolic processes, thereby providing therapeutic benefits to patients suffering from such conditions.

[0237] It may be provided that said polypeptide is for use in the prevention or treatment of inflammatory and metabolic diseases in a subject or patient in need thereof according to claim 9, wherein the inflammatory and metabolic diseases are selected from the group consisting of type 2 diabetes, insulin resistance, obesity, cardiovascular disease, Inflammatory Bowel Disease (IBD) including Crohn's disease and ulcerative colitis, and colitis. Colitis refers to inflammation of the colon. One advantage of this arrangement is the specificity with which the polypeptide can target and treat a range of inflammatory and metabolic diseases, potentially improving patient outcomes across multiple conditions.

[0238] The dispersin variant of the present invention may be for use in one embodiment to stabilize the healthy microflora of animals, in particular livestock. An aspect of the invention is the use of a dispersin variant of the invention or the animal feed additive of the invention to to stabilize the healthy microflora of animals, in particular livestock.

[0239] The feed addtivie of the present invention may for example be used to stabilize or support the healthy microflora of animals such as pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks and chicken (including but not limited to broiler chicks, layers); horses (including but not limited to hotbloods, coldbloods and warm bloods), and young calves.

[0240] The feed additive of the present invention may for example be used to stabilize the healthy microflora of animals such as fish include but are not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, sea bass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout, tuna, turbot, vendace, walleye and whitefish. Examples of crustaceans include but are not limited to shrimps and prawns.

[0241] The animal feed of the invention may, without being bound to a particular theory, stabilize the healthy microflora of animals by suppressing growth / intestinal colonization of bacterial pathogens such as Enterococcus cecorum, Enterococcus faecalis, Enterococcus faecium, Listeria species, Campylobacter species, Clostridium perfringens, Escherichia coli, Campylobacter coli, Campylobacter hyointestinalis and Campylobacter jejuni, Yersinia ssp., Treponema suis, Brachyspira hyodysenteriae, Lawsonia intracellularis and Salmonella, such as Salmonella enterica, Salmonella typhimurium, Salmonella mbandaka, Salmonella enterica serovar Typhi and typhimurium.

[0242] In a further embodiment a dispersin variant of the present invention is used as a feed additive, where it may provide a positive effect on the microbial balance of the chicken digestive tract and in this way improve animal performance.

[0243] The one or more dispersin variants of the present invention may also be used in animal feed as feed enhancing enzymes that improve feed digestibility to increase the efficiency of its utilization according to WO 00 / 21381 and WO 04 / 026334.

[0244] In a further embodiment a dispersin variant of the present invention may be used as a feed additive, where it may provide a positive effect on the animals’ digestive tract and in this way improve animal performance in accordance to weight gain, feed conversion ratio (FOR), or improved animal health such as decreased mortality rate. FOR is calculated as the feed intake in g / animal relative to the weight gain in g / animal.

[0245] In the use according to the invention the dispersin variants can be fed to the animal before, after, or simultaneously with the diet. The latter is preferred.

[0246] In a particular embodiment, the form of the dispersin variants when it is added to the feed or when it is included in a feed additive is well-defined. Well-defined means that the dispersin variants preparation is at least 50% pure as determined by Size-exclusion chromatography (see Example 12 of WO 01 / 58275). In other particular embodiments the dispersin variants preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.

[0247] A well-defined dispersin variant preparation is advantageous. For instance, it is much easier to dose correctly to the feed a dispersin variant that is essentially free from interfering or contaminating other dispersin variants. The term dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimizing dosage based upon the desired effect.

[0248] For the use in animal feed, however, the dispersin variants need not be pure; it may e.g. include other enzymes, in which case it could be termed a dispersin variant preparation.

[0249] The dispersin variants preparation can be (a) added directly to the feed, or (b) it can be used in the production of one or more intermediate compositions such as feed additives or premixes that is subsequently added to the feed (or used in a treatment process). The degree of purity described above refers to the purity of the original dispersin variant preparation, whether used according to (a) or (b) above.

[0250] A further aspect of the invention is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving intestinal health of an animal. An aspect of the invention is direct to a method of improving the intestinal health of an animal comprising feeding the animal the polypeptide or the animal feed feed additive of the invention. A further aspect of the invention is directed to a method of increasing the relative and / or absolute level of beneficial gut microbes in an animal. Beneficial gut microbes may be selected from the group Lactic acid bacteria, Bifidiobacteria and Corynebacterium sp.

[0251] A further aspect of the invention is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing the relative and / or the absolute level of Enterococcus cecorum in the intestine and / or the relative and / or the absolute level of Escherichia coli in the intestine. Another aspect is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for the reduction of pathogenic bacteria present in faeces. In one embodiment, the reduction of pathogenic species relates to a reduction of E.coli in faeces.

[0252] The ratio of microbes within the intestinal microbiome is important for intestinal health. A further aspect of the invention is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the ratio of lactic acid bacteria to Enterococcus species. The lactic acid bacteria may be any Lactobacillaceae including Lactobacillus, Ligilactobacillus and Limosilactobacilus species. The invention is furthermore directed to method of reducing the relative and / or absolute level / abundance of Enterococcus cecorum in the intestine of an animal comprising feeding the animal the polypeptide or the animal feed additive of the invention. Another aspect of the invention is directed to a method of increasing the ratio of lactic acid bacteria to Enterococcus species ratio (Lactic acid bacteria: Enterococcus species ratio) of an animal comprising feeding the animal the polypeptide or the animal feed additive of the invention. Stated otherwise, the invention is directed to a method of decreasing the ratio of Enterococci to Lactobacillaceae taxa in an animal, as demonstrated in Example 7. The lactic acid bacteria may be any Lactobacillaceae including Lactobacillus, Ligilactobacillus and Limosilactobacilus species, and Enterococcus species were compared, i.e. Lactobacillaceae abundance / Enterococcus abundance).

[0253] A further aspect of the invention is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the relative and / or absolute level of Bifidobacteria, such as Bifidobacterium pseudoIongum, Bifidobacterium bifidum, Bifidobacterium long urn, Bifidobacterium thermophilium, Bifidobacterium inf antis and Bifidobacterium suis in the intestinal microbiome of an animal. A further aspect of the invention is directed to a method of increasing the relative and / or absolute level of Bifidobacteria, a beneficial gut microbe, such as Bifidobacterium pseudoIongum, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium thermophilium, Bifidobacterium inf antis and Bifidobacterium suis in the intestinal microbiome of an animal, said method comprising feeding the animal the polypeptide or animal feed additive of the invention.

[0254] A further aspect of the invention is directed to a method of improving the relative and / or absolute level of Corynebacterium sp in an animal comprising feeding the animal the polypeptide or animal feed additive of the invention. In an alternative aspect, the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving the relative and / or absolute level of Corynebacterium sp.

[0255] A further aspect of the invention is directed to a method of preventing or reducing the prevalence of cellulitis in poultry, said method comprising feeding the animal the polypeptide or the animal feed additive of the invention. In the alternative, the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preventing or reducing the prevalence of cellulitis in poultry.

[0256] A further aspect of the invention is directed to a method of maintaining / preserving intestinal paracellular permeability or gut integrity in an animal comprising feeding the animal the polypeptide or the animal feed additive of the invention. A further aspect of the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for maintaining or preserving intestinal paracellular permeability or gut integrity in an animal.

[0257] A further aspect of the invention is directed to a method of reducing coccidiosis levels in chickens or preventing the spread of coccidosis in a poultry population or reducing the levels of Eimeria in a poultry population, typically broiler chickens. A related as pect of the invention is directed to a method of reducing the level of Eimeria or preventing or treating Eimeria, comprising feeding the animal the polypeptide or the animal feed additive of the invention.

[0258] A further aspect of the invention is directed to a method of reducing the levels of Avian Pathogenic Escherichia coli (APEC) in a poultry population comprising feeding the animal the polypeptide or the animal feed additive of the invention.

[0259] A further aspect of the invention is directed to a method of reducing the abundance of bacteria selected from Salmonella, Listeria species, E. coli and Campylobacter in a poultry population) comprising feeding the animal the polypeptide or the animal feed additive of the invention. A further aspect of the invention is directed to a method of reducing the dose of antibiotics in an animal feed or in poultry farming comprising feeding the animal the polypeptide or the animal feed additive of the invention.

[0260] A further aspect of the invention is directed to a method of preventing or reducing the level of skin infections in poultry comprising feeding the animal the polypeptide or the animal feed additive of the invention. Typically, the skin infections may be related or caused by E. coli or Staphylococcus infections, as known to the person skilled in the art.

[0261] An aspect of the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving the performance of an animal. Accordingly, a further aspect of the invention is directed to a method of improving the performance of an animal comprising feeding the animal the polypeptide or the animal feed additive of the invention, said performance selected from the group consisting of a. a reduced feed conversion ratio (FCR), and b. an increased average body weight gain comprising feeding the animal the polypeptide or the animal feed additive of the invention. The increase in performance is preferably in an animal selected from the group consisting of fowl or swine, such as a pig, sow, piglet, poultry, turkey, duck and goose, typically broiler chickens, layer chickens, sows and piglets.

[0262] Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for increasing the relative and / or absolute level of Bifidobacteria, such as Bifidobacterium pseudoIongum, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium thermophilium, Bifidobacterium inf antis and Bifidobacterium suis in the intestinal microbiome of an animal, (a beneficial gut microbe).

[0263] A further aspect of the invention is directed to a method of reducing the infection levels of microbial species that generates exopolysaccharides containing (3-1 ,6-linked poly-N- acetylglucosamine (polyGIcNAc). These microbial species have as a mechanism of action to translocate intestinal epithelial monolayer surfaces in an animal. Accordingly, the invention is directed to the use of a polypeptide of the invention or the animal feed additive of the invention for lowering intestinal levels of microbial species that generate exopolysaccharides containing p- 1 ,6-linked poly-N-acetylglucosamine (polyGIcNAc) in animals or lowering intestinal levels of microbial species that make use of or otherwise exploit the exopolysaccharides containing p-1 ,6- linked poly-N-acetylglucosamine (polyGIcNAc) produced by other microbes. Microbial species that produce exopolysaccharides containing p-1 ,6-linked poly-N-acetylglucosamine (polyGIcNAc) can be selected from the group consisting of E. coli, Enterococcus faecalis, Enterococcus faecium, Campylobacter jejuni, Salmonella enterica serovar Typhi and typhimurium. A related aspect of the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the release of N-acetyl glucosamine (NAG) in the gastrointestinal tract. An aspect of the invention is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the intestinal hydrolysis of PNAG. Alternatively defined, a related aspect of the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the liberation N-acetyl glucosamine (NAG) in the gastrointestinal tract. However, the liberated PNAG may be consumed or otherwis utilized by healthy bacteria, thereby reducing the levels. In one embodiment, is directed to the use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 to reduce bacterial load in faeces. This has the further use of reducing bacterial infections on a farm and thus of great value to a farmer.

[0264] A further relates aspect of the invention is directed to use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 to promote mucin production in the gastrointestinal tract of an animal.

[0265] Preparation of Variants

[0266] An aspect of the invention is directed to a method of increasing the gastric stability of a dispersin comprising the step of mutating the amino acid residue at three or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1 , such as five or more positions, seven or more positions, nine or more positions, eleven or more positions, thirteen or more positions, fifteen or more positions, seventeen or more positions, such as at each of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324.

[0267] The present invention also relates to methods for obtaining a variant having hexoaminidase activity, comprising: (a) introducing into a parent dispersin having at least 70% sequence identity to SEQ ID NO: 1 a substitution, an insertion or a deletion, preferably a substitution or deletion at one or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ I D NO: 1 , wherein the variant has hexoaminidase activity; and (b) recovering the variant. The variants can be prepared using any mutagenesis procedure known in the art, such as site-directed mutagenesis, synthetic gene construction, semi-synthetic gene construction, random mutagenesis, shuffling, etc.

[0268] Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a polynucleotide encoding the parent.

[0269] Site-directed mutagenesis can be accomplished in vitro by PCR involving the use of oligonucleotide primers containing the desired mutation. Site-directed mutagenesis can also be performed in vitro by cassette mutagenesis involving the cleavage by a restriction enzyme at a site in the plasmid comprising a polynucleotide encoding the parent and subsequent ligation of an oligonucleotide containing the mutation in the polynucleotide. Usually the restriction enzyme that digests the plasmid and the oligonucleotide is the same, permitting sticky ends of the plasmid and the insert to ligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 7Q: 4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18: 7349-4966.

[0270] Site-directed mutagenesis can also be accomplished in vivo by methods known in the art. See, e.g., US 2004 / 0171154; Storici et al., 2001 , Nature Biotechnol. 19: 773-776; Kren et al., 1998, Nat. Med. 4: 285-290; and Calissano and Macino, 1996, Fungal Genet. Newslett. 43: 15- 16.

[0271] Any site-directed mutagenesis procedure can be used in the present invention. There are many commercial kits available that can be used to prepare variants.

[0272] Synthetic gene construction entails in vitro synthesis of a designed polynucleotide molecule to encode a polypeptide of interest. Gene synthesis can be performed utilizing a number of techniques, such as the multiplex microchip-based technology described by Tian et al., 2004, Nature 432: 1050-1054, and similar technologies wherein oligonucleotides are synthesized and assembled upon photo-programmable microfluidic chips.

[0273] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0274] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. Semi-synthetic gene construction is accomplished by combining aspects of synthetic gene construction, and / or site-directed mutagenesis, and / or random mutagenesis, and / or shuffling. Semi-synthetic construction is typified by a process utilizing polynucleotide fragments that are synthesized, in combination with PCR techniques. Defined regions of genes may thus be synthesized de novo, while other regions may be amplified using site-specific mutagenic primers, while yet other regions may be subjected to error-prone PCR or non-error prone PCR amplification. Polynucleotide subsequences may then be shuffled.

[0275] The present invention also relates to polynucleotides encoding a variant of the present invention.

[0276] The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.

[0277] In an aspect, the polynucleotide is isolated.

[0278] In another aspect, the polynucleotide is purified.

[0279] The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a variant of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0280] The polynucleotide may be manipulated in a variety of ways to provide for expression of a variant. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0281] The control sequence may be a promoter, a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a variant of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the variant. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0282] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3’-terminus of the polynucleotide encoding the variant. Any terminator that is functional in the host cell may be used in the present invention.

[0283] The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

[0284] Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94 / 25612) and a Bacillus subtilis SP82 gene (Hue etal., 1995, J. Bacteriol. 177: 3465-3471).

[0285] Examples of mRNA stabilizer regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4): 812-824, and in Morozov et al., 2006, Eukaryotic Ce / / 5(11): 1838-1846. The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5’-terminus of the polynucleotide encoding the variant. Any leader that is functional in the host cell may be used.

[0286] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used.

[0287] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a variant and directs the variant into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the variant. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the variant. However, any signal peptide coding sequence that directs the expressed variant into the secretory pathway of a host cell may be used.

[0288] The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a variant. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active variant by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0289] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a variant and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

[0290] It may also be desirable to add regulatory sequences that regulate expression of the variant relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.

[0291] The control sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of the transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. The transcription factor may function alone and / or together with one or more other polypeptides or transcription factors in a complex by promoting or blocking the recruitment of RNA polymerase. Transcription factors are characterized by comprising at least one DNA-bindmg domain which often attaches to a specific DNA sequence adjacent to the genetic elements which are regulated by the transcription factor. The transcription factor may regulate the expression of a protein of interest either directly, i.e., by activating the transcription of the gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor which regulates the transcription of the gene encoding the protein of interest, such as by binding to the promoter of the further transcription factor. Suitable transcription factors for fungal host cells are described in WO 2017 / 144177. Suitable transcription factors for prokaryotic host cells are described in Seshasayee et al., 2011 , Subcellular Biochemistry 52: 7- 23, as well in Balleza et al., 2009, FEMS Microbiol. Rev. 33(1): 133-151.

[0292] The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a variant of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the variant at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0293] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0294] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0295] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0296] For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).

[0297] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0298] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0299] The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a variant of the present invention.

[0300] A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra- chromosomal vector as described earlier. The choice of a host cell will to a large extent depend upon the gene encoding the variant and its source. The recombinant host cell may comprise a single copy, or at least two copies, e.g., three, four, five, or more copies of the polynucleotide of the present invention.

[0301] The host cell may be any cell useful in the recombinant production of a variant of the invention, e.g., a prokaryotic cell or a fungal cell.

[0302] The host cell may be any microbial cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryotic cell or a fungal cell.

[0303] Methods for introducing DNA into prokaryotic host cells are well-known in the art, and any suitable method can be used including but not limited to protoplast transformation, competent cell transformation, electroporation, conjugation, transduction, with DNA introduced as linearized or as circular polynucleotide. Persons skilled in the art will be readily able to identify a suitable method for introducing DNA into a given prokaryotic cell depending, e.g., on the genus. Methods for introducing DNA into prokaryotic host cells are for example described in Heinze et al., 2018, BMC Microbiology 18:56, Burke et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 6289-6294, Choi et al., 2006, J. Microbiol. Methods 64: 391-397, and Donald et al., 2013, J. Bacteriol. 195(11): 2612- 2620.

[0304] The present invention also relates to methods of producing a variant of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the variant; and optionally (b) recovering the variant.

[0305] The host cell is cultivated in a nutrient medium suitable for production of the variant using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the variant to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the variant is secreted into the nutrient medium, the variant can be recovered directly from the medium. If the variant is not secreted, it can be recovered from cell lysates.

[0306] The variant may be detected using methods known in the art that are specific for the variant, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an enzyme assay determining the relative or specific activity of the variant.

[0307] The variant may be recovered from the medium using methods known in the art, including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, a cell- free fermentation broth comprising the polypeptide is recovered.

[0308] The variant may be purified by a variety of procedures known in the art to obtain substantially pure variants and / or fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science-, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0309] In an alternative aspect, the variant is not recovered.

[0310] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0311] Preferred Embodiments

[0312] The invention is further defined by the following numbered paragraphs:

[0313] 1. An animal feed additive comprising a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1. 2. The animal feed additive according to paragraph 1 , having a residual activity at pH 3 of at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96, at least 97, at least 98, or at least 99%, wherein the residual activity is measured after 30 minutes at a temperature of 40 °C according to Residual Activity Assay as described in Example 4

[0314] 3. The animal feed additive according to paragraph 1 , having a residual activity as measured at pH 3 and upon pepsin exposure, preferably at pH 3 and at a pepsin concentration of 2775 U / rnL wherein the residual activity is measured after 30 minutes at a temperature of 40 °C according to Residual Activity Assay as described in Example 4

[0315] 4. The animal feed additive according to paragraph 1 or 2, wherein said polypeptide has at least 75% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1.

[0316] 5. The animal feed additive according to paragraphs 1 to 3, where said polypeptide comprises a substitution at one or more, such as two or more, such as three or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1 , such as five or more positions, seven or more positions, nine or more positions, eleven or more positions, thirteen or more positions, fifteen or more positions, seventeen or more positions, such as at each of positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324.

[0317] 6. The animal feed additive according to any of the paragraphs 1 to 4 comprising three or more substitutions, such as five or more substitutions, seven or more substitutions, nine or more substitutions, eleven or more substitutions, thirteen or more substitutions, fifteen or more substitutions, seventeen or more substitutions selected from the group consisting of Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q.

[0318] 7. The animal feed additive according to paragraphs 1 to 5, selected from the group consisting of a. a polypeptide having at least 80% sequence identity to SEQ ID NO: 2; b. a polypeptide having at least 80% sequence identity to SEQ ID NO: 3; c. a polypeptide having at least 80% sequence identity to SEQ ID NO: 4; d. a polypeptide having at least 80% sequence identity to SEQ ID NO: 5; e. a polypeptide having at least 80% sequence identity to SEQ ID NO: 6; f. a polypeptide having at least 80% sequence identity to SEQ ID NO: 7; g. a polypeptide having at least 80% sequence identity to SEQ ID NO: 8; h. a polypeptide having at least 80% sequence identity to SEQ ID NO: 9; i. a polypeptide having at least 80% sequence identity to SEQ ID NO: 10; j. a polypeptide having at least 80% sequence identity to SEQ ID NO: 11 ; k. a polypeptide having at least 80% sequence identity to SEQ ID NO: 12; l. a polypeptide having at least 80% sequence identity to SEQ ID NO: 13; m. a polypeptide having at least 80% sequence identity to SEQ ID NO: 14; n. a polypeptide having at least 80% sequence identity to SEQ ID NO: 15; and o. a polypeptide having at least 80% sequence identity to SEQ ID NO: 16. An animal feed additive comprising a polypeptide having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 and comprising mutations selected from the group consisting of i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q ix. Q1G Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aG; xii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324al; xiii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q L324*; xiv. *1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; and xv. Q1* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q The animal feed additive according to any of the preceding claims further comprising an ingredient selected from the group consisting of a. at least one fat soluble vitamin, and / or a. at least one water soluble vitamin, and / or a. at least one trace mineral. . The animal feed additive according to any of the preceding paragraphs in granule form. . A method of increasing the gastric stability of a dispersin comprising the step of mutating the amino acid residue at three or more positions corresponding to positions 3, 15, 49, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309, and 312 of the polypeptide of SEQ ID NO: 1 , such as five or more positions, seven or more positions, nine or more positions, eleven or more positions, thirteen or more positions, fifteen or more positions, seventeen or more positions, such as at each of positions 3, 15, 49, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309, and 312. . A method of improving intestinal health of an animal comprising feeding the animal the polypeptide or the animal feed additive of paragraphs 1 to 9 . A method of reducing the relative and / or absolute level / abundance of Enterococcus cecorum in the intestine of an animal comprising feeding the animal the animal feed additive of paragraphs 1 to 9. . A method of increasing the ratio of lactic acid bacteria to Enterococcus species ratio of an animal comprising feeding the animal the animal feed additive of paragraphs 1 to 9 (Lactobacillaceae (Lactobacillus, Ligilactobacillus and Limosilactobacilus species) and Enterococcus species were compared, i.e. Lactobacillaceae abundance I Enterococcus abundance) . A method of increasing the relative and / or absolute level of Bifidobacteria, such as Bifidobacterium pseudoIongum, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium thermophilium, Bifidobacterium inf antis and Bifidobacterium suis in the intestinal microbiome of an animal, (a beneficial gut microbe) comprising feeding the animal the animal feed additive of paragraphs 1 to 9. . A method of preventing or reducing the prevalence of cellulitis in poultry, said method comprising feeding the animal the animal feed additive of paragraphs 1 to 9. . A method of maintaining / preserving intestinal paracellular permeability or gut integrity in an animal comprising feeding the animal the animal feed additive of claims 1-9 comprising feeding the animal the animal feed additive of paragraphs 1 to 9. . A method of improving the relative and / or absolute level of Corynebacterium sp in an animal comprising feeding the animal the animal feed additive of paragraphs 1 to 9. 9. A method of improving the performance of an animal comprising feeding the animal the polypeptide or the animal feed additive of the invention, said performance selected from the group consisting of a. reduced feed conversion ratio (FCR), and b. an increased average body weight gain

[0319] 20. A method of reducing coccidiosis levels in broiler chickens comprising feeding the animal the animal feed additive of paragraphs 1 to 9.

[0320] 21. A method of reducing the levels of Eimeria in a poultry population comprising feeding the animal the animal feed additive of paragraphs 1 to 9.

[0321] 22. A method of reducing the levels of Avian Pathogenic Escherichia coli (APEC) in a poultry population comprising feeding the animal the animal feed additive of paragraphs 1 to 9.

[0322] 23. A method of reducing the abundance of bacteria selected from Salmonella, Listeria species, E. coli and Campylobacter (such as in a chicken or swine population) comprising feeding the animal the animal feed additive of paragraphs 1 to 9.

[0323] 24. A method of reducing the dose of antibiotics in poultry farming comprising feeding the animal the animal feed additive of paragraphs 1 to 9.

[0324] 25. A method of preventing or reducing the level of skin infections in poultry comprising feeding the animal the animal feed additive of paragraphs 1 to 9.

[0325] 26. The method according to paragraph 24 wherein the skin infections are from Staphylococcus infections.

[0326] 27. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving intestinal health of an animal.

[0327] 28. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing the relative and / or the absolute level of Enterococcus cecorum in the intestine and / or the relative and / or the absolute level of Escherichia coli n the intestine.

[0328] 29. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the ratio of lactic acid bacteria to Enterococcus species (Lactobacillaceae (Lactobacillus, Ligilactobacillus and Limosilactobacilus species) and Enterococcus species were compared, i.e. Lactobacillaceae abundance I Enterococcus abundance)

[0329] 30. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for increasing the relative and / or absolute level of Bifidobacteria, such as Bifidobacterium pseudoIongum, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium thermophilium, Bifidobacterium infantis and Bifidobacterium suis in the intestinal microbiome of an animal, (a beneficial gut microbe) 31. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preventing or reducing the prevalence of cellulitis in poultry, said method comprising feeding the animal the animal feed additive of paragraphs 1-9.

[0330] 32. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for maintaining / preserving intestinal paracellular permeability or gut integrity in an animal.

[0331] 33. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving the relative and / or absolute level of Corynebacterium sp

[0332] 34. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving the performance of an animal , said performance selected from the group consisting of a. a reduced feed conversion ratio (FCR), and b. an increased body weight gain.

[0333] 35. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing coccidiosis in broiler chickens infected with Eimeria,

[0334] 36. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing the abundance of bacteria selected from Salmonella, Listeria species, E. coli and Campylobacter.

[0335] 37. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing the dose of antibiotics in poultry farming

[0336] 38. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preventing or reducing the level of skin infections in poultry comprising feeding the animal the animal feed additive of paragraphs 1-9.

[0337] 39. The use according to paragraph 37 wherein the skin infections are from Staphylococcus infections.

[0338] 40. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for improving intestinal health of an animal.

[0339] 41. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for reducing the relative and / or the absolute level of Enterococcus cecorum in the intestine and / or the relative and / or the absolute level of Escherichia colijn the intestine. 42. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for increasing the ratio of lactic acid bacteria to Enterococcus species (Lactobacillaceae (Lactobacillus, Ligilactobacillus and Limosilactobacilus species) and Enterococcus species were compared, i.e. Lactobacillaceae abundance I Enterococcus abundance)

[0340] 43. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for increasing the relative and / or absolute level of Bifidobacteria, such as Bifidobacterium pseudoIongum, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium thermophilium, Bifidobacterium inf antis and Bifidobacterium suis in the intestinal microbiome of an animal.

[0341] 44. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for increasing the intestinal hydrolysis of PNAG.

[0342] 45. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for maintaining / preserving intestinal paracellular permeability or gut integrity in an animal.

[0343] 46. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for improving the relative and / or absolute level of Corynebacterium sp

[0344] 47. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for improving the performance of an animal.

[0345] 48. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for for preparing an animal feed additive reducing coccidiosis in broiler chickens infected with Eimeria

[0346] 49. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for reducing the abundance of bacteria selected from Salmonella, Listeria species, E. coli and Campylobacter in an animal population such as in a chicken population.

[0347] 50. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for reducing the dose of antibiotics in poultry farming. 51 . Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for release of N- acetyl glucosamine (NAG) 2. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 to promote mucin production.

[0348] 53. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for preparing an animal feed additive for the prevention or reduction of Avian Pathogenic Escherichia coli (APEC) in a poultry population.

[0349] 54. A dispersin variant, comprising a substitution at one or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1 , wherein the variant has hexosaminidase activity and wherein the variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1 , wherein the variant optionally comprises an extension of one or more amino acids at the N-terminal and / or C-terminal ends or a truncation of one or more amino acids at the N-terminal and / or C-terminal ends.

[0350] 55. A dispersin variant, comprising a substitution at one or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1 , preferably 5 or more positions, such as 10 or more, such as 12 or more, such as 15 or more positions, wherein the variant has hexosaminidase activity.

[0351] 56. A dispersin variant, comprising a substitution at one or more positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1 , preferably three or more positions, such as five or more positions, typically, seven or more positions, such as ten or more positions, such as twelve or more positions, preferably fifteen or more positions, having an amino acid sequence which has at most 30% sequence differences compared to the parent, wherein the variant has hexosaminidase activity.

[0352] 57. The variant of any one of paragraphs 54-56, which comprises a substitution of the amino acid residue at position 3 with I.

[0353] 58. The variant of any one of paragraphs 54-57, which comprises a substitution of the amino acid residue at position 15 with Y.

[0354] 59. The variant of any one of paragraphs 54-58, which comprises a substitution of the amino acid residue at position 49 with W. 60. The variant of any one of paragraphs 54-59, which comprises a substitution of the ammo acid residue at position 59 with E.

[0355] 61 . The variant of any one of paragraphs 54-60, which comprises a substitution of the amino acid residue at position 163 with P.

[0356] 62. The variant of any one of paragraphs 54-61 , which comprises a substitution of the amino acid residue at position 186 with R.

[0357] 63. The variant of any one of paragraphs 54-62, which comprises a substitution of the amino acid residue at position 225 with G.

[0358] 64. The variant of any one of paragraphs 54-63, which comprises a substitution of the amino acid residue at position 227 with T.

[0359] 65. The variant of any one of paragraphs 54-64, which comprises a substitution of the amino acid residue at position 232 with D.

[0360] 66. The variant of any one of paragraphs 54-65, which comprises a substitution of the amino acid residue at position 235 with W.

[0361] 67. The variant of any one of paragraphs 54-66, which comprises a substitution of the amino acid residue at position 252 with P.

[0362] 68. The variant of any one of paragraphs 54-67, which comprises a substitution of the amino acid residue at position 260 with Q.

[0363] 69. The variant of any one of paragraphs 54-68, which comprises a substitution of the amino acid residue at position 272 with V.

[0364] 70. The variant of any one of paragraphs 54-69, which comprises a substitution of the amino acid residue at position 282 with P.

[0365] 71. The variant of any one of paragraphs 54-70, which comprises a substitution of the amino acid residue at position 308 with Q.

[0366] 72. The variant of any one of paragraphs 54-71 , which comprises a substitution of the amino acid residue at position 309 with E.

[0367] 73. The variant of any one of paragraphs 54-72, which comprises a substitution of the amino acid residue at position 312 with Q.

[0368] 74. A dispersin variant, comprising one or more substitutions selected from the group consisting of Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q of the polypeptide of SEQ ID NO: 1 , preferably 5 or more substitutions, such as 10 or more substitutions, such as 12 or more substitutions, such as 15 or more substitutions, wherein the variant has hexosaminidase activity. The dispersin variant according to any of paragraphs 54 to 74, comprising substitutions of SEQ ID NO: 1 selected from the group consisting of: i. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309Q K312Q; iv. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; v. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q vi. Q1G Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aA; viii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aG; ix. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324al; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q L324*; xi. *1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; and xii. QI* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q. The dispersin variant according to any of paragraphs 54 to 75 selected from the group consisting of a. a variant having at least 80% sequence identity to SEQ ID NO: 3; b. a variant having at least 80% sequence identity to SEQ ID NO: 6; c. a variant having at least 80% sequence identity to SEQ ID NO: 7; d. a variant having at least 80% sequence identity to SEQ ID NO: 8; e. a variant having at least 80% sequence identity to SEQ ID NO: 9; f. a variant having at least 80% sequence identity to SEQ ID NO: 10; g. a variant having at least 80% sequence identity to SEQ ID NO: 11 ; h. a variant having at least 80% sequence identity to SEQ ID NO: 12; i. a variant having at least 80% sequence identity to SEQ ID NO: 13; j. a variant having at least 80% sequence identity to SEQ ID NO: 14; k. a variant having at least 80% sequence identity to SEQ ID NO: 15; and l. a variant having at least 80% sequence identity to SEQ ID NO: 16.

[0369] 77. A granule, which comprises:

[0370] (a) a core comprising the polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 ; and

[0371] (b) a coating consisting of one or more layer(s) surrounding the core, said coating selected from a salt coating or a hydrophobic coating.

[0372] 78. A granule, which comprises:

[0373] (a) a core, and

[0374] (b) a coating consisting of one or more layer(s) surrounding the core, wherein the coating comprises polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 .

[0375] 79. A composition comprising the variant of any one of paragraphs 54 to 76.

[0376] 80. A polynucleotide encoding the variant of any one of paragraphs 54 to 76.

[0377] 81. The polynucleotide of paragraph 80, which is isolated.

[0378] 82. The polynucleotide of paragraph 80 or 81 , which is purified.

[0379] 83. A nucleic acid construct or expression vector comprising the polynucleotide of any one of paragraphs 80-82.

[0380] 84. A recombinant host cell transformed with the polynucleotide of any one of paragraphs 80-82.

[0381] 85. The recombinant host cell of paragraph 84, which comprises at least two copies, e.g., three, four, or five, or more copies of the polynucleotide of any one of paragraphs 80-82.

[0382] 86. The recombinant host cell of paragraph 84 or 85, which is a yeast recombinant host cell, e.g., a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0383] 87. The recombinant host cell of paragraph 84 or 85, which is a filamentous fungal recombinant host cell, e.g., an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Fili basidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell, in particular, an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0384] 88. The recombinant host cell of paragraph 84 or 85, which is a prokaryotic recombinant host cell, e.g., a Gram-positive cell selected from the group consisting of Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces cells, or a Gram-negative bacteria selected from the group consisting of Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma cells, such as Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0385] 89. The recombinant host cell of any one of paragraphs 84-88, which is isolated.

[0386] 90. The recombinant host cell of any one of paragraphs 84-89, which is purified.

[0387] 91. A method of producing a dispersin variant, comprising: i. cultivating the host cell of any one of paragraphs 84-89 under conditions suitable for expression of the variant; and ii. recovering the variant.

[0388] 92. A dispersin variant, comprising a substitution at one or more positions corresponding to positions 3, 15, 49, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309, and 312 of the polypeptide of SEQ ID NO: 1 , wherein the variant has hexosaminidase activity and wherein the variant has at least 75% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1 , wherein the variant optionally comprises an extension of one or more amino acids at the N-terminal and / or C- terminal ends or a truncation of one or more amino acids at the N-terminal and / or C-terminal ends and wherein the variant has hexosaminidase activity. Examples

[0389] Example 1 : Generation of dispersin variants containing several mutations

[0390] Dispersin variants containing several mutations listed in table X were constructed as described in WO 2020 / 207944

[0391] Table 1.1

[0392] Example 2. Gastric stability of dispersin variants and biofilm assay

[0393] Gastric challenge:

[0394] 20ppm of purified enzyme solution of each of SEQ ID 1 to 16 were incubated for 15 minutes at 40°C at pH3 in 0.2M citric acid in the presence and absence of pepsin (500 ll / rnl, Sigma # P7000). Next, enzyme samples were adjusted to pH 7 with 0,3M Na2HPO4 and incubated on ice. In parallel, control samples were prepared by incubating 20ppm of the purified enzyme at pH 7 in 50mM Hepes and incubated for 15 minutes at 40°C, followed by incubation on ice.

[0395] Subsequently, all enzyme samples were diluted in water hardness at 0.3ppm and tested for biofilm removal activity in the biofilm assay against Staphylococcus aureus at 2x serial dilutions performed in water hardness as described in WO 2017 / 186943 A1. All enzymes were assayed per duplicate.

[0396] The lowest concentration of each enzyme that could visibly reduce the biofilm of S. aureus biofilm after 1-hour incubation in enzyme solution was determined as MBRC (Minimal Biofilm Reduction Concentration (see Table 2.1).

[0397] Water hardness composition:

[0398] 4mM NaHCO3+ 2mM CaCI2+ 0,5mM MgCI2,6H20

[0399] Table 2.1. Minimal concentration of enzyme that can visibly reduce the biofilm of S. aureus (MBRC) after 1-hour incubation in enzyme solutions previously incubated at either pH3+pepsin, pH3 or pH7 for 15 minutes at 40°C.

[0400] As shown in the table, all dispersin variants had lower MBRCs compared to dispersin wild type backbone (SEQ ID NO 1) after 15 minutes incubation at pH3 in the presence and absence of pepsin. Thus, the dispersin variants had improved the gastric stability at pH3, since less enzyme was required to reduce the biofilm of S. aureus.

[0401] Example 3: Thermal shift assay (TSA) for determination of TmD

[0402] Protein thermal unfolding of dispersin polypeptides with SEQ ID NO:1 to 17 was monitored with Sypro Orange (Invitrogen, S-6650) using a real-time PCR instrument (Applied Biosystems; Step-One-Plus). Purified enzyme samples were diluted to 0.2 mg / mL in milliQ water. TSA reaction buffers were prepared by diluting Sypro Orange protein stain (#S6650, Invitrogen) 200-fold into pH-multi buffers (100 mM acetic, 100 mM MES, 100 mM HEPES, 100 mM Glycine adjusted to pH 3, 4, 5, 6, 7,8, 9 and 10 with HCI or NaOH)

[0403] Next, 5 pl enzyme sample were mixed with 10 pl TSA reaction buffer in a 384-well qPCR plate (# A36931 , Applied Biosystems / Thermo Fischer Scientific) and the plate sealed with optical adhesive film (#4311971, Applied Biosystems / Thermo Fischer Scientific). The melt curve was determined by running a melt curve protocol (temperature ramp 3.2 °C / min, excision / emission filters: x1 (470 + / - 15 nm) / m3586.5 + / - 10 nm) in the QuantStudio 7 Flex Real-time 384-well PCR system. The melting temperature (TmD) was extracted from the melt curves by the Protein Thermal Shift Software (Applied Biosystems, version 1.4).

[0404] Table 3.1. TSA, Thermal shift analysis of dispersin wt and variants

[0405] Results shown in table 3.1 reveal that all dispersin variants SEQ ID NO: 2 to 16 have a higher thermal stability compared to wild-type GH45 of SEQ ID NO:1 and wild-type GH27 (DispersinB) of SEQ ID NO: 17 at all pH tested, pH3-10. Example 4 - Dispersin Gastric Conditions Stress / Residual Activity Assay Assay Assay purpose

[0406] This protocol describes the assay used to determine the stability of dispersin WTs and variants under gastric stress conditions. Gastric condition stress is defined as: 30 min at pH 3, 40 degC, with exposure to Pepsin protease. Stability of dispersin is evaluated by measuring dispersin activity before and after exposure to stress. Results are given as percent Residual Activity (%RA) defined as the activity of a given sample after stress relative to the same samples’ unstressed activity, i.e. activity before stress is defined as 100%.

[0407] Enzyme samples are diluted in buffer (10 mM MES, 0.01vol% TritonX-100, pH 6)

[0408] Assay protocol

[0409] Split each sample in three different aliquots and dilute to 6 pM in Universal Stress Buffer (40mM Acetic Acid, 40mM MES, 40mM HEPES, 40mM Glycine) at pH 7, pH 3 and pH 3 + pepsin (2775 U / mL) each in a total volume of 100 pL. After heat exposure, measure activity of all three samples by mixing 40 pL sample with 160 pL Activity Assay Solution (7.5 mM 4-Nitrophenyl N-acetyl-p-D- glucosaminide (CAS: 3459-18-5) in 250 mM MES, pH 6) and immediately start reading change in absorbance at 405 nm for 30 min.

[0410] Data analysis

[0411] Determine the initial rate (OD / min) for each sample and calculate percent Residual Activity (%RA) using the initial rate for the sample dissolved at pH 7 as index 100%

[0412] Assay data: Residual Activity of samples after 15 min stress at 40 degC with varying pH and incl / excl pepsin protease. Disp27 and Disp45 wildtypes show no activity (0 %RA) after stress at pH 3 with pepsin protease, whereas variants engineered towards gastric stress tolerance show up to 97 % residual activity.

[0413] Table 4.1

[0414] Example 5. Enzymatic hydrolysis measurements of Extracellular Polymeric Substances (EPS) from Pseudomonas fluorescens

[0415] Enzymatic hydrolysis measurements of Extracellular Polymeric Substances

[0416] Crude EPS extracts from Pseudomonas fluorescens cultures known to produce (3-1 ,6-linked N- acetylglucosamine (PNAG) and its enzymatic hydrolysis was carried out as described in WO 2017 / 186943 A1 with some modifications. The assay is based on staining EPS with the fluorescently labelled lectin, wheat germ agglutinin (WGA-Alexafluor488; Thermo Fischer Scientific, #W11261), known to bind N-Acetyl-D-glucosamine (GIcNAc).

[0417] In this study, round sterile swatches (wfk 20A, polyester / cotton 65% / 35%) were placed in wells of a 96 well nunc Maxisorp black plate (ThermoScientific #437111). Next, 50 l of EPS extracts were spotted on the swatches and incubated 15 minutes at room temperature. Swatches were then rinsed with 100 pl distilled water and dried 15 minutes at room temperature. 50pl of either 50mM HEPES / 100mM NaCI buffer pH 7 (control) or 40 ppm enzyme solution of SEQ ID Nos: 1 to 16 was applied to the wells containing swatches and incubated 1 hour at 30 °C to allow enzymatic hydrolysis of EPS. The enzyme solution was then removed from each well and swatches were rinsed with 100pl of water. Finally, staining of N-Acetyl-D-glucosamine (GIcNAc) remaining in the swatches, was performed by adding 50 pl of WGA-Alexa fluor488 dye as described in W02020 / 207944 Fluorescence emission measurements using the SpectraMax i3 (Molecular Devices) instrument are listed below in Table 5.1 (excitation / emission maxima -495 / 519 nm).

[0418] Table 5.1. Fluorescence emission measurements of WGA-Alexa Fluor488 dye of crude EPS extracts from P. fluorescens treated with either buffer, or dispersin enzyme of the invention

[0419] Results from fluorescence emission measurements show that the EPS extracts from P. fluorescens were stained with WGA-Alexa Fluor488 indicating that the EPS extracts contain N- Acetyl-D-glucosamine residues and are sensitive to dispersin hydrolysis by dispersins of the invention. Thus, enzyme treated EPS samples release GIcNAc and result with lower fluorescence emission numbers. Example 6: Microbiome changes in broiler chickens fed hexosaminidase day 1-35 The study was conducted in the Poultry Research Farm of the University of Arkansas. Seventeen hundred and ten one-day-old male (Flock 399 with 46-week-old RXR3 hens) broiler chicks obtained from a local hatchery were used in this study. Chicks were vaccinated at the hatchery with Embrex Marek vaccine HVT 1 dose and Zoetis Newcastle-bronchitis [B1 / B1 Mass / Conn]) vaccine full dose. Upon arrival, chicks were weighed individually and sorted by weight. Chicks with body weight between 41 - 52g were selected to be included in the trial. An allocation analysis with the selected weights was performed to homogenize initial bird BW among pens and treatments. Chicks were equally allocated in pens to evaluate 4 treatments with 19 replicate pens each (18 chicks per pen) as describe in Table 6.1.

[0420] Table 6.1

[0421] Diets

[0422] A diet based on wheat, rye, and rice hulls was used as a nutritional enteric stressor throughout the trial (Starter [d0-d7]; Grower [d8-d21 ] ; and finisher [d22-d35]) for all treatments. The diet was formulated to meet or exceed NRC Nutrient Requirements for Poultry Ninth Edition (1994) guidelines with no coccidiostat on it. Feed was in mashed form presentation, and it was provided ad libitum throughout the experimental period. Diet composition is described in Table 6.2.

[0423] Table 6.2

[0424] HiPhos 20000 GT included at 50 ppm

[0425] Conditions

[0426] The birds used in the trial were cared for using standard procedures approved by the University of Arkansas Institutional Animal Care and Use Committee (IACUC#21057). Briefly, this trial was a production trial; so, no replacement for mortality during the trial was performed. All mortality was weighed and recorded before removing them from the facility in a biosecurity manner. Flock was monitored daily for morbidity and health. Sick or birds in poor health (culls) were removed from the pens at the judgment of the study coordinator or animal caretaker. As bedding material, new “Kiln-dried” pine shaving was used as litter (from 2 to 4 inches deep) and any wet spots that occurred during the trial were removed from the pen and replaced with new bedding material.

[0427] Birds were kept in 24h light program during the three days and from day 4 to the end of the experiment light program was 20h light with 4h darkness (10:00 pm to 2:00 am) of the next day.

[0428] Microbiome samples On D24 and D35, one chick per pen from pen treatments T1 , T2, and T4 was removed, After the chicks being humanely euthanized, a section of the GIT (mid ileum to ceca junction) was removed and a sample of digesta was collected in a 2-ml cryovial. Samples were immediately snap frozen in liquid nitrogen and transported to the lab on dry ice. At the lab, samples were stored at -80°C until processing for DNA extraction. Microbiome analysis performed by full-length ribosomal operon amplicon sequencing on Oxford Nanopore platform. Amplicon sequence data was classified with Kraken2 against the NCBI Refseq index, and feature classifications and feature taxonomy data was saved in biom formatted file.

[0429] Results Broilers fed daily 25 ppm SEQ ID NO: 5 produced significant and measurable changes in the ileal digesta microbial diversity (q=0.006) and was responsible for 11.5% of compositional variance between chicks. The taxa that strongly associated with a shift point to an enrichment of Bifidobacterium pseudoIongum, Streptococcus equinus and L. salivarius', and a depletion of several other Lactobacillaceae members. The most pronounced and statistically significant shift was a 5.5-fold increase in the abundance of Bifidobacterium pseudoIongum (q=0.033).

[0430] Table 6.3 Ileal microbiome. Relative abundance at day 35, Control (T1)

[0431]

[0432] Table 6. 4 Ileal microbiome. Relative abundance at day 35, SEQ ID NO: 5 (T4)

[0433] Table 6.5 Differential Relative abundance of Lactobacillus on day 35

[0434] Table 6.6 Differential Relative abundance of Bifidobacterium pseudoIongum at day 35

[0435] Table 6.7 Differential abundance. Natural log-fold change of taxa in SEQ ID NO: 5 (T2) from Control (T1) on day 35. Positive values for enrichment, negative values for depletion

[0436] Conclusion- The study showed a 5.5-fold increase in Bifidobacterium pseudoIongum, known for promoting gut health as well as a decrease in Enterococcus cecorum and certain Lactobacillaceae members, which could imply reduced gut disturbance. The Dispersin (SEQ ID NO: 5) led to favorable shifts in gut microbiota, suggesting similar potential benefits for maintaining microbiome balance and promoting gut health. Example 7 Microbiome changes in broiler chickens fed hexosaminidase day 22-35

[0437] The study was conducted in the Poultry Research Farm of the University of Arkansas. Day-of hatch male broiler chicks were obtained from a local hatchery. Chicks were equally allocated in pens with 19 chicks / pen to evaluate 2 treatments (Table 7.1). Birds had free access to control and experimental diets (Table 7.1) dosed with Balancius and water throughout the trial (d0-d35).

[0438] Table 7.1

[0439] Diets

[0440] A Corn-based optimized diet with wheat and rye for Ross308 was formulated with three diet phases (Starter DO-7; Grower D8-21 ; and finisher D22-35) for T1. Diets were formulated to meet or exceed NRC Nutrient Requirements for Poultry Ninth Edition (1994) guidelines with no coccidiostat on them. All diets were in mash presentation. Feed was provided ad libitum during the trial. A commercial phytase was added at the specified dose. Diet composition is described in table 7.2. SEQ ID NO: 5 was sprayed on the finisher diet to reach 25ppm, mixed for 6 minutes and given on day 22 to day35.

[0441] Table 7.2 Common diets before adding treatment (to Finisher)

[0442] HiPhos 20000 GT included at 50 ppm

[0443] Conditions

[0444] The birds used in the trial were cared for using standard procedures approved by the University of Arkansas Institutional Animal Care and Use Committee (IACUC#21057). Briefly, this trial was a production trial; so, no replacement for mortality during the trial was performed. As bedding material, new “Kiln-dried” pine shaving was used as litter (from 2 to 4 inches deep) and any wet spots that occurred during the trial were removed from the pen and replaced with new bedding material. Birds were kept in 24h light program during the three days and from day 4 to the end of the experiment light program was 20h light with 4h darkness (10:00 pm to 2:00 am) of the next day.

[0445] Microbiome samples

[0446] On day 35, 8 chicks from T2 and from T1 Control will be humanely euthanized. After CO2 asphyxiation, three samples were collected from the same bird: Ileum digesta (2 samples) and ileum scraping mucosa (remove as much digesta as possible). Digesta samples and ileum scraping mucosa were collected in 5mL cryovials and flashed frozen in liquid nitrogen. Samples were transported and stored at -80°C until processing for DNA extraction. Microbiome analysis performed by full-length ribosomal operon amplicon sequencing on Oxford Nanopore platform. Amplicon sequence data was classified with Kraken2 against the NCBI Refseq index, and feature classifications and feature taxonomy data was saved in biom formatted file.

[0447] Results

[0448] In digesta material, the feed additive containing 25 ppm SEQ ID NO: 5 significantly reduced the absolute abundance of Enterococcus cecorum (natural log-fold = 2.67, or a 14.4-fold change, q=0.048) and enriched the abundance of two Corynebacterium species.

[0449] Table 7.3 Differential abundance. Natural log-fold change of taxa in SEQ ID NO: 5 diet (T2) from Control (T1). Positive values for enrichment, negative values for depletion

[0450] An important statistical tool for assessing microbiome shifts in relative abuandnce data, is the assessment of changes in the log-ratios of higher level taxa. The inventors theorize that SEQ ID NO: 5 has a general effect of depleting enterococcal species and enriching members of the Lactobacillaceae species. The inventors therefore compared differences in the natural log-ratios of Lactobacillaceae (Lactobacillus, Ligilactobacillus and Limosilactobacilus species) and Enterococcus species, i.e. \r\(Lactobacillaceae abundance I Enterococcus abundance).

[0451] In digesta, the shift in the ratio of lactic acid bacteria and enterococcal species between 25 ppm SEQ ID NO: 5 and Control groups was large (18-fold change) and significant (p=0.046). Table 7.4 Ratio of Enterococci to Lactobacillaceae taxa on natural log scale, digesta

[0452] The specific effect of dispersin on Enterococcus cecorum is seen as a more than 2 log reduction of relative abundance compared to Control, both in the digesta and mucosa. The positive effect of dispersin on the abundance of Bifidobacterium pseudoIongum (previous example) is confirmed in this trial as B pseudoIongum is only detected in mucosa from dispersin treatment and not in Control mucosa.

[0453] Table 7.5 Relative abundance of Enterococci, mucosa

[0454] Table 7.6 Relative abundance of Bifidobacterium pseudoIongum, mucosa

[0455] Conclusion: The study demonstrated a significant impact of Dispersin (SEQ ID NO: 5) on the gut microbiome of broiler chickens. A notable reduction in the abundance of Enterococcus cecorum was observed, achieving a natural log-fold change of 2.67, indicative of a 14.4-fold reduction. This suggests potential benefits in reducing gut disturbances. Furthermore, the presence of beneficial bacteria such as Bifidobacterium pseudoIongum was confirmed, supporting findings from previous studies. The enrichment of Lactobacillaceae and specific Corynebacterium species underscores Dispersin's ability to promote a balanced gut microbiota.

[0456] Example 8 Escherichia coli (ETEC) challenge trial in pigs

[0457] Objective: To investigate the effects of Dispersin (SEQ ID NO:5) on the E.coli shedding and diarrhea score of weaning pigs during a 21-day Escherichia coli (ETEC) challenge trial. Table 8.1 Summary of Information

[0458] Table 8.2 Treatment Details

[0459] Oral ETEC Challenge: The 18 pigs weaned at 21 days of age are orally administered with sub- clinical dose (6.7 x 108CFU / ml) of F18 strain of E. coli using a needleless syringe on day 1 of the study and fed 4 experimental diets for 21 days.

[0460] Materials and Methods

[0461] 1. Feed Mixing and Formulation Table 8.3 - Diet Formulation

[0462] Diets and water were offered ad libitum.

[0463] 3. Feeding Schedule Phase 1 ; d 0-7

[0464] Phase 1 ; d 8-21

[0465] Feed and water were available ad libitum for the duration of the study.

[0466] 4. Experimental Design

[0467] Pigs are assigned to pens according to weight and sex. There are an equal number of males and females in each pen.

[0468] 5. Sampling Methods

[0469] Diarrhea Score: Fecal consistency on a per pig basis was recorded daily from day 0 to

[0470] 14. Scoring is conducted as described by Marquardt, R. R., Jin, L. Z., Kim, J. W., Fang, L., Frohlich, A. A., & Baidoo, S. K. (1999). Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets. FEMS Immunology & Medical Microbiology, 23(4), 283-288. https: / / doi.org / 10.1016 / S0928- 8244(99)00060-9 ETEC shedding: Fecal samples are collected on days 3, 7, 14 and 21 for E. coli enumeration. The method of E. coli enumeration as described by Lin, J., Lee, I. S., Frey, J., Slonczewski, J. L., & Foster, J. W. (2013). Comparative analysis of extreme acid survival in Salmonella enterica, Escherichia coli, and Shigella flexneri: effects on pH homeostasis and protein expression. Applied and Environmental Microbiology, 62(9), 3094-3100. https: / / doi.Org / 10.1128 / AEM.62.9.3094-3100.1996.

[0471] Table 8.4. Effect of dietary treatment and E. coli challenge on fecal score Table 8.5. Effect of dietary treatment and E. coli challenge on fecal E. coli count on different days. zDiet 2 tended to have greater (P = 0.058) fecal E. coli count than diet 4.

[0472] Conclusions of the study: 1 When analyzing microbial counts, by day 21 there was a significant effect of dispersin on

[0473] ETEC shedding with 3.5 log reduction compared to challenged control group (Table 8.5 and Figure 2).

[0474] 2. Dispersin influenced fecal scores between days 8 and 14, resulting in lower scores in the challenged group treated with dispersin compared to the challenged control group (Table 8.4 and Figure 3).

Claims

CLAIMS1. An animal feed additive comprising a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.

2. The animal feed additive according to claim 1 , having a residual activity at pH 3 of at least 60%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, wherein the residual activity is measured after 30 minutes at a temperature of 40 °C.

3. The animal feed additive according to claim 1 or 2, wherein said polypeptide has at least 75% such as at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, but less than 100% sequence identity, to the polypeptide of SEQ ID NO: 1 .

4. The animal feed additive according to claims 1 to 3, where said polypeptide comprises a substitution at one or more, positions corresponding to positions 1 , 3, 15, 49, 59, 111 , 124, 148, 163, 171 , 186, 225, 227, 232, 235, 249, 252, 260, 272, 272, 279, 281 , 308, 309, 312, and 324 of the polypeptide of SEQ ID NO: 1.

5. The animal feed additive according to claims 1 to 4, selected from the group consisting of a. a polypeptide having at least 80% sequence identity to SEQ ID NO: 2; b. a polypeptide having at least 80% sequence identity to SEQ ID NO: 3; c. a polypeptide having at least 80% sequence identity to SEQ ID NO: 4; d. a polypeptide having at least 80% sequence identity to SEQ ID NO: 5; e. a polypeptide having at least 80% sequence identity to SEQ ID NO: 6; f. a polypeptide having at least 80% sequence identity to SEQ ID NO: 7; g. a polypeptide having at least 80% sequence identity to SEQ ID NO: 8; h. a polypeptide having at least 80% sequence identity to SEQ ID NO: 9; i. a polypeptide having at least 80% sequence identity to SEQ ID NO: 10; j. a polypeptide having at least 80% sequence identity to SEQ ID NO: 11 ; k. a polypeptide having at least 80% sequence identity to SEQ ID NO: 12; l. a polypeptide having at least 80% sequence identity to SEQ ID NO: 13; m. a polypeptide having at least 80% sequence identity to SEQ ID NO: 14; n. a polypeptide having at least 80% sequence identity to SEQ ID NO: 15; and o. a polypeptide having at least 80% sequence identity to SEQ ID NO: 16.

856. An animal feed additive comprising a polypeptide having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 and comprising mutations selected from the group consisting of i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q ix. Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324aG; xii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q *324al; xiii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q L324*; xiv. *1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q; and xv. Q1* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281 P K308Q K309E K312Q.

7. The animal feed additive according to any of the preceding claims further comprising an ingredient selected from the group consisting of a. at least one fat soluble vitamin, and / or b. at least one water soluble vitamin, and / or86c. at least one trace mineral.

8. Use of a dispersin variant polypeptide having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 or the animal feed additive as defined in claims 1 to 7, to stabilize the healthy microflora of animals, in particular livestock.

9. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for improving intestinal health of an animal or maintaining / preserving intestinal paracellular permeability or gut integrity in an animal.

10. A method of increasing the ratio of lactic acid bacteria to Enterococcus species ratio of an animal comprising feeding the animal a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or the animal feed additive of claims 1 to 7.

11. A method of increasing the relative and / or absolute level of beneficial gut microbes in an animal, wherein beneficial gut microbes are selected from the group Lactic acid bacteria, Bifidiobacteria and Corynebacterium sp.

12. Use of a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for reducing the relative and / or the absolute level of Enterococcus cecorum in the intestine and / or the relative and / or the absolute level of Escherichia coli in the intestine.

13. A method of improving the performance of an animal comprising feeding the animal a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ I D NO: 1 or the animal feed additive as defined by claims 1 to 7, said performance selected from the group consisting of a. a reduced feed conversion ratio (FCR), and b. an increased average body weight gain.

14. A method of reducing the intestinal levels of microbial species that generates exopolysaccharides containing (3-1 ,6-linked poly-N-acetylglucosamine (polyGIcNAc) comprising feeding the animal a polypeptide having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 for or the animal feed additive as defined by claims 1 to 7.

15. A dispersin variant having hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 comprising a substitution at positions 3, 15, 59, 163, 186, 225, 227, 232, 235, 252, 260, 272, 279, 281 , 308, 309 and 31287and further comprising a substitution, an insertion or a deletion at a position selected from the group consisting of 1, 49, 111, 124, 148, 171, 249, and 324.

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