Polypeptides having lysozyme activity and polynucleotides encoding the same

Abstract

The present invention relates to thermostable polypeptides having lysozyme activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides, as well as methods of making and using the polypeptides.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0002] The present invention relates to polypeptides having lysozyme activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides, as well as methods of making and using the polypeptides. [Background technology]

[0003] Lysozyme is an O-glycosyl hydrolase produced by many organisms as a defense mechanism against bacteria. This enzyme causes hydrolysis of bacterial cell walls by cleaving glycosidic bonds in peptidoglycan, an important structural molecule in bacteria. After the cell wall is weakened by lysozyme action, the bacterial cell lyses due to osmotic pressure.

[0004] Lysozyme is present in many organisms, including viruses, plants, insects, birds, reptiles, and mammals. In mammals, lysozyme has been isolated from nasal secretions, saliva, tears, intestines, urine, and milk. This enzyme cleaves the glycosidic bond between carbon 1 of N-acetylmuramic acid and carbon 4 of N-acetyl-D-glucosamine. In vivo, these two carbohydrates are polymerized to form cell wall polysaccharides.

[0005] Lysozyme is typically produced by many organisms, including viruses, plants, insects, birds, reptiles, and mammals, as a defense mechanism against bacteria. This enzyme causes hydrolysis of bacterial cell walls by cleaving glycosidic bonds in peptidoglycan, a key structural molecule in bacteria. After the cell wall is weakened by lysozyme action, the bacterial cell lyses due to osmotic pressure. There has been growing interest in the potential of lysozyme enzymes as antimicrobial agents. For example, lysozyme activity has been demonstrated against pathogens such as Streptococcus pneumoniae, Bacillus anthracis, Enterococcus faecium, Bacillus stearothermophilus, Clostridium botulinum, Clostridium butyricum, Clostridium perfringens, Clostridium sporogenes, Clostridium tyrobutyricum, and Listeria monocytogenes.

[0006] Lysozymes have been classified into five distinct glycoside hydrolase (GH) families (CAZy, www.cazy.org): hen egg white lysozyme (GH22), goose egg white lysozyme (GH23), bacteriophage T4 lysozyme (GH24), Sphingomonas flagellar protein (GH73), and Chalaropsis lysozyme (GH25). Lysozymes of families GH23 and GH24 are known to be derived primarily from bacteriophages and have not been identified in fungi. Lysozyme family GH25 has been found to be structurally unrelated to other lysozyme families.

[0007] Uses of lysozyme have been proposed in animal feed (see, e.g., WO 00 / 21381 and WO 04 / 026334), cheese making (see, e.g., WO 05 / 080559), food preservation (Hughey and Johnson (1987) Appl Environ Microbiol 53:2165), detergents (see, e.g., U.S. Pat. No. 5,041,236 and EP 0 425 016), oral care (see, e.g., U.S. Pat. No. 4,355,022, WO 04 / 017988, and WO 08 / 124764), cosmetology and dermatology, contraception, urology, and gynecology (see, e.g., WO 08 / 124764).

[0008] GH25 lysozyme has been reported from Chalaropsis (Felsch JW, Ingagami T, and Hash JH. (1975) The N,O-Diacetylmuramidase of Chalaropsis species; V The complete amino acid sequence. JBC. 250:10 pp 3713-3720). Chicken egg white lysozyme, the main product available on the commercial market, for example, does not cleave N,6-O-diacetylmuramidase in Streptococcus aureus cell walls and therefore is unable to lyse this important human pathogen, among others.

[0009] It has been observed that different lysozymes have different specificities for different microorganisms. Therefore, it is desirable to have several lysozymes available so that the appropriate enzyme can be selected for each specific application. Therefore, new polypeptides with lysozyme activity are desirable. For example, many applications in which lysozyme is used involve high-temperature processes, so thermostable lysozymes are needed. Summary of the Invention [Means for solving the problem]

[0010] The present invention relates to polypeptides having lysozyme activity and nucleotide sequences encoding the polypeptides. In one aspect, the polypeptides include substitutions at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO: 1, wherein the polypeptide has at least 60%, e.g., at least 65%, 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%, but less than 100%, sequence identity to the polypeptide of SEQ ID NO: 1 or amino acids 20 to 227 of SEQ ID NO: 2. The polypeptide has improved thermal stability compared to the polypeptide having amino acids 20 to 227 of SEQ ID NO:1 or SEQ ID NO:2.

[0011] In further aspects, the invention relates to polynucleotides encoding the polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and methods for producing the polypeptides.

[0012] The present invention further relates to compositions comprising the polypeptides defined herein.

[0013] An interesting aspect of the present invention relates to an animal feed additive comprising a polypeptide of the invention and at least one fat-soluble vitamin and / or at least one water-soluble vitamin and / or at least one trace mineral.

[0014] In yet another aspect, the present invention also relates to the use of the polypeptides of the present invention in animal feed.

[0015] One aspect of the present invention is a polypeptide of the present invention, i. In animal feed, ii. In animal feed additives, iii. in the preparation of a composition for use in animal feed, and / or iv. To improve the intestinal health of animals; Regarding use.

[0016] Sequence Listing Overview SEQ ID NO: 1 is the amino acid sequence of lysozyme isolated from Sodiomyces alcalophilus.

[0017] SEQ ID NO: 2 is the mature amino acid sequence of lysozyme isolated from Sodiomyces alcalophilus.

[0018] SEQ ID NO: 3 is the nucleic acid sequence of lysozyme isolated from Sodiomyces alcalophilus. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows a graph demonstrating the residual activity of polypeptides of the invention and wild type under steam box conditions. DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention relates to polypeptides having lysozyme activity and polynucleotides encoding the polypeptides. Polypeptides having lysozyme activity can be defined as variants of SEQ ID NO: 1 or SEQ ID NO: 2, comprising substitutions at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO: 1, wherein the polypeptide has at least 60% sequence identity with amino acids 20 to 227 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0021] definition 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 indicates otherwise.

[0022] Unless otherwise defined 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.

[0023] Lysozyme: The term "lysozyme" refers to a peptidoglycan N-acetylmuramoyl hydrolase (EC 3.2.1.17) that catalyzes the hydrolysis of 1,4-beta bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin.

[0024] Lysozyme activity: The term "lysozyme activity" is defined herein as peptidoglycan N-acetylmuramoyl hydrolase activity (EC 3.2.1.17) that catalyzes the hydrolysis of 1,4-beta bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin.

[0025] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a spliced ​​mature 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 emerging as a spliced ​​mature mRNA.

[0026] Coding sequence: The term "coding sequence" refers to a polynucleotide that directly specifies the amino acid sequence of a variant. The boundaries of the coding sequence are generally determined by an open reading frame, which generally 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 can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0027] Control sequence: The term "control sequence" refers to a nucleic acid sequence involved in regulating the expression of a polynucleotide in a particular organism or in vitro. Each control sequence may be native (i.e., derived from the same gene) or heterologous (i.e., derived from different genes) to the polynucleotide encoding the variant, and may be native or heterologous to each other. Such control sequences include, but are not limited to, leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcriptional or translational initiator and terminator sequences. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding the variant.

[0028] Expression: The term "expression" includes all steps involved in the production of a variant (for example, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion).

[0029] Expression vector: An "expression vector" refers to a linear or circular DNA construct containing a DNA sequence encoding a variant, the coding sequence 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 initiate transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on mRNA, an enhancer, and sequences that control the termination of transcription and translation.

[0030] Extended: The term "extended" refers to the addition of one or more amino acids to the amino and / or carboxyl termini of a variant, such that the "extended" variant has lysozyme activity.

[0031] Fragment: The term "fragment" refers to a variant in which one or more amino acids are absent from the amino and / or carboxyl terminus of the variant, which fragment has lysozyme activity.

[0032] Fusion polypeptide: The term "fusion polypeptide" refers to a polypeptide in which a polypeptide is fused at the N-terminus and / or C-terminus of a variant of the present invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art and involve ligating coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same promoter and terminator. Fusion polypeptides can also be constructed using intein technology, in which the fusion polypeptide is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779). Fusion polypeptides can further include a cleavage site between the two polypeptides. Upon secretion of the fusion protein, this site is cleaved to release the two polypeptides. Examples of cleavage sites include those described in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3:568-576; Svetina et al., 2000, J. Biotechnol. 76:245-251; Rasmussen-Wilson et al., 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. al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0033] Heterologous: The term "heterologous" with respect to a host cell means that the polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" with respect to a polypeptide or nucleic acid means that the regulatory sequences of the polypeptide or nucleic acid, e.g., a promoter, are not naturally associated with the polypeptide or nucleic acid, i.e., the regulatory sequences are derived from a gene other than the gene encoding the mature polypeptide.

[0034] Host strain or host cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct containing a polynucleotide encoding a variant has been introduced. Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts made from the cells.

[0035] Improved thermostability: The term "improved thermostability" is defined herein as a variant enzyme that exhibits retention of enzymatic activity after an incubation period at elevated temperatures compared to the parent enzyme. Such variants may or may not exhibit an altered thermoactivity profile compared to the parent. For example, a variant may not be active at elevated temperatures, but may maintain its three-dimensional structure and subsequently regain activity upon returning to a lower temperature. Alternatively, a variant may have an improved ability to refold after incubation at elevated temperatures compared to the parent enzyme.

[0036] In one embodiment, the thermal stability of the lysozyme variant is improved such that the variant can withstand elevated temperatures, e.g., 45°C to 110°C, preferably 50°C to 100°C, more preferably 60°C to 90°C, and even more preferably 70°C to 80°C. Preferably, the variant lysozyme maintains at least 40%, preferably at least 50%, 60%, 70%, or 80%, more preferably at least 90%, and even more preferably at least 95% of its residual activity after 1 hour incubation at a given elevated temperature, compared to the variant simultaneously maintained at room temperature. Preferably, the residual activity of the variant lysozyme is at least 1.5-fold, preferably at least 2-fold, more preferably at least 5-fold, most preferably at least 7-fold, and even most preferably at least 20-fold higher than the residual activity of the parent lysozyme treated under the same conditions. Preferably, activity is tested at temperature deviations to the desired elevated temperature using the lysozyme activity assay described in the "Examples" section.

[0037] Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell means "transfection," "transformation," or "transduction," as known in the art.

[0038] Isolated: The term "isolated" refers to a variant, nucleic acid, cell, or other specified substance or component that is separated from at least one other substance or component, including, but not limited to, other proteins, nucleic acids, cells, etc. Thus, an isolated polypeptide, nucleic acid, cell, or other substance is in a form that does not occur in nature. Isolated polypeptides include, but are not limited to, culture broths containing secreted variants expressed in host cells.

[0039] Mature polypeptide: The term "mature polypeptide" refers to the mature form of a polypeptide after N-terminal and / or C-terminal processing (eg, removal of a signal peptide).

[0040] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide that encodes a mature polypeptide having lysozyme activity.

[0041] Mutant: The term "mutant" refers to a polynucleotide that encodes a variant.

[0042] Native: The term "native" refers to a nucleic acid or polypeptide that is naturally present in a host cell.

[0043] Nucleic Acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding variants. Nucleic acids may be single-stranded or double-stranded and may be chemically modified. 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 specified, nucleic acid sequences are presented in the 5' to 3' direction.

[0044] Nucleic Acid Construct: The term "nucleic acid construct" refers to a nucleic acid molecule, either single-stranded or double-stranded, that is isolated from a naturally occurring gene or that has been modified to contain a segment of nucleic acid in a manner not normally occurring in nature, or that is synthetic, and that includes one or more regulatory sequences operably linked to the nucleic acid sequence.

[0045] Operably linked: The term "operably linked" means that the specified components are in a relationship (including, but not limited to, a juxtaposition) permitting them to function in their intended manner. For example, a regulatory sequence is operably linked to a coding sequence so that expression of the coding sequence is under the control of the regulatory sequence.

[0046] Parent or Parent Lysozyme: The terms "parent" or "parent lysozyme" refer to the lysozyme to which modifications are made to produce the enzyme variant of the present invention.

[0047] Purified: The term "purified" refers to 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, a chromatographic eluate, and / or a medium subjected to density gradient centrifugation). A purified nucleic acid or variant is at least about 50% pure, and typically at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or more pure (e.g., percent by weight or molar concentration). In a related sense, a composition is enriched with respect to a molecule if there is a substantial increase in the concentration of that molecule after application of a purification or concentration 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 higher relative or absolute concentration compared to the starting composition.

[0048] In one aspect, the term "purified," as used herein, refers to a variant or cell that is essentially free of components, particularly insoluble components, from the producing organism. In another aspect, the term "purified" refers to a variant that is essentially free of insoluble components, particularly insoluble components, from the natural organism from which it is obtained. In one aspect, the variant has been separated from a portion of the soluble components of the organism and culture medium from which it is recovered. The variant may be purified (i.e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0049] Thus, a variant may be purified such that only trace amounts of other proteins (particularly other polypeptides) are present. The term "purified," as used herein, may refer to the removal of other components (particularly other proteins, most particularly other enzymes) present in the cell of origin of the polypeptide. A variant may also be "substantially pure," i.e., free from other components from the organism in which it is produced, e.g., the host organism of a recombinantly produced variant. In one aspect, the polypeptide is at least 40% pure by weight of total polypeptide material present in the formulation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80%, or 90% pure by weight of total polypeptide material present in the formulation. As used herein, a "substantially pure polypeptide" can refer to a polypeptide preparation that contains up to 10%, preferably up to 8%, more preferably up to 6%, more preferably up to 5%, more preferably up to 4%, more preferably up to 3%, even more preferably up to 2%, most preferably up to 1%, and even most preferably up to 0.5% by weight of other polypeptide material with which the polypeptide is naturally or recombinantly associated.

[0050] Thus, a 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, and most preferably at least 99.5% pure, by weight of the total polypeptide material present in the formulation. The variants of the invention are preferably in substantially pure form (i.e., the preparation is essentially free of other polypeptide material with which it is naturally or recombinantly associated). This can be achieved, for example, by preparing the variant by well-known recombinant methods or by classical purification methods.

[0051] Recombinant: The term "recombinant" is used in its conventional sense to refer to the manipulation, e.g., cutting and recombining, of nucleic acid sequences to form constellations that differ from those found in nature. The term recombinant refers to a cell, nucleic acid, variant, or vector that has been modified from its natural state. Thus, for example, a recombinant cell expresses genes that are not found within the native form of the cell (non-recombinant), or expresses native genes at levels or under conditions that are different from those found in nature. The term "recombinant" is synonymous with "genetically modified" and "transgenic."

[0052] Recover: The term "recover" or "recovery" refers to the removal of a polypeptide from at least one fermentation broth component selected from the list of cells, nucleic acids, or other specified materials, e.g., recovery of a polypeptide from a whole fermentation broth or from a cell-free fermentation broth, by collecting polypeptide crystals, by filtration, e.g., depth filtration (using filter aids or packed filter media, chamber filter fabric filtration, rotary drum filtration, drum filtration, rotary vacuum drum filter, candle filter, horizontal leaf filter, or similar, with seed or pad filtration in a framework or modular setup), 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 a decanter centrifuge, disc centrifuge, hydrocyclone, or similar), or by precipitating the polypeptide and using particle size classification separation using an appropriate solid-liquid separation method. Recovery encompasses isolation and / or purification of the polypeptide.

[0053] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity."

[0054] For the purposes of the present invention, sequence identity between two amino acid sequences is determined as the "longest identity" output 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 were 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 on the command line. The Needle output labeled "longest identity" is calculated as follows: (Identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0055] For purposes of the present invention, sequence identity between two polynucleotide sequences is preferably determined as the "longest identity" output 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), version 6.6.0 or later. The parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and an EDNAFULL (the EMBOSS version in NCBI NUC4.4) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified on the command line. The Needle output labeled "longest identity" is calculated as follows: (identical deoxyribonucleotides x 100) / (length of alignment - total number of gaps in the alignment).

[0056] Signal peptide: A "signal peptide" is a sequence of amino acids attached to the N-terminal portion of a protein that facilitates secretion of the protein outside the cell. The mature form of the extracellular protein lacks the signal peptide, which is cleaved during the secretion process.

[0057] Subsequence: The term "subsequence" refers to a polynucleotide having one or more nucleotides missing at the 5' and / or 3' end of the mature polypeptide coding sequence, wherein the subsequence encodes a fragment that has lysozyme activity.

[0058] Variant: The term "variant" refers to a polypeptide having lysozyme activity, which contains a substitution, an insertion (including an extension), and / or a deletion (e.g., a truncation) at one or more positions. A substitution refers to the replacement of an amino acid occupying a position with a different amino acid; a deletion refers to the removal of an amino acid occupying a position; and an insertion refers to the addition of 1 to 5 amino acids (e.g., 1 to 3 amino acids, particularly 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0059] Wild-type: The term "wild-type" in reference to an amino acid sequence or a nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to something that is found in nature (e.g., a protein, amino acid, or nucleic acid sequence). Conversely, the term "non-naturally occurring" refers to something that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modifications of a wild-type sequence).

[0060] Rules for Designating Polypeptides of the Invention For the purposes of the present invention, the polypeptide disclosed in SEQ ID NO: 1 is used to determine the corresponding amino acid position in another lysozyme. The amino acid sequence of another lysozyme is aligned with the polypeptide disclosed in SEQ ID NO: 1, and based on this 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 in 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 were a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.

[0061] In describing the polypeptides of the present invention, the following nomenclature is adopted for ease of reference: accepted IUPAC single-letter or three-letter amino acid abbreviations are used.

[0062] Substitutions. For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Thus, a substitution of threonine at position 226 with alanine is designated "Thr226Ala" or "T226A." Multiple mutations are separated by a symbol ("+"), e.g., "Gly205Arg+Ser411Phe" or "G205R+S411F" represent a substitution of glycine (G) with arginine (R) and serine (S) with phenylalanine (F) at positions 205 and 411, respectively.

[0063] Multiple Alterations. Variants containing multiple alterations are separated by a symbol ("+"), e.g., "Arg170Tyr+Gly195Glu" or "R170Y+G195E" represent substitutions of arginine and glycine with tyrosine and glutamic acid at positions 170 and 195, respectively.

[0064] Different modifications. When different modifications can be introduced at a certain position, the different modifications are separated by a comma, for example, "Arg170Tyr,Glu" represents the substitution of arginine at position 170 with tyrosine or glutamic acid. Thus, "Tyr167Gly,Ala + Arg170Gly,Ala" designates: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly" and "Tyr167Ala+Arg170Ala".

[0065] The present invention relates to polypeptides having lysozyme activity, which polypeptides comprise substitutions at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO:1, and which have at least 60%, e.g., at least 65%, 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 to SEQ ID NO:1, but less than 100% sequence identity.

[0066] The polypeptide may further comprise an extension of one or more amino acids at the N-terminus and / or C-terminus.

[0067] Alternatively, the polypeptide may further comprise a truncation of one or more amino acids at the N-terminus and / or C-terminus.

[0068] Polypeptides of the Invention In another embodiment, the polypeptide has at least 60%, such as at least 65%, 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%, such as at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100%, sequence identity to SEQ ID NO:1 or amino acids 20 to 227 of SEQ ID NO:2.

[0069] In one aspect the polypeptide of the invention comprises between 1 and 20 alterations, such as between 1 and 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 alterations.

[0070] In another aspect, the polypeptide comprises substitutions at one or more positions corresponding to 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO: 1. In another aspect, the polypeptide comprises substitutions at two positions corresponding to any of positions 10, 13, 14, 143, 166, and 176 of SEQ ID NO: 1. In another aspect, the polypeptide comprises substitutions at three positions corresponding to any of 10, 13, 14, 37, 101, 153, 166, 176, and 202 of SEQ ID NO: 1. In another embodiment, the polypeptide comprises substitutions at four positions corresponding to any of 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 63, 65, 101, 118, 122, 125, 129, 131, 166, 175, 176, and 202 of SEQ ID NO: 1. In another embodiment, the polypeptide comprises substitutions at five positions corresponding to any of 1, 10, 13, 14, 118, 122, 166, 176 of SEQ ID NO: 1.

[0071] In another aspect, the polypeptide comprises or consists of a substitution at a position corresponding to position 1 of SEQ ID NO:1. In another aspect, the amino acid at the position corresponding to position 1 of SEQ ID NO:1 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Thr or Gln. In another aspect, the polypeptide comprises or consists of the substitution R1T of SEQ ID NO:1. In yet another aspect, the polypeptide comprises or consists of SEQ ID NO:1 substituted at position 1, i.e., a substitution at position 1 such as R1Q. The substitution at position 1 of SEQ ID NO:1 shown in Tables 2 and 4 may have a negative effect on thermostability with some substitutions, but it provides the advantage of preventing glycosylation of muramidase. In some embodiments of the substitution at position 1 of SEQ ID NO:1, both increased thermostability and decreased glycosylation are observed.

[0072] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 2 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 2 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Val. In yet another embodiment, the polypeptide is SEQ ID NO: 1 comprising or consisting of a substitution at position 2, i.e., a substitution at position 2, e.g., I2V.

[0073] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 6 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 6 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ser. In yet another embodiment, the polypeptide comprises or consists of SEQ ID NO: 1 substituted at position 6, i.e., a substitution at position 6, for example D6S.

[0074] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 9 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 9 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro. In yet another embodiment, the polypeptide comprises or consists of SEQ ID NO: 1 substituted at position 9, i.e., a substitution at position 9, e.g., G9P.

[0075] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 10 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr or Val, preferably Ala, Asp or Arg. In another embodiment, the polypeptide comprises or consists of the substitution W10A, W10D or W10R of SEQ ID NO: 1.

[0076] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 13 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 13 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably Asn. In yet another embodiment, the polypeptide is SEQ ID NO: 1 comprising or consisting of a substitution at position 13, i.e., a substitution at position 13, e.g., T13N.

[0077] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 14 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 14 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably Val. In another embodiment, the polypeptide comprises or consists of the substitution T14V of SEQ ID NO: 1.

[0078] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 20 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 20 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val, preferably Trp. In another embodiment, the polypeptide comprises or consists of the substitution Y20W of SEQ ID NO: 1.

[0079] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 36 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 36 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably Glu. In another embodiment, the polypeptide comprises or consists of the substitution T36E of SEQ ID NO: 1.

[0080] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 37 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 37 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asn or Tyr. In another embodiment, the polypeptide comprises or consists of the substitution F37N or F37Y of SEQ ID NO: 1.

[0081] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 40 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 40 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably Pro. In another embodiment, the polypeptide comprises or consists of the substitution S40P of SEQ ID NO: 1.

[0082] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 41 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 41 of SEQ ID NO: 1 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Tyr. In another embodiment, the polypeptide comprises or consists of the substitution A41Y of SEQ ID NO: 1.

[0083] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 63 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 63 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg. In another embodiment, the polypeptide comprises or consists of the substitution Q63R of SEQ ID NO: 1.

[0084] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 65 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 65 of SEQ ID NO: 1 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asp. In another embodiment, the polypeptide comprises or consists of the substitution A65D of SEQ ID NO: 1.

[0085] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 101 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 101 of SEQ ID NO: 1 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ser. In another embodiment, the polypeptide comprises or consists of the substitution N101S of SEQ ID NO: 1.

[0086] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 103 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 103 of SEQ ID NO: 1 is substituted with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Ser. In another embodiment, the polypeptide comprises or consists of the substitution A103S of SEQ ID NO: 1.

[0087] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 118 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 118 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Arg. In another embodiment, the polypeptide comprises or consists of the substitution E118R of SEQ ID NO: 1.

[0088] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 122 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 122 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, preferably Pro or Asp. In another embodiment, the polypeptide comprises or consists of the substitution T122P or T122D of SEQ ID NO: 1.

[0089] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 125 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 125 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro or Lys. In another embodiment, the polypeptide comprises or consists of the substitution H125P or H125K of SEQ ID NO: 1.

[0090] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 126 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 126 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asn or Pro. In another embodiment, the polypeptide comprises or consists of the substitution G126N or G126P of SEQ ID NO: 1.

[0091] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 129 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 129 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Thr, Trp, Tyr, or Val, preferably Gly. In another embodiment, the polypeptide comprises or consists of the substitution S129G of SEQ ID NO: 1.

[0092] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 131 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 131 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, preferably Cys or Tyr. In another embodiment, the polypeptide comprises or consists of the substitution V131C or W131Y of SEQ ID NO: 1.

[0093] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 143 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 143 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asn. In another embodiment, the polypeptide comprises or consists of the substitution Q143N of SEQ ID NO: 1.

[0094] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 153 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 153 of SEQ ID NO: 1 is substituted with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ser. In another embodiment, the polypeptide comprises or consists of the substitution N153S of SEQ ID NO: 1.

[0095] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 155 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 155 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Trp. In another embodiment, the polypeptide comprises or consists of the substitution C155W of SEQ ID NO: 1.

[0096] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 166 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 166 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Tyr, preferably Pro. In another embodiment, the polypeptide comprises or consists of the substitution V166P of SEQ ID NO: 1.

[0097] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 174 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 174 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro. In another embodiment, the polypeptide comprises or consists of the substitution G174P of SEQ ID NO: 1.

[0098] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 175 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 175 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Ser or Asn. In another embodiment, the polypeptide comprises or consists of the substitution F175S or F175N of SEQ ID NO: 1.

[0099] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 176 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 176 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp or Val, preferably Trp. In another embodiment, the polypeptide comprises or consists of the substitution Y176W of SEQ ID NO: 1.

[0100] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 188 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 188 of SEQ ID NO: 1 is substituted with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Pro. In another embodiment, the polypeptide comprises or consists of the substitution G188P of SEQ ID NO: 1.

[0101] In another embodiment, the polypeptide comprises or consists of a substitution at a position corresponding to position 202 of SEQ ID NO: 1. In another embodiment, the amino acid at a position corresponding to position 202 of SEQ ID NO: 1 is substituted with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Glu. In another embodiment, the polypeptide comprises or consists of the substitution R202E of SEQ ID NO: 1.

[0102] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10 and 143 of SEQ ID NO: 1, such as those described above. In another embodiment, the polypeptide comprises or consists of substitutions W10D and Q143E of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Arg.

[0103] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to 13 and 176 of SEQ ID NO:1. In another embodiment, the polypeptide comprises or consists of substitutions T13N and Y176W of SEQ ID NO:1.

[0104] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10 and 14 of SEQ ID NO: 1, such as those described above. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Ala. In another embodiment, the polypeptide comprises or consists of substitutions W10A and T14V of SEQ ID NO: 1.

[0105] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10 and 176 of SEQ ID NO: 1. In another embodiment, the polypeptide comprises or consists of the substitutions W10R and Y176W of SEQ ID NO: 1.

[0106] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10 and 166 of SEQ ID NO: 1. In another embodiment, the polypeptide comprises or consists of substitutions W10R and V166P of SEQ ID NO: 1.

[0107] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to 166 and 176 of SEQ ID NO: 1. In another embodiment, the polypeptide comprises or consists of substitutions V166P and Y176W of SEQ ID NO: 1.

[0108] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 13, and 37 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Arg, and the amino acid at the position corresponding to position 37 is preferably substituted with Tyr.

[0109] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 13, 14, and 37 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 37 is preferably substituted with Tyr.

[0110] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 13, and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Arg.

[0111] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 166, and 176 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Asp.

[0112] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 13, and 14 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Ala.

[0113] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 14, and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Arg.

[0114] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 101, and 153 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Asp.

[0115] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 14, and 37 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Ala, and the amino acid at the position corresponding to position 37 is preferably substituted with Tyr.

[0116] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 13, 14 and 202 of SEQ ID NO:1, such as those described above.

[0117] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 14, and 153 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Ala.

[0118] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 13, 166, and 176 of SEQ ID NO:1, such as those described above.

[0119] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 14, 166, and 176 of SEQ ID NO:1, such as those described above.

[0120] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 166, and 176 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Ala.

[0121] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 166, and 176 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0122] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10, 14, 166, and 176 of SEQ ID NO:1, such as those described above.

[0123] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 101, 166, and 176 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0124] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 14, 118, and 122 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Asp. In another embodiment, the amino acid at the position corresponding to position 122 of SEQ ID NO: 1 is preferably substituted with Asp.

[0125] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 166, 176 and 202 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 is preferably substituted with Asp.

[0126] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 13, 118, and 122 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Ala. In another embodiment, the amino acid at the position corresponding to position 122 of SEQ ID NO: 1 is preferably substituted with Asp.

[0127] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 37, 166, 176 and 202 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 37 of SEQ ID NO: 1 is preferably substituted with Tyr.

[0128] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 118, 122, and 202 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Ala. In another embodiment, the amino acid at the position corresponding to position 122 of SEQ ID NO: 1 is preferably substituted with Asp.

[0129] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 6, 10, 101 and 166 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0130] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 6, 9, 10 and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0131] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 6, 10, 36 and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0132] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 9, 10, 101 and 175 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Asp. In another embodiment, the amino acid at the position corresponding to position 175 is preferably substituted with Asn.

[0133] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 9, 10, 101 and 129 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0134] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 36, 37, and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Asp. In another embodiment, the amino acid at the position corresponding to position 37 of SEQ ID NO: 1 is preferably substituted with Glu.

[0135] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 40, 101 and 175 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Asp. In another embodiment, the amino acid at the position corresponding to position 175 of SEQ ID NO: 1 is preferably substituted with Asn.

[0136] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 20, 101 and 175 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Asp. In another embodiment, the amino acid at the position corresponding to position 175 of SEQ ID NO: 1 is preferably substituted with Asn.

[0137] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 2, 9, 10 and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0138] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 63, 65, and 101 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Asp.

[0139] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10, 101, 125, and 131 of SEQ ID NO: 1, such as those described above. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Asp. In another embodiment, the amino acid at the position corresponding to position 125 of SEQ ID NO: 1 is preferably substituted with Lys. In another embodiment, the amino acid at the position corresponding to position 131 of SEQ ID NO: 1 is preferably substituted with Tyr.

[0140] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 13, 14, 166 and 176 of SEQ ID NO:1, such as those described above.

[0141] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 13, 166, and 176 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Ala.

[0142] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 37, 166, 176 and 202 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 37 of SEQ ID NO: 1 is preferably substituted with Tyr.

[0143] In another embodiment, the polypeptide comprises or consists of substitutions, such as those described above, at positions corresponding to positions 10, 166, 176 and 202 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is substituted, preferably with Ala.

[0144] In another embodiment, the polypeptide comprises or consists of substitutions such as those described above at positions corresponding to positions 10, 37, 166, and 176 of SEQ ID NO: 1. In another embodiment, the amino acid at the position corresponding to position 10 of SEQ ID NO: 1 is preferably substituted with Ala. In another embodiment, the amino acid at the position corresponding to position 37 of SEQ ID NO: 1 is preferably substituted with Tyr.

[0145] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10, 118, 122, 166 and 176 of SEQ ID NO:1, such as those described above.

[0146] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10, 13, 14, 166 and 176 of SEQ ID NO:1, such as those described above.

[0147] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 10, 13, 122, 166 and 176 of SEQ ID NO:1, such as those described above.

[0148] In another embodiment, the polypeptide comprises or consists of substitutions at positions corresponding to positions 1, 10, 13, 14, 166 and 176 of SEQ ID NO:1, such as those described above.

[0149] In another aspect, the polypeptide comprises or consists of one or more substitutions selected from the group consisting of R1T, R1Q, W10A, W10D, W10R, T14V, F37N, F37Y, A41Y, A103S, E118R, T122P, H125P, G126N, G126P, V131C, Q143N, N153S, C155W, G174P, F175S, G188P, R202E of SEQ ID NO:1.

[0150] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+Q143E of SEQ ID NO:1.

[0151] In another embodiment, the polypeptide comprises or consists of the substitutions V166P+Y176W of SEQ ID NO:1.

[0152] In another embodiment, the polypeptide comprises or consists of the substitution T13N+Y176W of SEQ ID NO:1.

[0153] In another embodiment, the polypeptide comprises or consists of W10A+T14V of SEQ ID NO:1.

[0154] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+T14V of SEQ ID NO:1.

[0155] In another embodiment, the polypeptide comprises or consists of the substitutions W10R+Y176W of SEQ ID NO:1.

[0156] In another embodiment, the polypeptide comprises or consists of the substitutions W10R+V166P of SEQ ID NO:1.

[0157] In another embodiment, the polypeptide comprises or consists of W10R+T13N+F37Y of SEQ ID NO:1.

[0158] In another embodiment, the polypeptide comprises or consists of the substitutions T13N+T14V+F37Y of SEQ ID NO: 1. In another embodiment, the polypeptide comprises or consists of the substitutions W10D+T13N+N101S of SEQ ID NO: 1.

[0159] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+V166P+Y176W of SEQ ID NO:1.

[0160] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T13N+T14V of SEQ ID NO:1.

[0161] In another embodiment, the polypeptide comprises or consists of the substitutions W10R+T14V+N101S of SEQ ID NO:1.

[0162] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+N101S+N153S of SEQ ID NO:1.

[0163] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T14V+F37Y of SEQ ID NO:1.

[0164] In another embodiment, the polypeptide comprises or consists of the substitutions T13N+T14V+R202E of SEQ ID NO:1.

[0165] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T14V+N153S of SEQ ID NO:1.

[0166] In another embodiment, the polypeptide comprises or consists of the substitutions T13N+V166P+Y176W of SEQ ID NO:1.

[0167] In another embodiment, the polypeptide comprises or consists of the substitutions T14V+V166P+Y176W of SEQ ID NO:1.

[0168] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+V166P+Y176W of SEQ ID NO:1.

[0169] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+V166P+Y176W of SEQ ID NO:1.

[0170] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T14V+V166P+Y176W of SEQ ID NO:1.

[0171] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+N101S+V166P+Y176W of SEQ ID NO:1.

[0172] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+T14V+E118R+T122D of SEQ ID NO:1.

[0173] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+V166P+Y176W+R202E of SEQ ID NO:1.

[0174] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T13N+E118R+T122D of SEQ ID NO:1.

[0175] In another embodiment, the polypeptide comprises or consists of the substitutions F37Y+V166P+Y176W+R202E of SEQ ID NO:1.

[0176] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+E118R+T122D+R202E of SEQ ID NO:1.

[0177] In another embodiment, the polypeptide comprises or consists of the substitutions D6S+W10D+N101S+V166P of SEQ ID NO:1.

[0178] In another embodiment, the polypeptide comprises or consists of the substitutions D6S+G9P+W10D+N101S of the polypeptide of SEQ ID NO:1.

[0179] In another embodiment, the polypeptide comprises or consists of the substitutions D6S+W10D+T36E+N101S of SEQ ID NO:1.

[0180] In another embodiment, the polypeptide comprises or consists of the substitutions G9P+W10D+N101S+F175N of SEQ ID NO:1.

[0181] In another embodiment, the polypeptide comprises or consists of the substitutions G9P+W10D+N101S+S129G of SEQ ID NO:1.

[0182] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+T36E+F37E+N101S of SEQ ID NO:1.

[0183] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+S40P+N101S+F175N of SEQ ID NO:1.

[0184] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+Y20W+N101S+F175N of SEQ ID NO:1.

[0185] In another embodiment, the polypeptide comprises or consists of the substitutions I2V+G9P+W10D+N101S of the polypeptide of SEQ ID NO:1.

[0186] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+Q63R+A65D+N101S of SEQ ID NO:1.

[0187] In another embodiment, the polypeptide comprises or consists of the substitutions W10D+N101S+H125K+W131Y of SEQ ID NO:1.

[0188] In another embodiment, the polypeptide comprises or consists of the substitutions T13N+T14V+V166P+Y176W of SEQ ID NO:1.

[0189] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T13N+V166P+Y176W of SEQ ID NO:1.

[0190] In another embodiment, the polypeptide comprises or consists of the substitutions F37Y+V166P+Y176W+R202E of SEQ ID NO:1.

[0191] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+V166P+Y176W+R202E of SEQ ID NO:1.

[0192] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+F37Y+V166P+Y176W of SEQ ID NO:1.

[0193] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+E118R+T122D+V166P+Y176W of SEQ ID NO:1.

[0194] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T13N+T14V+V166P+Y176W of SEQ ID NO:1.

[0195] In another embodiment, the polypeptide comprises or consists of the substitutions W10A+T13N+T122D+V166P+Y176W of SEQ ID NO:1.

[0196] In another embodiment, the polypeptide comprises or consists of the substitutions R1Q+W10A+T13N+T14V+V166P+Y176W of SEQ ID NO:1.

[0197] Amino acid changes can be minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering net charge or another function, e.g., a polyhistidine tract, an antigenic epitope, or a binding domain.

[0198] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and low molecular weight amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that generally do not change specific activity are known in the art and are described, for example, in H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0199] Instead, the amino acid changes are of such a nature that they alter the physicochemical properties of the polypeptide, e.g., they may improve the thermostability of the polypeptide, alter its substrate specificity, change its pH optimum, etc.

[0200] Essential amino acids in a polypeptide can be identified using procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for lysozyme activity to identify amino acid residues essential for the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Enzyme active sites or other biological interactions can also be determined by physical analysis of structures, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of amino acids at putative contact sites. See, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. Identification of essential amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides, or within a polypeptide or protein family, with polypeptides / proteins derived from a common ancestor (typically having similar three-dimensional structure, function, and significant sequence similarity). Additionally, or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or the active site of a polypeptide. See, e.g., Jumper et al., 2021, "Highly accurate protein structure prediction with AlphaFold," Nature 596:583-589.

[0201] The polypeptide can consist of 200 to 250 amino acids, for example, 210 to 240, 215 to 235, and 220 to 240 amino acids.

[0202] In one embodiment, the polypeptide has improved thermal stability compared to amino acids 20-227 of SEQ ID NO: 1 or SEQ ID NO: 2. Improved thermal stability is measured as at least 5°C, at least 5.5°C, at least 6°C, at least 6.5°C, at least 7°C, at least 7.5°C, at least 8°C, at least 8.5°C, at least 9°C, at least 9.5°C, at least 10°C compared to a polypeptide having amino acids 20-227 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0203] The polypeptide may be a fusion polypeptide comprising a polypeptide of the invention.

[0204] In one aspect, the polypeptide is isolated.

[0205] In another embodiment, the polypeptide is purified.

[0206] Wild-type lysozyme Wild-type lysozyme is SEQ ID NO: 1, or amino acids 20-227 of SEQ ID NO: 2 can be obtained from a microorganism of any genus. For purposes of the present invention, the term "obtained from," when used herein in reference to a given source, means that the wild-type encoded by the polynucleotide is produced by the source or strain into which the polynucleotide from the source is inserted. In one aspect, the wild-type is secreted extracellularly.

[0207] In another embodiment, the wild-type lysozyme is derived from Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysosporium keratinophilum, or the like. keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia phimeci fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededoniumspededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptides.

[0208] In another embodiment, the wild-type lysozyme is an Aspergillus aculeatus or Acremonium alcalophilum polypeptide, e.g., a polypeptide obtained from Aspergillus aculeatus CBS172.66 or Acremonium alcalophilum CBS114.92.

[0209] With respect to the aforementioned species, it will be understood that the invention encompasses both perfect and imperfect forms, as well as other taxonomic equivalents (e.g., anamorphs), regardless of the species name by which they are known. Those skilled in the art will readily recognize the equivalence of appropriate equivalents.

[0210] Strains of these species are readily available to the public in numerous culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0211] Wild-type lysozyme can be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.), using the probes described above. Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. A polynucleotide encoding a wild-type lysozyme polypeptide can then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or a mixed DNA sample. After detecting a polynucleotide encoding a wild-type lysozyme polypeptide using one or more probes, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sambrook et al., 1989, supra).

[0212] Preparation of the Polypeptides of the Invention The present invention also relates to a method for obtaining a polypeptide having lysozyme activity, comprising: (a) introducing substitutions at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO:1; and (b) recovering the polypeptide.

[0213] Polypeptides 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, and the like.

[0214] Site-directed mutagenesis is a technique in which one or more mutations are introduced at one or more defined sites in a parent coding polynucleotide.

[0215] Site-specific mutagenesis can be achieved in vitro by PCR, involving the use of oligonucleotide primers containing the desired mutation. Site-specific mutagenesis can also be performed in vitro by cassette mutagenesis, involving restriction enzyme cleavage at a site in a plasmid containing the parent encoding polynucleotide, followed by ligation of an oligonucleotide containing the mutation into the polynucleotide. Typically, the restriction enzymes used to digest the plasmid and the oligonucleotide are the same, allowing the sticky ends of the plasmid and the insert to be ligated together. See, e.g., Scherer and Davis, 1979, Proc. Natl. Acad. Sci. USA 76:4949-4955; and Barton et al., 1990, Nucleic Acids Res. 18:7349-4966.

[0216] Site-directed mutagenesis can also be achieved in vivo by methods known in the art (see, e.g., U.S. Patent Application Publication No. 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).

[0217] Any site-directed mutagenesis procedure can be used in the present invention. Many commercial kits are available that can be used to prepare the polypeptides of the present invention.

[0218] Synthetic gene construction methods involve the in vitro synthesis of polynucleotide molecules designed to encode a polypeptide of interest. Gene synthesis can be performed using a number of techniques, such as the multiplexed microchip-based technique described by Tian et al. (2004, Nature 432:1050-1054) and similar techniques in which oligonucleotides are synthesized and assembled on a photoprogrammable microfluidic chip.

[0219] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known mutagenesis, recombination, and / or shuffling methods, followed by associated screening procedures 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; U.S. Pat. No. 5,223,409; WO 92 / 06204), and region-specific mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0220] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues within a polypeptide.

[0221] Semisynthetic gene construction methods are performed by combining aspects of synthetic gene construction, site-directed mutagenesis, random mutagenesis, and / or shuffling. Semisynthetic construction methods typically utilize synthetic polynucleotide fragments in combination with PCR technology. Thus, defined regions of a gene may be synthesized de novo, while other regions may be amplified using site-directed mutagenesis primers, and still other regions may be subjected to error-prone or non-error-prone PCR amplification. The polynucleotide subsequences may then be shuffled.

[0222] Polynucleotides The present invention also relates to polynucleotides encoding the polypeptides of the present invention.

[0223] The polynucleotide can be genomic DNA, cDNA, synthetic DNA, synthetic RNA, mRNA, or a combination thereof.

[0224] In one embodiment, the polynucleotide is isolated.

[0225] In another embodiment, the polynucleotide is purified.

[0226] nucleic acid construct The present invention also relates to nucleic acid constructs comprising a polynucleotide encoding a polypeptide of the 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.

[0227] Polynucleotides can be manipulated in a variety of ways to result in expression of a polypeptide. It may be desirable or necessary to manipulate the polynucleotide depending on the expression vector prior to insertion into the vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0228] Promoter The control sequence may be a promoter, which is a polynucleotide recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the invention. The promoter contains transcriptional control sequences that mediate expression of the polypeptide. The promoter may be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host.

[0229] Examples of suitable promoters for directing transcription of the polynucleotides of the invention in filamentous fungal host cells are promoters obtained from Aspergillus, Fusarium, Rhizomucor, and Trichoderma cells, such as those described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0230] terminator The control sequence may also be a transcription terminator recognized by the host cell to terminate transcription. The terminator is operably linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator functional in the host cell may be used in the present invention.

[0231] Preferred terminators for filamentous fungal host cells can be obtained from Aspergillus or Trichoderma, such as those obtained from Aspergillus niger glucoamylase, Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, and Trichoderma reesei endoglucanase I, such as the terminators described in Mukherjee et al., 2013, "Trichoderma: Biology and Applications," and Schmoll and Dattenboeck, 2016, "Gene Expression Systems in Fungi: Advancements and Applications," Fungal Biology.

[0232] mRNA stabilizers A regulatory sequence can also be an mRNA stabilizing region downstream of a promoter and upstream of the coding sequence of a gene that increases expression of the gene.

[0233] Examples of suitable mRNA stabilization regions are obtained from the cryIIIA gene of Bacillus thuringiensis (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, J. Bacteriol. 177:3465-3471).

[0234] Examples of mRNA stabilization regions for fungal cells are described in Geisberg et al., 2014, Cell 156(4):812-824, and Morozov et al., 2006, Eukaryotic Cell 5(11):1838-1846.

[0235] Leader sequence The control sequence may also be a leader, untranslated 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 polypeptide. Any leader that is functional in the host cell may be used.

[0236] Preferred leaders for filamentous fungal host cells may be obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0237] Polyadenylation sequence The control sequence may also be a polyadenylation sequence, which is a sequence operably linked to the 3' end of a polynucleotide that, upon transcription, is recognized by a host cell as a signal for the addition of polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence functional in the host cell may be used.

[0238] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0239] signal peptide The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide, directing the polypeptide into the secretory pathway of a cell. The 5' end of a polynucleotide coding sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence encoding the polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence foreign to the coding sequence. A foreign signal peptide coding sequence may be required when the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.

[0240] Effective signal peptide coding sequences for filamentous fungal host cells are signal peptide coding sequences obtained from genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase, such as the signal peptides described by Xu et al., 2018, Biotechnology Letters 40:949-955.

[0241] Propeptide The regulatory sequence may also be a propeptide coding sequence that encodes a propeptide located at the N-terminus of a polypeptide. The resulting polypeptide is known as a proenzyme or propolypeptide (or sometimes a zymogen). Propolypeptides are generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding sequences 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.

[0242] When both a signal peptide and a propeptide sequence are present, the propeptide sequence is located adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located adjacent to the N-terminus of the propeptide sequence.

[0243] Regulatory sequences It may also be desirable to add regulatory sequences that control polypeptide expression relative to the growth of the host cell. Examples of regulatory sequences are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. In filamentous fungi, the Aspergillus niger glucoamylase promoter, the Aspergillus oryzae TAKA alpha-amylase promoter, the Aspergillus oryzae glucoamylase promoter, the Trichoderma reesei cellobiohydrolase I promoter, and the Trichoderma reesei cellobiohydrolase II promoter may be used. Other examples of regulatory sequences are sequences that allow for gene amplification.

[0244] transcription factors The regulatory sequence may also be a transcription factor, a polynucleotide encoding a polynucleotide-specific DNA-binding polypeptide that controls the rate of transcription of genetic information from DNA to mRNA by binding to a specific polynucleotide sequence. Transcription factors can function alone and / or 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 containing at least one DNA-binding domain, which often binds to a specific DNA sequence adjacent to the genetic element regulated by the transcription factor. Transcription factors can regulate the expression of a protein of interest directly, i.e., by activating the transcription of a gene encoding the protein of interest by binding to its promoter, or indirectly, i.e., by activating the transcription of a further transcription factor that 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, and Balleza et al., 2009, FEMS Microbiol. Rev. 33(1):133-151.

[0245] Expression vector The present invention also relates to recombinant expression vectors comprising a polynucleotide encoding a polypeptide of the invention, a promoter, and transcriptional and translational stop signals. Various nucleotide and control sequences can be ligated together to generate a recombinant expression vector, which may contain one or more convenient restriction sites to allow for insertion or substitution of a polynucleotide encoding a polypeptide at such sites. Alternatively, a polynucleotide can be expressed by inserting the polynucleotide, or a nucleic acid construct comprising the polynucleotide, into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed within the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0246] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can bring about expression of a polynucleotide. The choice of 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.

[0247] The vector may be a self-replicating vector, i.e., a vector whose replication exists as an extrachromosomal entity independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any means for ensuring self-replication. Alternatively, the vector may be one that is integrated into the genome when introduced into a host cell and replicated together with one or more chromosomes into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, that together contain the total DNA to be introduced into the genome of the host cell, may be used.

[0248] Vectors preferably contain one or more selectable markers which facilitate the selection of cells that have been transformed, transduced, transduced, etc. A selectable marker is a gene the product of which confers biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0249] Preferably, the vector contains at least one element that allows for integration of the vector into the genome of the host cell or autonomous replication of the vector within the cell independent of the genome.

[0250] For integration into the host cell genome, the vector may rely on the sequence of the polynucleotide 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).

[0251] For autonomous replication, the vector may further comprise an origin of replication that allows the vector to replicate autonomously in a host cell of interest. The origin of replication may be any plasmid replicator that mediates autonomous replication that functions within the cell. The term "origin of replication" or "plasmid replicator" refers to a polynucleotide that enables a plasmid or vector to replicate autonomously in vivo.

[0252] Two or more copies of a polynucleotide of the present invention can be inserted into a host cell to increase polypeptide production. For example, two, three, four, five or more copies can be inserted into the host cell. Increasing the copy number of a polynucleotide can be achieved by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene in the polynucleotide; cells containing an amplified copy of the selectable marker gene, and thus cells containing additional copies of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selection agent.

[0253] host cell The present invention also relates to recombinant host cells containing a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention.

[0254] Once the construct or vector containing the polynucleotide is introduced into a host cell, the construct or vector may be maintained as a chromosomal integrant or a self-replicating extrachromosomal vector, as described above. The choice of host cell will largely depend on the gene encoding the polypeptide and its source. The recombinant host cell may contain a single copy of the polynucleotide of the invention, or at least two copies, e.g., three, four, five, or more copies.

[0255] The host cell can be any cell useful for the recombinant production of a polypeptide of the invention, for example, a prokaryotic or fungal cell.

[0256] The host cell can be any microbial cell, such as a prokaryotic or fungal cell, useful for the recombinant production of the polypeptides of the invention.

[0257] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subdivision Euglena and Oomycota (as defined by Hawksworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus, and carbon catabolism can be fermentative.

[0258] Filamentous fungal host cells include those of the genera Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Nelumbo nucifera, and others. The cell may be a cell of the genus Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma. In preferred embodiments, the filamentous fungal host cell is a cell of the genus Aspergillus, Trichoderma, or Fusarium, hi even more preferred embodiments, the filamentous fungal host cell is a cell of Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, or Fusarium venenatum.

[0259] For example, filamentous fungal host cells may be selected from the group consisting of Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, and Ceriporiopsis girvescens. gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenstrandicum queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioidesbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochromium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestrisThe cells may be cells of Trichoderma terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0260] In one embodiment, the host cell is isolated.

[0261] In another embodiment, the host cells are purified.

[0262] Generation method The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) culturing a recombinant host cell of the present invention under conditions conducive to production of the polypeptide, and optionally (b) recovering the polypeptide.

[0263] The host cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultured in shake flask cultures or by small- or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermentors under conditions that allow for expression and / or isolation of the polypeptide in a suitable medium. Suitable media are available from commercial suppliers or can be prepared according to published compositions (e.g., catalogs of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, the polypeptide can be recovered from cell lysates.

[0264] Polypeptides can be detected using methods known in the art that are specific for the polypeptides, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or enzyme assays to determine the relative or specific activity of the polypeptide.

[0265] The polypeptide may be recovered from the culture medium using methods known in the art, including, but not limited to, harvesting, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the whole fermentation broth is recovered. In another aspect, the cell-free fermentation broth containing the polypeptide is recovered.

[0266] Polypeptides can be purified by various procedures known in the art to obtain substantially pure polypeptides 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).

[0267] In an alternative embodiment, the polypeptide is not recovered.

[0268] Lysozyme Granules The present invention also relates to enzyme granules / particles comprising the polypeptides of the present invention. In one embodiment, the granules comprise a core and optionally one or more coatings (outer layers) surrounding the core.

[0269] The core may have a diameter, measured as an equivalent spherical diameter (average particle size by volume), of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm, or 250 to 1200 μm. The core diameter, measured as an equivalent spherical diameter, may be determined using laser diffraction, such as using a Malvern Mastersizer and / or the method described under ISO 13320 (2020).

[0270] In certain embodiments, the core comprises a polypeptide of the invention.

[0271] The core may contain further substances such as fillers, fibrous materials (cellulose or synthetic), stabilizers, solubilizers, suspending agents, viscosity modifiers, light spheres, plasticizers, salts, lubricants and fragrances.

[0272] The core may include a binder such as a synthetic polymer, a wax, a fat, or a carbohydrate.

[0273] The core may include salts of multivalent cations, reducing agents, antioxidants, peroxide decomposition catalysts and / or acidic buffer components, typically as a homogeneous blend.

[0274] The core may comprise an inert particle into which the polypeptide is absorbed, or onto which the enzyme is applied, for example, by fluidized bed coating.

[0275] The core may have a diameter of 20 to 2000 μm, in particular 50 to 1500 μm, 100 to 1500 μm or 250 to 1200 μm.

[0276] The core may be surrounded by at least one coating, for example, to improve storage stability, reduce dust formation during handling, or to color the granules. Optional coatings may include salt coatings or other suitable coating materials, such as polyethylene glycol (PEG), methyl hydroxypropyl cellulose (MHPC), and polyvinyl alcohol (PVA).

[0277] The coating may be added in an amount of at least 0.1% by weight of the core, such as at least 0.5%, at least 1%, at least 5%, at least 10%, or at least 15%, which may be up to 100%, 70%, 50%, 40%, or 30%.

[0278] The coating is preferably at least 0.1 μm thick, particularly at least 0.5 μm, at least 1 μm, or at least 5 μm thick. In some embodiments, the coating thickness is less than 100 μm, for example less than 60 μm, or less than 40 μm.

[0279] The coating must encapsulate the core unit by forming a substantially continuous layer, which should be understood as a coating with few or no holes, so that there are few or no uncoated areas of the core unit. The layer or coating must in particular be uniform in thickness.

[0280] The coating may further contain other materials known in the art, such as fillers, anti-adherents, pigments, dyes, plasticizers and / or binders, such as titanium dioxide, kaolin, calcium carbonate or talc.

[0281] The salt coating may comprise at least 60% by weight salt, e.g., at least 65% by weight, at least 70% by weight, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, at least 95% by weight, or at least 99% by weight salt.

[0282] To provide acceptable protection, the salt coating is preferably at least 0.1 μm thick, e.g., at least 0.5 μm, at least 1 μm, at least 2 μm, at least 4 μm, at least 5 μm, or at least 8 μm thick. In certain embodiments, the salt coating is less than 100 μm thick, e.g., less than 60 μm or less than 40 μm thick.

[0283] The salt may be added from a salt solution in which the salt is completely dissolved or from a salt suspension in which the particulates are less than 50 μm, such as less than 10 μm or less than 5 μm.

[0284] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility in 100 g of water at 20° C. of at least 0.1 g, preferably at least 0.5 g per 100 g of water, such as at least 1 g per 100 g of water, for example at least 5 g per 100 g of water.

[0285] The salts may be inorganic salts, such as sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides, or carbonates, or salts of simple organic acids (having less than 10 carbon atoms, e.g., 6 or fewer carbon atoms), such as citrates, malonates, or acetates. Examples of cations in these salts include alkali or earth alkali metal ions, ammonium ions, or first transition series metal ions, such as sodium, potassium, magnesium, calcium, zinc, or aluminum. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate, or gluconate. In particular, alkali or earth alkali metal sulfates, sulfites, phosphates, phosphonates, nitrates, chlorides or carbonates or salts of simple organic acids such as citrates, malonates or acetates may be used.

[0286] The salt in the coating may have a constant humidity of more than 60%, in particular more than 70%, 80% or more than 85% at 20° C., or may be another hydrated form of such a salt (e.g., anhydrous). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0287] An example of a suitable salt is NaCl(CH 20℃ = 76%), Na2CO3(CH 20℃ = 92%), NaNO3(CH 20℃= 73%), Na2HPO4(CH 20℃ = 95%), Na3PO4(CH 25℃ = 92%), NH4Cl(CH 20℃ =79.5%), (NH4)2HPO4(CH 20℃ =93.0%), NH4H2PO4(CH 20℃ = 93.1%), (NH4)2SO4(CH 20℃ = 81.1%), KCl(CH 20℃ = 85%), K2HPO4(CH 20℃ = 92%), KH2PO4(CH 20℃ =96.5%), KNO3(CH 20℃ = 93.5%), Na2SO4(CH 20℃ = 93%), K2SO4(CH 20℃ = 98%), KHSO4(CH 20℃ = 86%), MgSO4(CH 20℃ = 90%), ZnSO4(CH 20℃ = 90%) and sodium citrate (CH 25℃ =86%). Other examples include NaH2PO4, (NH4)H2PO4, CuSO4, Mg(NO3)2, and magnesium acetate.

[0288] The salts may be in anhydrous form or may be hydrated salts, i.e., crystalline salt hydrates containing bound water of crystallization, such as those described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (NaSO), anhydrous magnesium sulfate (MgSO), magnesium sulfate heptahydrate (MgSO · 7H2O), zinc sulfate heptahydrate (ZnSO4 · 7H2O), dibasic sodium phosphate heptahydrate (Na2HPO4 · 7H2O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate, and magnesium acetate tetrahydrate.

[0289] Preferably, the salt is added as a solution of the salt, for example using a fluidized bed.

[0290] The coating material can be a wax coating material or a film-forming coating material. Examples of wax coating materials are poly(ethylene oxide) products (polyethylene glycol: PEG) with an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenols with 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains 12 to 20 carbon atoms and there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application by fluidized bed technology are given in GB 1,483,591.

[0291] The granules may optionally have one or more additional coatings. Examples of suitable coating materials are polyethylene glycol (PEG), methylhydroxypropylcellulose (MHPC), and polyvinyl alcohol (PVA). Examples of multi-coated enzyme granules are described in WO 93 / 07263 and WO 97 / 23606.

[0292] The cores can be prepared by granulating a blend of ingredients by methods including granulation techniques such as crystallization, precipitation, pan coating, fluidized bed coating, fluidized bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction, drum granulation, and / or high shear granulation.

[0293] Methods for preparing the cores can be found in Handbook of Powder Technology; Particle size enlargement by CECapes; Vol. 1; 1980; Elsevier. Preparation methods include known feed and granule formulation techniques, such as:

[0294] (a) Spray-dried products, in which a liquid enzyme-containing solution is atomized in a spray-drying tower to form droplets that are dried during passage through the drying tower to form enzyme-containing particulate material, thus producing microparticles (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker).

[0295] (b) Layered products, in which the enzyme is coated as a layer surrounding a preformed inert core particle, the enzyme-containing solution is atomized, typically in a fluidized bed apparatus, the preformed core particle is fluidized, the enzyme-containing solution adheres to the core particle, and the preformed core particle is completely dried, leaving a layer of dried enzyme on the surface of the core particle. If a useful core particle of the desired particle size can be found, particles of the desired particle size can be obtained in this way. Products of this type are described, for example, in WO 97 / 23606.

[0296] (c) Adsorbed core particles, in which the enzyme is adsorbed onto and / or into the core rather than having a layer of polypeptide coated around the core. Such a process is described in WO 97 / 39116.

[0297] (d) Extruded or pelletized products, in which a polypeptide-containing paste is pressed into pellets or extruded under pressure through small orifices and cut into particles, which are then dried. Such particles are usually of considerable size because the material from which the extrusion orifices are made (usually a perforated plate) limits the allowable pressure drop across the orifice. When small orifices are used, very high extrusion pressures also increase heat generation within the enzyme paste, which is detrimental to the enzyme (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pages 140-142; Marcel Dekker).

[0298] (e) A granulated product in which a polypeptide-containing powder is suspended in molten wax and the suspension is sprayed, for example, with a rotating disk atomizer, into a cooled chamber where the droplets rapidly solidify (Michael S. Showell (editor); Powdered detergents; Surfactant Science Series; 1998; Vol. 71; pp. 140-142; Marcel Dekker). The resulting product is one in which the polypeptide is uniformly distributed throughout the inert material instead of being concentrated on its surface. U.S. Pat. Nos. 4,016,040 and 4,713,245 describe this technique.

[0299] (f) Mixer-granulated products, in which a polypeptide-containing liquid is added to a dry powder composition of conventional granulation components. When the appropriate proportions of liquid and powder are mixed, as the moisture from the liquid is absorbed into the dry powder, the dry powder components begin to adhere and aggregate, resulting in particle deposition and the formation of enzyme-containing granules. Such processes are described in U.S. Pat. No. 4,106,991, European Patent Nos. 170360, 304332, and 304331, and International Publication Nos. 90 / 09440 and 90 / 09428. In certain embodiments of this process, various high-shear mixers can be used as granulators. Granules consisting of polypeptide, filler, and binder are mixed with cellulose fibers to reinforce the particles, producing so-called T-granules. The reinforced particles are more robust and less likely to release enzyme dust.

[0300] (g) Size reduction, in which cores are produced by crushing or grinding larger enzyme-containing particles, pellets, tablets, briquettes, etc. The desired core particle fraction is obtained by sieving the crushed or ground product. Oversized and undersized particles can be recycled. Size reduction is described in Martin Rhodes (editor); Principles of Powder Technology; 1990; Chapter 10; John Wiley & Sons.

[0301] (h) Fluidized Bed Granulation. Fluidized bed granulation involves suspending particles in an air stream and spraying a liquid through a nozzle onto the fluidized particles. Particles hit by the spray droplets become wet and sticky. The sticky particles collide with other particles, causing them to adhere together, forming granules.

[0302] (i) The cores may be subjected to drying, such as in a fluidized bed dryer. Other known methods for drying granules in the feed or enzyme industry may be used by those skilled in the art. Drying is preferably carried out at a product temperature of 25 to 90°C. For some enzymes, it is important that the polypeptide-containing cores contain a small amount of water before coating with salt. If a water-sensitive enzyme is coated with salt before removing the excess water, the excess water may become trapped within the core, adversely affecting the enzyme's activity. After drying, the cores preferably contain 0.1 to 10% water by weight.

[0303] Non-shattering granules may be prepared, for example, as disclosed in US Pat. Nos. 4,106,991 and 4,661,452, and optionally coated by methods known in the art.

[0304] The granules may further comprise one or more enzymes. Each enzyme will then be present in more granules, ensuring a more uniform enzyme distribution and reducing physical separation of the various enzymes due to different particle sizes. A method for making multi-enzyme co-granules is disclosed in ip.com disclosure IPCOM000200739D.

[0305] Another example of an enzyme formulation using co-granules is disclosed in WO 2013 / 188331.

[0306] The present invention also relates to protected enzymes made according to the methods disclosed in EP 238,216.

[0307] In one embodiment, the granules further comprise one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectin esterase, triacylglycerol lipase, xylanase, beta-xylosidase, or any combination thereof.

[0308] Liquid formulations The present invention also relates to liquid compositions comprising the polypeptides of the present invention. The compositions may contain enzyme stabilizers (examples include polyols such as propylene glycol or glycerol, sugars or sugar alcohols, lactic acid, reversible protease inhibitors, boric acid or boric acid derivatives, such as aromatic borate esters, or phenylboronic acid derivatives, such as 4-formylphenylboronic acid).

[0309] In some embodiments, fillers or carrier materials are included to increase the volume of such compositions. Suitable fillers and carrier materials include, but are not limited to, various salts such as sulfates, carbonates, and silicates, as well as talc, clay, and the like. Suitable fillers or carrier materials for liquid compositions include, but are not limited to, water or low molecular weight primary and secondary alcohols, including polyols and diols. Examples of such alcohols include, but are not limited to, methanol, ethanol, propanol, and isopropanol. In some embodiments, the compositions contain from about 5% to about 90% of such materials.

[0310] In one aspect, the liquid formulation comprises 20-80% by weight of a polyol. In one embodiment, the liquid formulation comprises 0.001-2% by weight of a preservative.

[0311] In another embodiment, the present invention provides (A) 0.001 to 25 wt % of the polypeptide of the present invention; (B) 20 to 80 wt. % of a polyol; (C) optionally 0.001 to 2% by weight of a preservative, and (D)Water The present invention relates to a liquid formulation comprising:

[0312] In another embodiment, the present invention provides (A) 0.001 to 25 wt % of the polypeptide of the present invention; (B) 0.001 to 2% by weight of a preservative; (C) optionally 20 to 80 wt. % of a polyol, and (D)Water The present invention relates to a liquid formulation comprising:

[0313] In another embodiment, the liquid formulation comprises one or more formulations selected from the group consisting of polyols, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, PVA, acetates, and phosphates, preferably sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate. In one embodiment, the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600, more preferably glycerol, sorbitol, and propylene glycol (MPG), or any combination thereof.

[0314] In another embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol. In one embodiment, the liquid formulation comprises 20% to 80% polyol, e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, where the polyol is selected from the group consisting of glycerol, sorbitol, propylene glycol (MPG), ethylene glycol, diethylene glycol, triethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, dipropylene glycol, polyethylene glycol (PEG) having an average molecular weight of less than about 600, and polypropylene glycol (PPG) having an average molecular weight of less than about 600. In one embodiment, the liquid formulation comprises 20% to 80% polyol (i.e., total amount of polyol), e.g., 25% to 75% polyol, 30% to 70% polyol, 35% to 65% polyol, or 40% to 60% polyol, wherein the polyol is selected from the group consisting of glycerol, sorbitol, and propylene glycol (MPG).

[0315] In another embodiment, the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof. In one embodiment, the liquid formulation comprises 0.02-1.5% by weight of the preservative, e.g., 0.05-1% by weight of the preservative, or 0.1-0.5% by weight of the preservative. In one embodiment, the liquid formulation comprises 0.001-2% by weight of the preservative (i.e., total amount of preservative), e.g., 0.02-1.5% by weight of the preservative, 0.05-1% by weight of the preservative, or 0.1-0.5% by weight of the preservative, wherein the preservative is selected from the group consisting of sodium sorbate, potassium sorbate, sodium benzoate, and potassium benzoate, or any combination thereof.

[0316] In another embodiment, the liquid formulation further comprises one or more additional enzymes, such as hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases. Preferably, the one or more additional enzymes are selected from the group consisting of acetyl xylan esterase, acylglycerol lipase, amylase, alpha-amylase, beta-amylase, arabinofuranosidase, cellobiohydrolase, cellulase, feruloyl esterase, galactanase, alpha-galactosidase, beta-galactosidase, beta-glucanase, beta-glucosidase, lysophospholipase, lysozyme, alpha-mannosidase, beta-mannosidase (mannanase), phytase, phospholipase A1, phospholipase A2, phospholipase D, protease, pullulanase, pectinesterase, triacylglycerol lipase, xylanase, beta-xylosidase, or any combination thereof.

[0317] composition In a still further aspect, the present invention relates to a composition comprising a polypeptide according to the invention having antimicrobial and / or lysozyme activity.

[0318] The composition may comprise a polypeptide according to the invention as the major enzymatic component, eg a mono-component composition.

[0319] The composition can be prepared according to methods known in the art and can be in the form of a liquid or dry composition. For example, the polypeptide composition can be in the form of granules or microgranules. The polypeptide contained in the composition can be stabilized according to methods known in the art.

[0320] Preferred examples of the use of the polypeptide composition according to the present invention are given below. The dosage of the polypeptide composition according to the present invention and other conditions under which the composition is used can be determined based on methods known in the art.

[0321] Animal feed and animal feed additives The lysozyme of the present invention is used in animal feed. In one embodiment, the present invention provides a method for preparing an animal feed composition, comprising adding a lysozyme of the present invention to one or more animal feed ingredients.

[0322] The present invention relates to polypeptides that have improved lysozyme activity against peptidoglycan found in the cell walls of undesirable intestinal microflora, making them suitable for use in animal feed to improve animal health. The lysozymes of the present invention have improved activity compared to the lysozymes described in WO 2013 / 076253. Surprisingly, a large subset of GH25 lysozymes has also been found to have lysozyme activity against Lactobacillus johnsonii, a key bacterium in the intestinal microflora of animals. Without being bound by theory, it is believed that the removal of dead Lactobacillus johnsonii cells from the intestinal microflora by enzymatic lysis of partially degraded bacterial cell walls is an important contribution to the intestinal health of animals.

[0323] One aspect of the present invention relates to a method for improving the intestinal health of an animal, including reducing the amount of dead cells or cell wall debris in the animal's digestive tract, comprising feeding the animal a feed or feed additive comprising a polypeptide as defined by the present invention.

[0324] The lysozyme according to the invention is effective against, for example, Clostridium perfringens, Escherichia coli and Salmonella, such as Salmonella enterica, Salmonella Typhimurium and Salmonella mbandaka. Lysozyme may be used to stabilize the healthy microflora of animals, particularly livestock such as, but not limited to, sheep, goats, cattle (including but not limited to beef cattle, dairy cattle, and calves), deer, pigs or wild boars (including but not limited to piglets, growing pigs, and sows), poultry (including but not limited to geese, turkeys, ducks, and chickens, e.g., broilers, chicks, and laying hens), horses, moose, and rabbits, as well as seafood (including but not limited to salmon, trout, tilapia, catfish, and carp, and shellfish (including but not limited to shrimp and prawns)), by inhibiting the growth / intestinal colonization of viral, bacterial, or parasitic pathogens such as Bacillus subtilis (B. spp.). In a preferred embodiment, lysozyme replaces antibiotics in animal feed. In a preferred embodiment, lysozyme is applied to chickens and has antimicrobial activity against Clostridium perfringens.

[0325] In a further embodiment, the lysozyme according to the invention is used as a feed additive to positively influence the microbial balance in the chicken digestive tract and thus improve animal productivity.

[0326] The lysozyme according to the invention can also be used in animal feed as a feed fortification enzyme to improve feed digestibility and increase its utilization efficiency in accordance with WO 00 / 21381 and WO 04 / 026334.

[0327] The present invention also relates to the use of a polypeptide according to the invention having lysozyme activity in animal feed, as well as to feed compositions and feed additives comprising a lysozyme according to the invention.

[0328] The polypeptides according to the invention can be used to inhibit, for example, viruses (such as members of the Coronaviridae family, porcine reproductive and respiratory syndrome virus (PRRSV), Persivirus, which causes bovine viral diarrhea, etc.), parasitic pathogens (such as the coccidia protozoan Eimeria maxima and Eimeria mitis), or bacterial pathogens, such as Clostridium perfringens, Escherichia coli, Campylobacter coli, C. hyointestinalis, and C. jejuni, Yersinia subsp., Treponema suis, Brachyspira hyodysenteriae, and the like. Salmonella, such as Salmonella enterica, Salmonella Typhimurium, and Salmonella mbandaka. mbandaka), thereby stabilizing the healthy microflora of animals, particularly livestock such as, but not limited to, sheep, goats, cattle (including but not limited to beef cattle, dairy cattle, and calves), deer, pigs or wild boars (including but not limited to piglets, growing pigs, and sows), poultry (including but not limited to, geese, turkeys, ducks, and chickens, e.g., broilers, chicks, and laying hens), horses, moose, and rabbits, as well as seafood (including but not limited to, salmon, trout, tilapia, catfish, and carp, and shellfish (including but not limited to, shrimp and prawns)).

[0329] One aspect of the present invention is a polypeptide of the present invention, v. In animal feed, vi. In animal feed additives; vii. in the preparation of a composition for use in animal feed, and / or viii. To improve the intestinal health of animals; Regarding use.

[0330] A further aspect of the present invention relates to a zootechnical additive for use in poultry or swine feed, the additive comprising a polypeptide as defined herein.

[0331] In the use according to the invention, lysozyme can be provided to the animal before, after or simultaneously with a meal, the latter being preferred.

[0332] In certain embodiments, the lysozyme is well-defined when added to feed or contained in a feed additive. Well-defined means that the lysozyme preparation is at least 50% pure as determined by size-exclusion chromatography (see Example 12 of WO 01 / 58275). In other specific embodiments, the lysozyme preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method.

[0333] A well-defined lysozyme formulation is advantageous. For example, it is much easier to accurately dose a lysozyme to a feed that does not essentially interfere with or contaminate other lysozymes. The term "dose" specifically refers to the goal of obtaining consistent and consistent results and the ability to optimize the dosage based on the desired effect.

[0334] However, for use in animal feed, lysozyme does not have to be pure: it may, for example, contain other enzymes, in which case it may be referred to as a lysozyme preparation.

[0335] The lysozyme preparation can be (a) added directly to the feed, or (b) used to produce one or more intermediate compositions, such as feed additives or premixes, which are subsequently added to the feed (or used in a treatment process). The degrees of purity described above refer to the purity of the original lysozyme preparation, whether used according to (a) or (b) above.

[0336] The lysozyme of the present invention can also be used to prevent necrotizing enterocolitis and / or Clostridium perfringens.

[0337] In one embodiment, the present invention also relates to a method for improving the performance of an animal, comprising administering to the animal the animal feed or animal feed additive of the present invention.

[0338] The term feed or feed composition refers to any compound, preparation, mixture, or composition suitable for or intended for ingestion by an animal. In the use according to the present invention, lysozyme can be provided to the animal before, after, or simultaneously with a meal, the latter being preferred. Such a lysozyme composition can, of course, be mixed with other enzymes.

[0339] The lysozyme can be added to the feed in any form, either as relatively pure lysozyme or mixed with other components intended for addition to animal feed, i.e. in the form of an animal feed additive, e.g. in the form of a so-called premix for animal feed. In a further aspect, the present invention relates to animal feed, e.g. animal feed, and compositions for use in animal feed additives, e.g. premixes.

[0340] Preferred examples of the use of lysozyme or compositions thereof according to the present invention are given below: The dosage of lysozyme and other conditions under which lysozyme is used can be determined based on methods known in the art.

[0341] In addition to the lysozyme according to the invention, the animal feed additive according to the invention contains at least one fat-soluble vitamin, and / or at least one water-soluble vitamin, and / or at least one trace mineral, and / or at least one macromineral.

[0342] Further optional feed additive ingredients are colorants, for example, carotenoids, such as beta-carotene, astaxanthin, and lutein, stabilizers, growth improvement additives, and aroma compounds / flavors, for example, creosol, anethole, deca-, undeca-, and / or dodecalactone, ionones, irones, gingerols, piperidine, propylidenephthalide, butylidenephthalide, capsaicin, and / or tannins, polyunsaturated fatty acids (PUFAs), reactive oxygen generating species, and a support may be used which may contain, for example, 40-50% by weight wood fiber, 8-10% by weight stearin, 4-5% by weight turmeric powder, 4-58% by weight rosemary powder, 22-28% by weight limestone, 1-3% by weight gum, for example gum arabic, 5-50% by weight sugar and / or starch, and 5-15% by weight water.

[0343] The feed or feed additive according to the invention may also comprise at least one other enzyme selected from phytase (EC 3.1.3.8 or 3.1.3.26), xylanase (EC 3.2.1.8), galactanase (EC 3.2.1.89), alpha-galactosidase (EC 3.2.1.22), protease (EC 3.4), phospholipase A1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), phospholipase C (3.1.4.3), phospholipase D (EC 3.1.4.4), amylase, such as alpha-amylase (EC 3.2.1.1); and / or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

[0344] Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of reactive oxygen generating species are chemicals such as perborates, persulfates, or percarbonates; and enzymes such as oxidases, oxygenases, or synthetases.

[0345] Typically, fat-soluble and water-soluble vitamins, as well as trace minerals, form part of so-called premixes intended for addition to the feed, while macrominerals are usually added separately to the feed. Any of these composition types, when enriched with the proteases of the present invention, are animal feed additives of the present invention.

[0346] In particular embodiments, the animal feed additive of the present invention is intended to be (or is specified to be) included in the animal's diet or feed at a level of 0.001 to 1.0%, more particularly 0.005 to 0.5%, or 0.02 to 0.1% (% meaning g of additive per 100 g of feed). This is particularly true in the case of premixes.

[0347] Animal feed typically includes plant-based material, which in some embodiments is selected from the group consisting of legumes, cereals, oats, rye, barley, wheat, corn, sorghum, switchgrass, millet, pearl millet, foxtail millet, soybean, wild soybean, pea, lupin, tepary bean, runner bean, slim jim bean, lima bean, green bean, broad bean (fava bean), chickpea, lentil, peanut, Spanish peanut, canola, rapeseed (rapeseed), rice, sugar beet, cabbage, sugar beet, spinach, quinoa, or pea, or any combination thereof, or processed forms thereof (such as soybean meal, rapeseed meal, etc.).

[0348] Animal feed compositions according to the present invention may contain at least one vegetable protein, including modified proteins and protein derivatives, such as those derived from or derived from vegetables. The plant protein may be derived from a plant protein source such as legumes and grains, e.g., material from plants of the Fabaceae (leguminous family), Cruciferaceae, Chenopodiaceae, and Poaceae families, e.g., soybean meal, lupin meal, and rapeseed meal; alternatively, the plant protein source is material from one or more plants of the Chenopodiaceae family, e.g., beet, sugar beet, spinach, or quinoa. Other examples of plant protein sources are rapeseed, sunflower seed, cottonseed, and cabbage, as well as grains such as barley, wheat, rye, oats, maize (corn), rice, triticale, and sorghum. The animal feed composition of the present invention may also contain animal protein such as meat and bone meal, feather meal, and / or fish meal, typically in an amount of 0 to 25%.The animal feed composition of the present invention may further contain distillers dried grains with solubles (DDGS), typically in an amount of 0 to 30%.

[0349] In still further specific embodiments, the animal feed composition of the present invention contains 0-80% corn; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% barley; and / or 0-30% oat; and / or 0-40% soybean meal; and / or 0-25% fish meal; and / or 0-25% meat and bone meal; and / or 0-20% whey.

[0350] Animal feed can be prepared, for example, as a mash feed (non-pelleted) or a pelleted feed. Typically, the ground feed material is mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications of the species in question. Enzymes can be added as solid or liquid enzyme preparations. For example, for mash feed, a solid or liquid enzyme preparation can be added before or during the raw material mixing step. For pelleted feed, a (liquid or solid) lysozyme / enzyme preparation can also be added before or during the feed raw material process. Typically, a liquid lysozyme / enzyme preparation is added after the pelleting step. Enzymes can also be incorporated into feed additives or premixes.

[0351] The final enzyme concentration in the feed is in the range of 0.01 to 200 mg enzyme protein per kg feed, for example, in the range of 0.5 to 25 mg enzyme protein per kg feed.

[0352] This invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

[0353] Preferred embodiments of the present invention are further defined by the following paragraphs.

[0354] 1. A polypeptide having lysozyme activity comprising a substitution at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO:1, wherein the polypeptide has at least 60%, at least 65%, 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% but less than 100% sequence identity to SEQ ID NO:1 or amino acids 20-227 of SEQ ID NO:2.

[0355] 2. The polypeptide of paragraph 1, wherein the polypeptide has improved thermal stability compared to a polypeptide having the sequence of amino acids 20 to 227 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0356] 3. The polypeptide of paragraph 1 or 2, wherein the improved thermal stability is measured to be at least 5°C, at least 5.5°C, at least 6°C, at least 6.5°C, at least 7°C, at least 7.5°C, at least 8°C, at least 8.5°C, at least 9°C, at least 9.5°C, or at least 10°C, compared to a polypeptide having amino acids 20 to 227 of SEQ ID NO:1 or SEQ ID NO:2.

[0357] 4. The polypeptide of any one of paragraphs 1 to 3, comprising 1 to 20 changes, such as 1 to 10 changes, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 changes.

[0358] 5. The polypeptide of any one of paragraphs 1 to 4, wherein the number of substitutions is, for example, 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions.

[0359] 6. The polypeptide of any one of paragraphs 1 to 5, comprising a substitution of the amino acid residue at position 1 of SEQ ID NO: 1 with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Thr or Gln.

[0360] 7. The polypeptide of any one of paragraphs 1 to 6, comprising a substitution of the amino acid residue at position 10 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val, preferably Ala, Asp, or Arg.

[0361] 8. The polypeptide of any one of paragraphs 1 to 7, comprising a substitution of the amino acid residue at position 14 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, preferably Val.

[0362] 9. The polypeptide of any one of paragraphs 1 to 8, comprising a substitution of the amino acid residue at position 37 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asn or Tyr.

[0363] 10. The polypeptide of any one of paragraphs 1 to 9, comprising a substitution of the amino acid residue at position 41 of SEQ ID NO: 1 with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Tyr.

[0364] 11. The polypeptide of any one of paragraphs 1 to 10, comprising a substitution of the amino acid residue at position 103 of SEQ ID NO: 1 with Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ser.

[0365] 12. The polypeptide of any one of paragraphs 1 to 11, comprising a substitution of the amino acid residue at position 118 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Arg.

[0366] 13. The polypeptide of any one of paragraphs 1 to 12, comprising a substitution of the amino acid residue at position 122 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr or Val, preferably Pro.

[0367] 14. The polypeptide according to any one of paragraphs 1 to 13, comprising a substitution of the amino acid residue at position 125 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Pro.

[0368] 15. The polypeptide of any one of paragraphs 1 to 14, comprising a substitution of the amino acid residue at position 126 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Asn or Pro.

[0369] 16. The polypeptide of any one of paragraphs 1 to 15, comprising a substitution of the amino acid residue at position 131 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Tyr, preferably Cys.

[0370] 17. The polypeptide of any one of paragraphs 1 to 16, comprising a substitution of the amino acid residue at position 143 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Asn.

[0371] 18. The polypeptide of any one of paragraphs 1 to 17, comprising a substitution of the amino acid residue at position 153 of SEQ ID NO: 1 with Ala, Arg, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ser.

[0372] 19. The polypeptide of any one of paragraphs 1 to 18, comprising a substitution of the amino acid residue at position 155 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, preferably Trp.

[0373] 20. The polypeptide of any one of paragraphs 1 to 19, comprising a substitution of the amino acid residue at position 174 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Pro.

[0374] 21. The polypeptide of any one of paragraphs 1 to 20, comprising a substitution of the amino acid residue at position 175 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr or Val, preferably Ser.

[0375] 22. The polypeptide of any one of paragraphs 1 to 21, comprising a substitution of the amino acid residue at position 188 of SEQ ID NO: 1 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Pro.

[0376] 23. The polypeptide according to any one of paragraphs 1 to 22, comprising a substitution of the amino acid residue at position 202 of SEQ ID NO: 1 with Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val, preferably Glu.

[0377] 24. The polypeptide of any one of paragraphs 1 to 23, wherein the polypeptide consists of 200 to 250 amino acids, for example, 210 to 240, 215 to 235, and 220 to 240 amino acids.

[0378] 25. The polypeptide of any one of paragraphs 1 to 24, comprising substitutions at least two positions corresponding to any of positions 10, 13, 14, 143, 166, and 176 of SEQ ID NO:1.

[0379] 26. The polypeptide of any one of paragraphs 1 to 25, comprising substitutions at least three positions corresponding to any of positions 10, 13, 14, 37, 101, 153, 166, 176, 202 of SEQ ID NO:1.

[0380] 27. The polypeptide of any one of paragraphs 1 to 26, comprising substitutions at least four positions corresponding to any of 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 63, 65, 101, 118, 122, 125, 126, 129, 131, 166, 175, 176, and 202 of SEQ ID NO:1.

[0381] 28. The polypeptide of any one of paragraphs 1 to 27, comprising substitutions at least five positions corresponding to any of positions 1, 10, 13, 14, 118, 122, 166, 176 of SEQ ID NO:1.

[0382] 29. The polypeptide of any one of paragraphs 1 to 28, comprising a substitution at position 1 of SEQ ID NO: 1, preferably at the position corresponding to R1Q.

[0383] 30. The polypeptide of any one of paragraphs 1 to 29, comprising substitutions at positions 166 and 176 of SEQ ID NO: 1, preferably at positions corresponding to V166P+Y176W.

[0384] 31. The polypeptide of any one of paragraphs 1 to 30, comprising substitutions at positions 10, 166 and 176 of SEQ ID NO: 1, preferably at positions corresponding to W10A+V166P+Y176W.

[0385] 32. The polypeptide of any one of paragraphs 1 to 31, comprising substitutions at positions 14, 166 and 176 of SEQ ID NO: 1, preferably at positions corresponding to T14V+V166P+Y176W.

[0386] 33. The polypeptide of any one of paragraphs 1 to 32, comprising substitutions at positions 10, 14, 166 and 176 of SEQ ID NO: 1, preferably at positions corresponding to W10A+T14V+V166P+Y176W.

[0387] 34. The polypeptide of any one of paragraphs 1 to 33, comprising substitutions at positions 10, 13, 14, 166, 176 of SEQ ID NO: 1, preferably at positions corresponding to W10A+T13N+T14V+V166P+Y176W.

[0388] 35. The polypeptide of any one of paragraphs 1 to 34, comprising substitutions at positions 1, 10, 13, 14, 166 and 176 of SEQ ID NO: 1, preferably at positions corresponding to R1Q+W10A+T13N+T14V+V166P+Y176W.

[0389] 36. The polypeptide comprises any of the following substitutions or combinations of substitutions in SEQ ID NO:1: R1T, R1Q, W10A, W10D, W10R, T14V, F37N, F37Y, A41Y, A103S, E118R, T122P, H1 25P, G126N, G126P, V131C, Q143N, N153S, C155W, G174P, F175S, G188P, R202E, V166P+Y176W, T14V+N101S+R202E, W10R+T13N+F37Y、 T13N+T14V+F37Y、 W10D+T13N+N101S、 W10D+N101S+V166P+Y176W、 W10D+V166P+Y176W、 W10A+T13N+T14V、 W10A+E118R+T122D+V166P+Y176W、 W10D+T14V+E118R+T122D、 W10D+V166P+Y176W+R202E、 W10R+T14V+N101S、 W10A+T13N+E118R+T122D、 W10D+N101S+N153S、 W10A+T14V+F37Y、 F37Y+V166P+Y176W+R202E、 W10A+E118R+T122D+R202E、 T13N+T14V+R202E、 W10A+T14V+N153S、 D6S+W10D+N101S+V166P D6S+G9P+W10D+N101S、 D6S+W10D+T36E+N101S、 G9P+W10D+N101S+F175N、 G9P+W10D+N101S+S129G、 W10D+T36E+F37E+N101S、 W10D+Q143E、 W10D+S40P+N101S+F175N、 W10D+Y20W+N101S+F175N、 I2V+G9P+W10D+N101S、 W10D+Q63R+A65D+N101S、 W10D+N101S+H125K+W131Y、 T13N+Y176W、 W10A+T14V、 W10D+T14V、 W10R+Y176W, W10R+V166P, T13N+V166P+Y176W, T13N+T14V+V166P+Y176W, W10A+T13N+V166P+Y176W, T14V+V166P+Y176W, W10A+V166P+Y176W, W10A+T14V+V166P+Y176W W10D+V166P+Y176W, W10A+T13N+T14V+V166P+Y176W, F37Y+V166P+Y176W+R202E, W10A+T13N+T122D+V166P+Y176W, W10A+V166P+Y176W+R202E, W10A+F37Y+V166P+Y176W, and R1Q+W10A+T13N+T14V+V166P+Y176W 36. The polypeptide of any one of paragraphs 1 to 35, comprising at least one of:

[0390] 37. The polypeptide of any one of paragraphs 1 to 36, comprising one or more of the following substitutions at positions corresponding to positions R1Q, W10A, T13N, T14V, V166P, Y176W in SEQ ID NO:1:

[0391] 38. A fusion polypeptide comprising a polypeptide according to any one of paragraphs 1 to 37 and a second polypeptide.

[0392] 39. Granules, (a) a core comprising the polypeptide of any one of paragraphs 1 to 37; and optionally, (b) a coating consisting of one or more layers surrounding the core; Granules containing

[0393] 40. Granules, (c) a core; (d) a coating consisting of one or more layers surrounding the core, the coating comprising a variant of any one of paragraphs 1 to 37; and Granules containing

[0394] 41. A liquid composition comprising the polypeptide of any one of paragraphs 1 to 37 and an enzyme stabilizer, e.g., a polyol, e.g., propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, a reversible protease inhibitor, boric acid or a boric acid derivative, e.g., an aromatic boric acid ester, or a phenylboronic acid derivative, e.g., 4-formylphenylboronic acid.

[0395] 42. The liquid composition according to paragraph 41, further comprising a filler or carrier material.

[0396] 43. The liquid composition according to paragraph 41 or 42, further comprising a preservative.

[0397] 44. A composition comprising a polypeptide according to any one of paragraphs 1 to 37, a granule according to paragraph 39 or 40, or a liquid composition according to any one of paragraphs 35 to 37.

[0398] 45. A polynucleotide encoding the polypeptide of any one of paragraphs 1 to 37, wherein the polynucleotide has 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% but less than 100% sequence identity to SEQ ID NO:3.

[0399] 46. ​​The polynucleotide of paragraph 45, which is isolated.

[0400] 47. The polynucleotide of paragraph 45 or 46, which is purified.

[0401] 48. A nucleic acid construct or expression vector comprising a polynucleotide according to any one of paragraphs 45 to 47.

[0402] 49. A recombinant host cell transformed with a polynucleotide according to any one of paragraphs 45 to 47.

[0403] 50. A recombinant host cell according to paragraph 49, comprising at least two copies, such as three, four or five or more copies of the polynucleotide of any one of paragraphs 45 to 47.

[0404] 51. Filamentous fungal recombinant host cells, such as Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastea Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma cells, in particular Aspergillus awamori awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirinaaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium meldarium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Talaromyces emersonii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum 51. The recombinant host cell of paragraph 49 or 50, which is a Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride cell.

[0405] 52. The recombinant host cell of any one of paragraphs 49 to 51, which is isolated.

[0406] 53. The recombinant host cell of any one of paragraphs 49 to 51, which is purified.

[0407] 54. a) culturing the host cell of any one of paragraphs 49 to 53 under conditions suitable for expression of the polypeptide; b) recovering the polypeptide; A method for producing a polypeptide, comprising:

[0408] 55. A method for obtaining a polypeptide, comprising introducing substitutions at one or more positions corresponding to 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO:1, wherein each modification is independently a substitution, and wherein the polypeptide has lysozyme activity; and recovering the polypeptide.

[0409] 56. A whole broth preparation or cell culture composition comprising the polypeptide of any of paragraphs 1-37.

[0410] 57. A composition comprising a polypeptide according to paragraphs 1-37.

[0411] 58. The composition of paragraph 57, wherein the composition further comprises one or more components.

[0412] 59. The composition of paragraph 58, wherein one or more components is an enzyme.

[0413] 60. The composition according to paragraphs 57 to 59, wherein the composition is a liquid and / or solid composition.

[0414] 61. The composition of paragraphs 57 to 60, wherein the composition is an animal feed composition.

[0415] 62. a) a polypeptide according to paragraphs 1 to 37, and b) at least one fat-soluble vitamin, and / or c) at least one water-soluble vitamin, and / or d) at least one trace mineral Animal feed additives, including

[0416] 63. The animal feed additive according to paragraph 62, further comprising one or more enzymes.

[0417] 64. Use of a polypeptide according to any of paragraphs 1 to 37 in animal feed.

[0418] 65. Use of a polypeptide according to any of paragraphs 1 to 37 for disrupting bacterial cell walls. [Example]

[0419] Chemicals used were commercially available products of at least reagent grade.

[0420] Example 1: Preparation of variants and determination of activity Preparation of muramidase variants Expression of muramidase variants in Aspergillus oryzae An Aspergillus expression vector was constructed using a construct containing the S. alcalophilus muramidase variant gene of the present example. The Aspergillus expression vector consisted of an expression cassette based on the Aspergillus niger neutral amylase II promoter fused to the Aspergillus nidulans triosephosphate isomerase untranslated leader sequence (Pna2 / tpi) and the Aspergillus niger amyloglycosidase terminator (Tamg). The plasmid also contained the Aspergillus selection marker pyrG from Aspergillus nidulans, allowing growth of pyrG-minus Aspergillus strains on minimal media. Expression plasmids for the muramidase variants were transformed into Aspergillus as described by Lassen et al. (2001), Applied and Environmental Microbiology, 67, 4701-4707. For each construct, four to six strains were isolated, purified, and grown in microtiter plates. Expression was determined using SDS-PAGE analysis. The best-producing strains were fermented in shake flasks.

[0421] Purification of S. alcalophilus muramidase variants The fermentation supernatant containing the muramidase variant was filtered through a Fast PES bottle-top filter with a 0.22 μm cutoff. The resulting solution (100–150 ml) was diluted to 500 ml with MQ water and the pH was adjusted to 4.5 with acetic acid.

[0422] After pretreatment, the muramidase variants were purified by chromatography on approximately 30 ml of S Sepharose in an XK26 column using 50 mM sodium acetate pH 4.5 as buffer A and 50 mM sodium acetate + 1 M NaCl pH 4.5 as buffer B. Fractions from the column were pooled based on the chromatogram monitoring the absorbance at 260 and 280 nm.

[0423] The molecular weight estimated by SDS-PAGE was approximately 22 kD, and the purity was >95%. The molecular weight of the variant confirmed the expected mutation (+ / - 1 Da). Lysozyme activity assay using fluorescein-conjugated peptidoglycan Fifty microliters of lysozyme sample (diluted to 0.01 mg / ml with 0.01% Triton) was added to 50 μl of fluorescein-conjugated peptidoglycan (12.5 μg / ml) in 140 mM NaHPO, 40 mM citric acid, 0.01% Triton, pH 6, in wells of a microtiter plate. The plate was incubated at 30°C for 30 minutes. During incubation, the rate of the reaction was monitored at 485 nm (excitation) / 528 nm (emission) for a 96-well plate at 2-minute intervals on a 15 SPECTRAMAX plate reader (Molecular Devices LLC). All variants were found to be active on fluorescein-conjugated peptidoglycan.

[0424] Example 2: Screening variants for thermostability Filtered fermentation broth was used to test single and multi-position variants for thermostability by nanodifferential scanning fluorescence (nanoDSF).

[0425] The nanoDSF monitors the intrinsic tryptophan (Trp) fluorescence of proteins as a function of temperature at 330 and 350 nm. The thermal stability of a protein can be expressed by its Tm (the temperature at which equal populations of folded and unfolded molecules exist), which is found at the inflection point of the fluorescence signal.

[0426] NanoDSF was performed using a nanoDSF Prometheus NT.48 instrument (NanoTemper Technologies GmbH, München, Germany). Muramidase variant samples (purified as described in Example 1, all in 50 mM sodium acetate, pH 4.5) were loaded into nanoDSF standard-grade capillaries (NanoTemper Technologies GmbH; catalog number PR-C002) by capillary action. Three capillaries were loaded for each sample. The capillaries were then placed into the instrument (up to 48 single capillaries can be loaded in a single run), and the laser intensity required for optimal signal generation was determined. Samples were run with the following experimental settings: a temperature ramp of 2°C / min, a starting temperature of 20°C, and an ending temperature of 95°C.

[0427] In some combination variations, nanoDSF is performed in closed capillaries, where the end temperature can be extended to 110°C.

[0428] [Table 1]

[0429] [Table 2]

[0430] [Table 3]

[0431] Example 3: Thermal stability of variants in the pH range 3.0-8.0 The purified variants were tested for thermal stability over the pH range 3.0-8.0 using NanoDSF (described above). Variant samples were in 0.1 M glycine, 0.1 M acetic acid, 0.1 M Bis-Tris and adjusted to the desired pH with either 0.5 M HCl or 0.1 M NaOH. The temperature gradient in this experiment was 3.33 °C / min. The results are shown in Table 2.

[0432] [Table 4]

[0433] The variant has an increased unfolding temperature at all pH values ​​tested.

[0434] The present invention as described and claimed herein should not be limited in scope by the specific embodiments disclosed herein, since these embodiments are intended to be illustrative of some aspects of the invention. All equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description.

[0435] Example 4: Muramidase activity assay The fluorescent assay described herein measures active wild-type (WT) and variant muramidases. Muramidase activity is measured using an in-house synthesized substrate consisting of fluorescein isothiocyanate (FITC)-labeled peptidoglycan (PGN). Micrococcus lysodeikticus ATCC No. 4698 PGN was obtained from Sigma (Cat. No. M3770). Alternatively, the Invitrogen™ EnzChek™ Lysozyme Assay Kit (Cat. No. E22013) can be used.

[0436] Assay principle: FITC-PGN is labeled to such an extent that its fluorescence is quenched. Muramidase action can relieve this quenching, resulting in a dramatic increase in fluorescence that is proportional to muramidase activity.

[0437] Muramidase activity is measured as the increase in fluorescence signal over 1-10 minutes using a standard plate reader in kinetic mode. Fluorescence from a no-enzyme control and a known muramidase reference is measured in parallel with the enzyme sample. Initial reaction rates for the sample muramidase, reference muramidase, and no-enzyme control are calculated as the maximum rate of change in fluorescence over time from t = 0 to t = 1-10 minutes. The no-enzyme control is used to correct for background signal in both the sample and control by subtracting the no-enzyme initial rate value from the sample and control initial rates. The calculated initial rates are normalized by the known muramidase concentration to obtain concentration-normalized activity. Finally, the sample muramidase activity is divided by the reference muramidase activity to obtain the reported relative muramidase activity (RelAct).

[0438] Selected materials: In-house FITC-labeled Micrococcus lysodeikticus ATCC No. 4698 made from Sigma M3770 material.

[0439] Assay Protocol: Prepare muramidase enzyme stock samples: 1 ppm in Milli-Q® water (containing 0.01 v% Triton® X-100). Further dilute the three muramidase stocks 10-fold to obtain four enzyme samples at 1.0, 0.1, -0.01, and 0.001 ppm. Prepare FITC-PGN substrate by dissolving FITC-PGN powder in an appropriate buffer (e.g., 140 mM NaHPO, 40 mM citric acid, 0.01 v% Triton X-100, pH 6.0) to a concentration of 12 μg / mL. Mix 50 μL of each enzyme sample with 50 μL of substrate. Read the fluorescent signal on a Biotek Synergy H1 plate reader using a fluorescence gain setting of 75 and excitation / emission of 485 / 528 nm for 30 minutes.

[0440] Data values ​​with an assay response in the linear range are used to evaluate the data as described above, using muramidase of SEQ ID NO: 1 as a reference.

[0441] The results for Sodiomyces alcalophilus wild type (SEQ ID NO: 1) and variants are shown in Table 4 below:

[0442] [Table 5]

[0443] Example 5: Steam Box Treatment Four samples were prepared as listed in Table 3 below.

[0444] [Table 6]

[0445] Four samples were subjected to a laboratory-scale steaming box, exposing the samples to temperatures of 90°C (°C) and 95°C, 95% relative humidity for a conditioning time of 90 seconds.

[0446] FIG. 1 shows a graph demonstrating the residual activity of the polypeptides of the invention and the wild type under steam box conditions.

[0447] Results: Sample 1 is very stable under steam box conditions. Sample 1 exhibits approximately a 50% improvement over Sample 2 when exposed to a temperature of 90° C. and approximately an 85% improvement when exposed to a temperature of 95° C. Sample 1 exhibits approximately a 27% improvement over Sample 4 when exposed to 95° C., while Sample 1 exhibits approximately an 8% improvement over Sample 4 when exposed to 90° C.

[0448] Conclusion: The substitutions of the present invention resulted in an increase in Tm and residual activity, thereby allowing either a concomitant increase in activity (sample 1 vs. sample 4) or a cheaper formulation with dramatically higher activity in the same formulation (sample 1 vs. sample 2).

Claims

1. 1. A polypeptide having lysozyme activity comprising substitutions at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO:1, wherein the polypeptide has at least 60%, at least 65%, 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%, but less than 100%, sequence identity to SEQ ID NO:1 or amino acids 20-227 of SEQ ID NO:

2.

2. 2. The polypeptide of claim 1, wherein the polypeptide has improved thermal stability compared to a polypeptide having amino acids 20 to 227 of SEQ ID NO: 1 or SEQ ID NO:

2.

3. 3. The polypeptide of claim 1 or 2, wherein the improved thermal stability is measured to be at least 5°C, at least 5.5°C, at least 6°C, at least 6.5°C, at least 7°C, at least 7.5°C, at least 8°C, at least 8.5°C, at least 9°C, at least 9.5°C, or at least 10°C compared to SEQ ID NO:1 or a polypeptide having amino acids 20 to 227 of SEQ ID NO:

2.

4. 4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises one or more of the following substitutions at positions corresponding to positions 1, 10, 13, 14, 166, 176 of SEQ ID NO: 1:

5. The polypeptide has any of the following substitutions or combinations of substitutions in SEQ ID NO:1: R1Q, R1T, W10A, W10D, W10R, T14V, F37N, F37Y, A41Y, A103S, E118R, T122P, H1 25P, G126N, G126P, V131C, Q143N, N153S, C155W, G174P, F175S, G188P, R202E, V166P+Y176W, T14V+N101S+R202E, W10R+T13N+F37Y, T13N+T14V+F37Y, W10D+T13N+N101S, W10D+N101S+V166P+Y176W, W10D+V166P+Y176W, W10A+T13N+T14V, W10A+E118R+T122D+V166P+Y176W, W10D+T14V+E118R+T122D, W10D+V166P+Y176W+R202E, W10R+T14V+N101S, W10A+T13N+E118R+T122D, W10D+N101S+N153S, W10A+T14V+F37Y, F37Y+V166P+Y176W+R202E, W10A+E118R+T122D+R202E, T13N+T14V+R202E, W10A+T14V+N153S, D6S+W10D+N101S+V166P D6S+G9P+W10D+N101S, D6S+W10D+T36E+N101S, G9P+W10D+N101S+F175N, G9P+W10D+N101S+S129G, W10D+T36E+F37E+N101S, W10D+Q143E, W10D+S40P+N101S+F175N, W10D+Y20W+N101S+F175N, I2V+G9P+W10D+N101S, W10D+Q63R+A65D+N101S, W10D+N101S+H125K+W131Y, T13N+Y176W, W10A+T14V, W10D+T14V, W10R+Y176W, W10R+V166P, T13N+V166P+Y176W, T13N+T14V+V166P+Y176W, W10A+T13N+V166P+Y176W, T14V+V166P+Y176W, W10A+V166P+Y176W, W10A+T14V+V166P+Y176W, W10D+V166P+Y176W, W10A+T13N+T14V+V166P+Y176W, F37Y+V166P+Y176W+R202E, W10A+T13N+T122D+V166P+Y176W, W10A+V166P+Y176W+R202E, W10A + F37Y + V166P + Y176W, and R1Q+W10A+T13N+T14V+V166P+Y176W 5. The polypeptide of claim 1, wherein the polypeptide has lysozyme activity and has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% but less than 100% sequence identity to the polypeptide of SEQ ID NO: 1 or amino acids 20-227 of SEQ ID NO:

2.

6. 6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide comprises one or more of the following substitutions at positions corresponding to positions R1Q, W10A, T13N, T14V, V166P, Y176W in SEQ ID NO: 1:

7. 7. A polynucleotide encoding the polypeptide of any one of claims 1 to 6, wherein the polynucleotide has 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% but less than 100% sequence identity to SEQ ID NO:

3.

8. A nucleic acid construct comprising the polynucleotide of claim 7.

9. An expression vector comprising the polynucleotide of claim 7.

10. A host cell comprising the polynucleotide of claim 7.

11. A method for producing the polypeptide according to any one of claims 1 to 6, comprising: a. culturing the host cell of claim 10 under conditions conducive to production of said polypeptide; b. recovering the polypeptide; A method comprising:

12. A method for obtaining a polypeptide, comprising: (a) introducing substitutions at one or more positions corresponding to positions 1, 2, 6, 9, 10, 13, 14, 20, 36, 37, 40, 41, 63, 65, 101, 103, 118, 122, 125, 126, 129, 131, 143, 153, 155, 166, 174, 175, 176, 188, and 202 of SEQ ID NO:1, wherein the polypeptide has lysozyme activity and has at least 60%, e.g., at least 65%, 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 to amino acids 20-227 of SEQ ID NO:1 or SEQ ID NO:2; (b) optionally recovering said polypeptide; A method comprising:

13. A composition comprising the polypeptide of any one of claims 1 to 6.

14. A polypeptide according to claims 1 to 6, and / or at least one fat-soluble vitamin, and / or at least one water-soluble vitamin, and / or At least one trace mineral Animal feed additives including:

15. Use of a polypeptide according to any one of claims 1 to 6 in animal feed.

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