Gastric stable dispersin variants

Abstract

The present invention relates to probiotic dispersin variants that are gastric stable, to the gastric stable polypeptides and to the polynucleotides expressing the variants. Further, the invention relates to the polymers for use in therapy. Further, the invention relates to use in prevention or treatment of inflammatory and metabolic diseases.

Description

[0001] GASTRIC STABLE DISPERSIN VARIANTS

[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 and their use in therapy 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).

[0008] The present disclosure thus pertains to the field of enzymology, specifically to enzymes designed for the degradation of biofilms. Biofilms are structured communities of bacterial cells enclosed in a selfproduced polymeric matrix that adheres to both biotic and abiotic surfaces. These biofilms present significant challenges across various sectors including healthcare, food production, and industrial applications. Biofilm have also been found attached to various surfaces including medical devices such as implants but also infections. W004061117 A2 (Kane Biotech INC) describes 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.

[0009] WO 2017 / 186943 discloses the parent wild-type GH45 dispersin of SEQ ID NO: 1. WO 2020 / 207944 discloses 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 in human or animal to prevent infections.

[0010] 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 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. 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 medicine, bacterial colonization on surfaces such as implants and valves can lead to persistent infections that are resistant to standard antibiotic therapies. Similarly, in the food industry, biofilm formation on equipment poses risks of contamination, while in industrial settings, biofilms can cause machine fouling, leading to operational inefficiencies.

[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), thereby offering an alternative approach to managing biofilm-related issues across various fields.

[0012] Summary of the Invention

[0013] 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.

[0014] 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.

[0015] 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 (Males, A.; Moroz, O.V.; Blagova, E.; Munch, A.; Hansen, G.H.; Johansen, A.H.; Ostergaard, L.H.; Segura, D.R.; Eddenden, A.; Due, A.V.; Gudmand, M.; Salomon, J.; Sorensen, S.R.; Franco Cairo, J.P.L.; Nitz, M.; Pache, R.A.; Vejborg, R.M.; Bhosale, S.; Vocadlo, D.J.; Davies, G.J.; Wilson, K.S. ‘Expansion of the diversity of dispersin scaffolds,’ Acta Crystallogr D Struct Biol, 2025, 81(3): 130-146).

[0016] 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.

[0017] 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 anti-inflammatory compounds that further contribute to reducing inflammation in the gut.

[0018] 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.

[0019] 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.

[0020] An aspect of the invention is directed to 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.

[0021] 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.

[0022] In one embodiment 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.

[0023] In another embodiment 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.

[0024] In yet another embodiment said polypeptide is 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.

[0025] In a further embodiment the polypeptide further comprises mutations selected from the group consisting of i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q ix. Q1G Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P 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.

[0026] 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.

[0027] It may be provided that a polypeptide having hexosaminidase activity according to the invention is for use in therapy. Therapy may be understood as the treatment intended to relieve or heal a disorder. One advantage of this arrangement is the potential for the polypeptide to be used in various therapeutic applications, targeting different diseases and conditions through its biochemical properties.

[0028] It may be provided that the polypeptides according to the invention is for use in prevention or treatment of diseases caused by biofilm in a subject or patient in need thereof

[0029] It may be provided that a polypeptide according to the invention is for use in the prevention of a disease. It may be provided that said polypeptide may prevent gut diseases by promoting the growth of Bifidobacterium and / or Lactobacillus in the gut.

[0030] It may be provided that that a polypeptide according to the invention is effective in reducing pathogenic bacterial loads in feces, which can contribute to controlling disease spread.

[0031] 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. 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.

[0032] 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. It may be provided that said polypeptide is for use in the treatment or prevention of Escherichia coli, Clostridioides difficile or Helicobacter pylori infections or other PNAG producing infections in a patient 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. Without being bound by theory, PNAG 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

[0033] It may be provided that said polypeptide is for use in the treatment or prevention of dental caries caused by biofilm-forming bacteria like Streptococcus mutans, periodontal disease caused by Porphyromonas gingivalis, or halitosis including prevention of biofilm-forming bacteria on the tongue and in periodontal pockets. One advantage of this arrangement is the polypeptide's ability to disrupt biofilms, thus preventing and treating oral health conditions effectively by targeting the microbial communities responsible for these diseases.

[0034] It may be provided that the polypeptide according to the invention is for use in the treatment or prevention of urinary tract infections (UTIs) such as Escherichia coli and / or Klebsiella pneumoniae, strains of Enterococcus such as E. faecalis and E. faecium, and / or strains of Streptococcus spp. such as S. aureus, and / or other PNAG producers, vaginal infections including bacterial vaginosis such as Gardnerella vaginalis, and recurrent yeast infections such as Candida albicans, reducing recurrence. Urinary tract infections and vaginal infections are common and can be difficult to treat. One advantage of this arrangement is the polypeptide's potential to prevent and treat these infections by disrupting pathogenic biofilms and supporting beneficial microbial communities.

[0035] It may be provided that said polypeptide according to the invention is for use in the treatment or prevention of acne in a patient in need thereof by promoting the growth of Bifidobacterium and / or Lactobacillus in the gut. Acne is a skin condition often influenced by gut health. One advantage of this arrangement is the polypeptide's ability to enhance gut bacterial populations such as Bifidobacterium, thereby potentially reducing acne symptoms by improving overall gut health and immune function.

[0036] It may be provided that the use of a dispersin variant or polypeptide of the invention having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 is to stabilize the healthy microflora of humans. Healthy microflora refers to the beneficial microbial communities residing in the human body. One advantage of this arrangement is the maintenance of a balanced microbiome, which is essential for overall health and wellbeing. By ensuring the stability of these beneficial microbes, the polypeptide may contribute to the prevention of various diseases and support general health. It may be provided that a polypeptide with hexosaminidase activity according to the invention, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 , is used for improving intestinal health of a human or an animal or maintaining or preserving intestinal paracellular permeability or gut integrity in a human or an animal. A polypeptide may be understood as a polymer consisting of amino acids linked by peptide bonds, which can perform a variety of biological functions, including enzymatic activity. Hexosaminidase activity refers to the enzymatic function of cleaving hexose sugars from larger molecules, such as glycosaminoglycans, a process that is significant in the degradation of complex carbohydrates. Intestinal health involves the proper functioning and balance of the gastrointestinal system, and intestinal paracellular permeability refers to the control of the passage of substances across the intestinal epithelium. Gut integrity signifies the maintenance of the structural and functional integrity of the gastrointestinal barrier. One advantage of this arrangement is that the polypeptide's hexosaminidase activity can help maintain or restore balance in the gut microbiome, supporting overall intestinal health and ensuring the integrity of the gut barrier.

[0037] It may be provided that a method of supporting intestinal barrier function and maintaining gut integrity in a human or animal comprises administering a polypeptide with hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 . Administration refers to the process of delivering a substance into the body. Supporting intestinal barrier function involves enhancing the barrier properties of the gut lining to prevent the translocation of harmful substances, while maintaining gut integrity ensures the proper functioning and resilience of the gastrointestinal tract. One advantage of this arrangement is that the polypeptide can enhance the gut's natural defenses, promoting better health outcomes by preventing conditions associated with a compromised intestinal barrier, such as leaky gut syndrome and related inflammatory responses.

[0038] It may be provided that a method according to the invention increases 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.

[0039] It may be provided that the non-therapeutic use of the polypeptide according to the invention is as a dietary supplement. A dietary supplement may be understood as a product taken orally that contains one or more ingredients intended to supplement the diet and provide additional nutritional value. One advantage of this arrangement is that individuals can incorporate the polypeptide into their daily regimen to support gut health and overall wellbeing. By maintaining a healthy balance of gut microbiota, the dietary supplement can contribute to better digestive health, improved nutrient absorption, and enhanced immune function. It may be provided that a method of reducing the intestinal levels of microbial species that generate exopolysaccharides containing (3-1 ,6-linked poly-N-acetylglucosamine (polyGIcNAc) comprises administering 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 a human or animal in need thereof. Exopolysaccharides are high-molecular-weight polymers secreted by microorganisms into their environment, contributing to biofilm formation and microbial adherence. Reducing these microbial species helps prevent biofilm-related infections and maintains gut health. One advantage of this arrangement is that the polypeptide can effectively degrade specific exopolysaccharides, disrupting harmful biofilms and reducing the prevalence of pathogenic bacteria in the intestines. This can lead to improved gut health, reduced risk of infections, and overall better gastrointestinal function. Incorporating the dispersin variant having hexosaminidase activity into a functional food, such as a functional infant food, has the further advantages of offering additional protection to the dispersin variant having hexosaminidase activity, enhancing its survival through the digestive system and ensuring its effectiveness upon reaching the targeted site of action.

[0040] Additionally, functional foods can improve the palatability of the dispersin variant having hexosaminidase activity, making it more appealing and increasing consumer acceptance compared to traditional supplement forms.

[0041] Furthermore, functional foods offer convenience, providing a familiar and easy way for individuals to incorporate the dispersin variant having hexosaminidase activity into their daily diet without the need for separate supplementation.

[0042] The present invention relates to a functional food comprising a polypeptide according to the invention having hexosaminidase activity. The present invention also relates to a functional infant food comprising a dispersin variant having hexosaminidase activity.

[0043] Brief Description of the Figures

[0044] Figure 1 is an alignment of the polypeptides of SEQ ID NOs: 1 to 16.

[0045] Figure 2 presents a bar chart illustrating the reduction in E. coli shedding over time, comparing challenged and unchallenged groups as well as enzyme-treated groups.

[0046] Figure 3 shows line graphs depicting trends in fecal consistency scores across different trial phases, highlighting the impact of treatment on diarrhea severity. Scoring system: normal = 0, soft feces = 1 , mild diarrhea = 2, severe diarrhea = 3.

[0047] Sequence Listing

[0048] 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.

[0049] SEQ ID NO:2 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1. SEQ ID N0:3 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0050] SEQ ID NO:4 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0051] SEQ ID NO:5 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1

[0052] SEQ ID NO:6 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1

[0053] SEQ ID NO:7 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0054] SEQ ID NO:8 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0055] SEQ ID NO:9 is the amino acid sequence of the mature polypeptide of a variant of SEQ ID NO: 1.

[0056] SEQ I D NO: 10 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0057] SEQ I D NO: 11 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0058] SEQ I D NO: 12 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0059] SEQ I D NO: 13 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0060] SEQ I D NO: 14 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0061] SEQ I D NO: 15 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0062] SEQ I D NO: 16 is the amino acid sequence of the mature polypeptide of a variant of SEQ I D NO: 1.

[0063] SEQ ID NO: 17 is the amino acid sequence of the mature polypeptide of wild-type Dispersin B, also known as Dispersin 27 from Aggregatibacter actinomycetemcomitans, wherein MNYIKKIILSLFLLGLFSVLNC is the signal peptide which is SEQ ID NO: 34.

[0064] SEQ ID NO: 18 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 1.

[0065] SEQ ID NO: 19 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 2.

[0066] SEQ ID NO: 20 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 3.

[0067] SEQ ID NO: 21 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 4.

[0068] SEQ ID NO: 22 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 5.

[0069] SEQ ID NO: 23 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 6.

[0070] SEQ ID NO: 24 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 7.

[0071] SEQ ID NO: 25 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 8.

[0072] SEQ ID NO: 26 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 9.

[0073] SEQ ID NO: 27 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 10. SEQ ID NO: 28 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 11.

[0074] SEQ ID NO: 29 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 12.

[0075] SEQ ID NO: 30 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 13.

[0076] SEQ ID NO: 31 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 14.

[0077] SEQ ID NO: 32 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 15.

[0078] SEQ ID NO: 33 is the polynucleotide sequence encoding for the mature polypeptide SEQ ID NO: 16.

[0079] SEQ ID NO: 34 is the polynucleotide sequence MNYIKKIILSLFLLGLFSVLNC.

[0080] Detailed description of the invention

[0081] Definitions

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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 .

[0086] 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.

[0087] 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.

[0088] The term “composition” refers to a composition comprising a carrier and at least one enzyme of the present invention.

[0089] The terms ‘effective amount,’ ‘effective concentration,’ or ‘effective dosage’ refer to the amount, concentration, or dosage of the enzyme(s) that is sufficient to achieve the intended biological effect, such as preventing, improving, or maintaining gut health, supporting the immune system, or influencing body weight in a human or animal. This includes but is not limited to: Modulating intestinal microbial populations or composition; Reducing intestinal permeability or supporting gut barrier integrity; Enhancing the production of beneficial metabolites, such as short-chain fatty acids (SCFAs); Supporting immune maturation or reducing inflammation; Modulating weight or weight-related parameters, such as improving energy metabolism, reducing fat deposition, or promoting healthy weight gain or loss as appropriate. The specific effective amount, concentration, or dosage may vary depending on factors such as the species, age, weight, health condition, dietary habits, and the specific enzyme(s) or formulation used. Such amounts can be determined by routine experimentation, animal models, or clinical studies, as understood by those skilled in the art.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] The term “catalytic domain” means the region of an enzyme containing the catalytic machinery of the enzyme.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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).

[0102] The term “mutant” means a polynucleotide encoding a variant.

[0103] The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0104] 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.

[0105] The term “parent” or “parent dispersin’’ means a dispersin to which an alteration is made to produce the enzyme variants of the present invention.

[0106] 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. 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.

[0107] 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.

[0108] 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 i.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.

[0109] 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 (nonrecombinant) 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”.

[0110] 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.

[0111] The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”.

[0112] 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: (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0113] 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:

[0114] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0115] 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.

[0116] 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).

[0117] The term "functional food” is defined as a food product that has been formulated to provide health benefits beyond basic nutrition, often containing additional biologically active components such as vitamins, minerals, herbs, or other ingredients known to have beneficial effects on human health when consumed as part of a regular diet. In the context of the invention, the "functional food” comprises at least one dispersin variant having hexosaminidase activity of the present invention. The functional food may be a beverage or a nutriment in solid and / or dried form. The functional food as disclosed herein is intended to be consumed by a human subject. Functional foods are suitable for a wide range of consumers, including adults, children, elderly individuals, athletes and fitness enthusiasts, as well as individuals with specific health conditions or dietary requirements. Additionally, functional foods cater to those seeking to improve their overall health and well-being. Thus, the human subject may be a subject at any age. In some embodiments, the functional food as described herein is intended to be consumed by an adult. In some embodiments, the functional food as described herein is intended to be consumed by an infant in which case the functional food may be referred to as a functional infant food. The term “infant” has its normal meaning which is well known and understood by those of skill in the art. In some embodiments, the infant is meant to comprise a baby of 0-12 months’ of age.

[0118] Conventions for Designation of Variants

[0119] 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 et al., 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.

[0120] 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.

[0121] 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. 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*”.

[0122] 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”.

[0123] 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:

[0124] 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”.

[0125] Nomenclature

[0126] 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.

[0127] 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

[0128] Gastric stable dispersins

[0129] 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. 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.

[0130] 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.

[0131] 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.

[0132] 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, 167, 170, 171 , 173, 174, 175, 178, 179, 181 , 185, 186, 187, 188, 189, 199, 203, 204, 205, 207, 210, 215, 221 , 225, 227, 232, 235, 244, 249, 252, 256, 260, 262, 263, 264, 265, 267, 270, 272, 273, 274, 276, 278, 279, 280, 281 , 282, 283, 284, 288, 290, 291 , 296, 303, 304, 305, 306, 308, 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.

[0133] 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 ID NO: 1 , and the variant has dispersin activity.

[0134] 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,

[0135] 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, D111 E, 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, D171E, D171K, D171L, 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, 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 ID NO: 1 .

[0136] 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, Y281 H, Y281 K, Y281 N, 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, 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, D171E, D171K, D171L, 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 ID NO: 1.

[0137] 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 Y281P K308Q K309E K312Q; iii. Q3I H15Y A49W N59E D111 R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281P 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.

[0138] 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.

[0139] 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.

[0140] 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).

[0141] 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 etal., 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.

[0142] In an aspect, the variant is isolated. In another aspect, the variant is purified.

[0143] Preparation of Variants

[0144] 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.

[0145] 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 ID NO: 1 , wherein the variant has hexoaminidase activity; and (b) recovering the variant.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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 a / ., 1988, DNA 7: 127).

[0153] 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.

[0154] 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. Semisynthetic 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.

[0155] The present invention also relates to polynucleotides encoding a variant of the present invention.

[0156] The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof.

[0157] In an aspect, the polynucleotide is isolated. In another aspect, the polynucleotide is purified.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94 / 25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacterid. 177: 3465-3471).

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] 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.

[0171] 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-binding 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.

[0172] 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.

[0173] 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.

[0174] 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 mini-chromosome, 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.

[0175] 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.

[0176] 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.

[0177] 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).

[0178] 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.

[0179] 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. 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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 etal., 2018, BMC Microbiology 18:56, Burke etal., 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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).

[0189] In an alternative aspect, the variant is not recovered.

[0190] Functional food of the Invention

[0191] The present invention relates to a functional food, such as a functional infant food, comprising a dispersin variant having hexosaminidase activity of the invention. Thus, the functional food of the invention comprises at least a dispersin variant having hexosaminidase activity.

[0192] When used in a functional food, the dispersin variant having hexosaminidase activity may be obtained as enzyme granules / particles comprising a polypeptide of the invention. In an embodiment, the enzyme granule comprises a core, and optionally one or more coatings (outer layers) surrounding the core. In an embodiment, the core comprises a dispersin variant having hexosaminidase activity of the present invention. The core may include additional materials such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants and fragrances. The core may include a binder, such as synthetic polymer, wax, fat, or carbohydrate. The core may include a salt of a multivalent cation, a reducing agent, an antioxidant, a peroxide decomposing catalyst and / or an acidic buffer component, typically as a homogenous blend. The core may include an inert particle with the polypeptide absorbed into it, or applied onto the surface, e.g., by fluid bed coating. The core can be prepared by granulating a blend of the ingredients, e.g., by a method comprising granulation techniques such as crystallization, precipitation, pancoating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation. Methods for preparing the core can be found in the Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Vol. 1 ; 1980; Elsevier. The core may be surrounded by at least one coating, e.g., to improve the storage stability, to reduce dust formation during handling, or for colouring the granule. The coating should encapsulate the core unit by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes, so that the core unit has few or no uncoated areas. The layer or coating should, in particular, be homogeneous in thickness. The optional coating(s) may include a salt coating, or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (P A). The granule may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC) and polyvinyl alcohol (PVA). Alternatively, for its use in functional food, the dispersin variant having hexosaminidase activity may be obtained as a liquid enzyme composition. In an embodiment, the liquid enzyme composition comprises an enzyme stabilizer. Examples of such enzyme stabilizers include, but are not limited to, 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). Additionally, the liquid enzyme formulation may further comprise suitable preservatives, such as sodium sorbate, potassium sorbate, sodium benzoate and potassium benzoate or any combination thereof, and formulating agents, such as 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, kaolin, cellulose, PVA, acetate, phosphate and combinations thereof. The skilled person will know how to formulate and design the dispersin variant for its use in functional food to meet the requirements for such foods. For example, the skilled person will know how to formulate and design the dispersin variant for its use in a functional infant food to meet the requirements for such foods.

[0193] The dispersin variant of the invention can be added as solid or liquid enzyme formulation. For example, a solid or liquid enzyme formulation may be added before or during the ingredient mixing step during manufacturing of the functional food. The enzyme may also be incorporated in the functional food after ingredient mixing, for example before sterilization and / or packaging of the functional food. Alternatively, the dispersin variant having hexosaminidase activity 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, and then adding to the functional food.

[0194] In an embodiment, the functional food comprises one or more additional enzymes. In an embodiment, the functional food comprises one or more microbes, such as one or more probiotics. In an embodiment, the functional food comprises one or more vitamins. In an embodiment, the functional food comprises one or more minerals. In an embodiment, the functional food comprises one or more amino acids. In an embodiment, the functional food comprises one or more preservatives. In an embodiment, the functional food comprises one or more prebiotics, such as one or more fructo-oligosaccharides, galactooligosaccharides, inulin, polydextrose and any mixtures thereof.

[0195] In an embodiment, the functional food further comprising one or more ingredients selected from the list comprising fat soluble vitamins, water soluble vitamins, minerals, amino acids, protein, fibre, probiotics, postbiotics, prebiotics, sugars, preservatives, water and any mixtures thereof. For example, the functional food may comprise a dispersin variant having hexosaminidase activity of the invention and one or more pro-, pre, and / or postbiotics. In an embodiment, the functional food comprises a dispersin variant of the invention and one or more probiotics. In an embodiment, the functional food comprises a dispersin variant of the invention and one or more probiotics, wherein the dispersin variant and the one or more probiotics have a synergistic effect on the health of the human subject consuming the functional food. In a preferred embodiment, a method of maintaining or supporting intestinal health, or improving intestinal health promoting growth of commensal bacteria, in a human subject comprises administering to said human subject a functional food comprising a polypeptide of the invention and one or more probiotics.

[0196] In an embodiment, the functional food is selected from the list comprising infant formula, follow-on formula, baby food formula, infant cereals formula, growing-up milk, infant or child's food supplement, fruit juice, fruit drink, flavoured water, soda, sports drink, tea-based beverage, coffee-based beverage, kombucha-based beverage, dairy-based yogurt beverage, dairy alternative yogurt beverage, dairy-based kefir beverage, dairy alternative kefir beverage, dairy-based milk beverages, dairy alternative beverages, clinical nutrition beverage, dairy-based stirred yogurt, dairy alternative stirred yogurt, dairy-based set-type yogurt, dairy alternative set-type yogurt, dairy-based strained yogurt, dairy alternative strain yogurt, dairybased ice cream, dairy alternative ice cream, protein bar, snack bar, meal replacement bar, cookie, biscuit, cracker, breakfast cereals, muesli, powdered milk, instant coffee, instant chocolate, instant cocoa, instant tea, instant soup, instant porridge, instant noodles, baby food, instant baby food, instant sauce mix, instant gravy, instant mashed potatoes, instant pudding, instant curry, instant tofu powder, powdered meals, field rations, and any suitable mixtures thereof.

[0197] In an embodiment, the functional food is a beverage. For example, the functional food may be an acidic functional beverage comprising at least the dispersin variant of the invention. An acidic beverage typically has a pH of 4.6 or lower. Juices and sodas frequently have a pH below 4.0. Acids and acidulants provide a tartness and tangy taste that helps balance the sweetness of sugar present in the beverage and are key factors in the taste of the beverage. Acids may also act as a preservative and can reduce the growth of bacteria and fungi in a beverage, thereby improving shelf-stability, also referred to as shelf-life. Weak acids and / or acids that naturally occur in fruits may be used to produce an acidic beverage. Citric acid, which occurs naturally in citrus, and malic acid, which occurs naturally occurs in apples, pears, and cherries, are added to many beverages including fruit drinks, sports drinks, and tea-based beverages. Phosphoric acid is added to cola drinks. Acetic acid, adipic acid, ascorbic acid, fumaric acid, butyric acid, gluconic acid, lactic acid, tartaric acid, and sorbic acid may also be used to produce an acidic functional beverage. In some embodiments, the pH of the acidic functional beverage is between 2.0 to 4.6, such as between pH 3.7 to 4.3.

[0198] In some embodiments, the functional food is a fruit juice, fruit drink, flavoured water, soda, sports drink, yogurt-based beverage, kombucha-based beverage, tea-based beverage, or coffee-based beverage.

[0199] In some embodiments, the functional food is a fruit juice, fruit juice cocktail, lemonade, cider, or a beverage flavoured with fruit juices. The fruit component may be orange, grapefruit, lemon, lime, apple, pear, peach, apricot, prune, plum, strawberry, raspberry, cranberry, blueberry, pineapple, banana, mango, grape, tomato, pomegranate, papaya, coconut, kiwi, lychee, yuzu, or any combination thereof.

[0200] In some embodiments, the functional food is a soda. A soda is a carbonated beverage, typically comprising carbonated water and sweeteners and natural and / or artificial flavourings. A soda may be a seltzer. A soda may be sugar-free, so that it comprises no sweeteners or may only comprise artificial sweeteners. A soda may be “zero sugar”, where it may not comprise carbohydrates. A soda may also comprise caffeine, colourings, preservatives, and optionally additional ingredients. A soda may also be referred to as a soft drink, seltzer, “pop”, or “soda pop”.

[0201] In some embodiments, the functional food is a flavoured water. The flavoured water may be carbonated, such as for example a sparkling water.

[0202] In some embodiments, the functional food is a sports drink. A sports drink comprises electrolytes, such as sodium, potassium, and chloride, and typically also comprises carbohydrates, frequently in the form of sugars such as glucose, sucrose, or high-fructose corn syrup. A sports drink may be sugar-free, so that it comprises no sweeteners or may only comprise artificial sweeteners. A sports drink may be “zero sugar”, where it may not comprise carbohydrates. A sports drink may comprise caffeine, vitamins, minerals, protein, amino acids, natural or artificial flavourings, and optionally additional ingredients. A sports drink may also be referred to as a vitamin water, a fitness water, an enhanced water, an energy drink, a hydration beverage or a ready-to-drink nutritional or sports beverage.

[0203] In some embodiments, the functional food is a tea or a tea-based beverage. The tea may be green tea, black tea, oolong tea, yellow tea, white tea, decaffeinated tea, herbal tea, or any combination thereof. The herbal tea may be rosehip tea, chamomile tea, jiaogulan tea, peppermint tea, rooibos tea, ginger tea, ginseng tea, lemongrass tea, or any combination thereof. In some embodiments, the tea-based beverage is a fermented tea. In further embodiments, the fermented tea is kombucha, or is a kombucha- based beverage. Kombucha is produced by symbiotic fermentation of sugared tea using a symbiotic culture of bacteria and yeast. Kombucha or kombucha-based beverages may further comprise fruit juices and / or spices. In some embodiments, kombucha or kombucha-based beverages may comprise ethanol, or be alcoholic. In other embodiments, the kombucha or kombucha-based beverage is non-alcoholic.

[0204] In some embodiments, the functional food is milk based. A milk-based beverage may be derived from fermented dairy products, such as kefir and yogurt.

[0205] In some embodiments, the functional food is a coffee or coffee-based beverage. The coffee may be roasted coffee, unroasted green coffee, decaffeinated coffee, or any combination thereof. The coffee or coffee-based beverage may further comprise a dairy component, such as milk or cream, and / or a plantbased substitute, such as oat, almond, soy, rice, or cashew milk, or any other non-dairy substitute known in the art. The coffee or coffee-based beverage may further comprise flavouring ingredients, such as cocoa, chocolate, artificial cocoa compositions, vanilla, artificial vanilla compositions, or any combination thereof.

[0206] The functional beverage may further comprise one or more additional ingredients to improve or alter the taste of the beverage. Additional ingredients include acidulants, thickeners, buffers or agents for pH adjustment, chelating agents, colorants, emulsifiers, excipient, flavorants, osmotic agents, acceptable carriers, preservatives, stabilizers, sugars, sweeteners, texturizers, minerals, and / or vitamins. The additional ingredients can be added in any suitable amount.

[0207] In an embodiment, the functional food is a functional infant food. The functional infant food may be intended for consumption by infants, such as babies at the age of 0-12 months. Depending on the age of the infant, the functional infant food may be designed accordingly. Thus, a functional infant food intended for an infant of age 0-6 months may be formulated differently than a functional infant food intended for an infant of age 6-12 months. In an embodiment, the functional infant food is an infant formula, follow-on formula, a baby food formula, an infant cereals formula, a growing-up milk, or an infant or child's food supplement. In a preferred embodiment, the functional infant food is a functional infant formula, such as a starter infant formula. For example, the functional infant formula may be in the form of a powder, a concentrated liquid, or a ready-to-use liquid. Such infant formula preferably contains ingredients which are designed to meet the nutritional needs of a human infant. The skilled person will know how to formulate and design the functional infant food to meet the requirements for such foods. Thus, in addition to a dispersin variant as disclosed herein, the functional infant food should preferably contain a lipid source, a carbohydrate source and other nutrients such as vitamins and minerals. Typically, animal oils, vegetable oils, starch, sucrose, lactose and / or corn syrup solids may be added to the functional infant food to supply part or all of the above nutrients.

[0208] It is preferred that a functional infant food comprising a dispersin variant of the invention is nutritionally complete. By the term "nutritionally complete" is meant that the composition contains adequate nutrients to sustain healthy human life for extended periods.

[0209] In an embodiment, the invention relates to a process for the preparation of a functional food, such as a functional infant food, which comprises the steps of obtaining a dispersin variant of the invention and which further comprises a step of including the dispersin variant into the functional food, such as the functional infant food.

[0210] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

[0211] Examples

[0212] Example 1 : Generation of dispersin variants containing several mutations

[0213] Dispersin variants containing several mutations listed in table X were constructed as described in WO 2020 / 207944

[0214] Table 1.1

[0215] Example 2. Gastric stability of dispersin variants and biofilm assay

[0216] Gastric challenge: 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.

[0217] 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.

[0218] 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).

[0219] Water hardness composition:

[0220] 4mM NaHCO3+ 2mM CaCI2+ 0,5mM MgCI2,6H20

[0221] 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.

[0222] 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.

[0223] Example 3: Thermal shift assay (TSA) for determination of TmD

[0224] 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).

[0225] 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)

[0226] 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) / m3 586.5 + / - 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).

[0227] T able 3.1. TSA, Thermal shift analysis of dispersin wt and variants

[0228] 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.

[0229] Example 4 - Dispersin Gastric Conditions Stress / Residual Activity Assay Assay

[0230] Assay purpose

[0231] 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%.

[0232] Enzyme samples are diluted in buffer (10 mM MES, 0.01vol% TritonX-100, pH 6)

[0233] Assay protocol

[0234] 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. Data analysis

[0235] 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%

[0236] 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.

[0237] Table 4.1

[0238] Example 5. Enzymatic hydrolysis measurements of Extracellular Polymeric Substances (EPS) from Pseudomonas fluorescens

[0239] Enzymatic hydrolysis measurements of Extracellular Polymeric Substances

[0240] 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).

[0241] 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 10OpI 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).

[0242] 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.

[0243] 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.

[0244] Example 6: Microbiome changes in broiler chickens fed hexosaminidase day 1-35

[0245] 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.

[0246] Table 6.1

[0247] Diets

[0248] 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.

[0249] Table 6.2

[0250] * HiPhos 20000 GT included at 50 ppm

[0251] Conditions 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. 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.

[0252] Microbiome samples

[0253] 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.

[0254] Results

[0255] 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 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).

[0256] Table 6.3 Ileal microbiome. Relative abundance at day 35, Control (T1)

[0257] Table 6. 4 Ileal microbiome. Relative abundance at day 35, SEQ ID NO: 5 (T4)

[0258]

[0259] Table 6.5 Differential Relative abundance of Lactobacillus on day 35

[0260] Table 6.6 Differential Relative abundance of Bifidobacterium pseudoIongum at day 35

[0261] 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

[0262] Conclusion

[0263] The study showed a 5.5-fold increase in Bifidobacterium pseudoIongum, known for promoting gut health as well as a decrease in 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.

[0264] Example 7: Microbiome changes in broiler chickens fed hexosaminidase day 22-35

[0265] 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).

[0266] Table 7.1

[0267] Diets

[0268] 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 day 35.

[0269] Table 7.2 Common diets before adding treatment (to Finisher)

[0270]

[0271] * HiPhos 20000 GT included at 50 ppm

[0272] Conditions

[0273] 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.

[0274] Microbiome samples

[0275] 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.

[0276] Results

[0277] 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.

[0278] 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

[0279] 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. We theorize that SEQ ID NO: 5 has a general effect of depleting enterococcal species and enriching members of the Lactobacillaceae species. We therefore compared differences in the natural log-ratios of Lactobacillaceae (Lactobacillus, Ligilactobacillus and Limosilactobacilus species) and Enterococcus species, i.e. ln(Lactobacillaceae abundance I Enterococcus abundance).

[0280] 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).

[0281] Table 7.4 Ratio of Enterococci to Lactobacillaceae taxa on natural log scale, digesta The specific effect of dispersin on Enterococcus cecorum is seen as a more than 2 log reduction of relative abundance compared to Contro, both in the digesta and mucosal. The positive effect of dispersin on the abundance of Bifidobacterium (B.) pseudoIongum (previous example) is confirmed in this trial as B. pseudoIongum is only detected in mucosa from dispersin treatment and not in Control mucosa.

[0282] Table 7.5 Relative abundance of Enterococci, mucosa

[0283] Table 7.6 Relative abundance of Bifidobacterium pseudoIongum, mucosa

[0284] Conclusion:

[0285] 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.

[0286] The enrichment of Lactobacillaceae and specific Corynebacterium species underscores Dispersin's ability to promote a balanced gut microbiota. Example 8: Escherichia coli (ETEC) challenge trial in pigs

[0287] Objective

[0288] Investigate the effects of Dispersin (SEQ ID NO:5) on the growth performance and diarrhea score of weaning pigs during a 21 -day Escherichia coli (ETEC) challenge trial.

[0289] Table 8.1 Summary of Information

[0290] Table 8.2 Treatment Details

[0291] Individually penned pigs; 36 pens total (9 reps / treatment; 4 treatments)

[0292] 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.

[0293] Materials and Methods

[0294] 1. Feed Mixing and Formulation

[0295] Table 8.3 Diet Formulation

[0296]

[0297] 2. Feed Sampling

[0298] Diets and water were offered ad libitium.

[0299] 3. Feeding Schedule

[0300] Phase 1; d 0-7

[0301] Phase 1; d 8-21

[0302] Feed and water were available ad libitum for the duration of the study.

[0303] 4. Experimental Design

[0304] Pigs are assigned to pens according to weight and sex. There are an equal number of males and females in each pen.

[0305] 5. Sampling Methods

[0306] Growth Performance; Performance: pigs were weighted at the start of the study (day 0), day 3, 7, 14 and 21.

[0307] Diarrhea Score: Fecal consistency on a per pig basis was recorded daily from day 0 to 14.

[0308] Scoring is conducted as described by Marquardt et al. (1999) 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

[0309] & Medical Microbiology, 23(4), 283-288. https: / / doi.org / 10.1016 / S0928-8244(99)00060-

[0310] 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 et al. (2013) 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.

[0311] 6. Methods of Analysis

[0312] Performance Parameters are measured on a per pig basis (BW) (Fl) at d 0, 3, 7, 14 and end of study (day 21)

[0313] • Body weight - BW (kg)

[0314] • Feed Intake - Fl (kg)

[0315] And the following performance parameters calculated for the same periods:

[0316] • Average Daily Gain - ADG (g)

[0317] • Average daily feed intake - ADFI (g)

[0318] • Feed conversion ratio - FCR (g / g)

[0319] • Gain-to-Feed Ratio - G:F (g / g)

[0320] Results:

[0321] Table 8.4. Effect of dietary treatment and E. coli challenge on fecal score

[0322]

[0323] Table 8.5. Effect of dietary treatment and E. coli challenge on fecal E. coli count on different days.

[0324] Conclusions of the study:

[0325] 1 . When analyzing microbial counts, by day 21 there was a significant effect of dispersin on ETEC shedding with 3.5 log reduction compared to challenged control group (Table 8.5 and Figure 2).

[0326] 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. 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 polypeptide according to claim 1 , 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.

3. The polypeptide according to claims 1 or 2, 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.

4. The polypeptide according to claims 1 to 3, 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.

5. The polypeptide according to claim 1 to 4, and further comprising mutations selected from the group consisting of i. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; ii. Q3I H15Y A49W N59E K148E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q;iii. Q3I H15Y A49W N59E D111R S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; iv. Q3I H15Y A49W N59E Y124H S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; v. Q3I H15Y A49W N59E S163P D171 H S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; vi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309Q K312Q; vii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W L249I N252P N260Q H272V S279D Y281P K308Q K309E K312Q; viii. *-1aG Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q ix. Q1G Q1G Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q; x. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q *324aA; xi. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q *324aG; xii. Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P 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 Y281P K308Q K309E K312Q; and xv. Q1* Q3I H15Y A49W N59E S163P S186R S225G N227T E232D G235W N252P N260Q H272V S279D Y281P K308Q K309E K312Q6. A polypeptide having hexosaminidase activity according to any of the previous claims 1 to 5 for use in therapy.

7. A polypeptide having hexosaminidase activity according to any of the previous claims 1 to 5 for use in prevention or treatment of diseases caused by biofilm in a subject or patient in need thereof. A polypeptide with hexosaminidase activity according to any of claims 1 to 5 for use in prevention or treatment of inflammatory and metabolic diseases in a subject or patient in need thereof.

609. The polypeptide with hexosaminidase activity according to any of claims 1 to 5 use in the prevention or treatment of inflammatory and metabolic diseases in a subject or patient in need thereof according to claim 8, 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, colitis.

10. A polypeptide having hexosaminidase activity according to any of claims 1 to 5 for use in treatment or prevention of Escherichia coli, Clostridioides difficile or Helicobacter pylori infections or other PNAG producing infections in a patient in need thereof.

11. The polypeptide having hexosaminidase activity according to any of claims 1 to 5 for use in treatment or prevention of dental caries (cavities) caused by biofilm-forming bacteria like Streptococcus mutans, periodontal disease (gum disease), Porphyromonas gingivalis or halitosis (bad breath) including prevention of biofilm-forming bacteria on the tongue and in periodontal pockets.

12. The polypeptide having hexosaminidase activity according to any of claims 1 to 5 for in treatment or prevention of urinary Tract Infections (UTIs) such as Escherichia coli, Klebsiella pneumoniae or Enterococcus spp., vaginal Infections including Bacterial vaginosis (BV) such as prevent Gardnerella vaginalis, yeast infections such as Candida albicans, and / or reducing recurrence of infection.

13. The polypeptide having hexosaminidase activity according to any of claims 1 to 5 for use in treatment or prevention of acne in a subject in need thereof by promoting growth of Bifidobacterium in the gut.

14. Use of a polypeptide having hexosaminidase activity according to any of claims 1 to 5, said polypeptide having at least 70% but less than 100% sequence identity to SEQ ID NO: 1 to stabilize the healthy microflora of a human.

15. Use of a polypeptide having hexosaminidase activity according to any of claims 1 to 5, 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 a human or maintaining or preserving intestinal paracellular permeability or gut integrity in a human.

16. A method of supporting intestinal barrier function and maintaining gut integrity in a human or animal, comprising administering a polypeptide with hexosaminidase activity, said polypeptide having at least 70% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1.

17. The method according to claim 16, wherein the relative and / or absolute level of beneficial gut microbes in the human or animal is increased, wherein beneficial gut microbes are selected from the group Lactic acid bacteria, Bifidobacteria and Corynebacterium sp.

18. Non-therapeutic use of the polypeptide with hexosaminidase activity according to any of claims 1-5 as a dietary supplement.

19. A method of reducing the intestinal levels of microbial species that generates exopolysaccharides containing (3-1 ,6-linked poly-N-acetylglucosamine (polyGIcNAc) comprising administering 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 a human or animal in need thereof.

20. 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 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.

21. A functional food comprising a polypeptide with hexosaminidase activity according to any of the previous claims.

Images