Polypeptides having lysozyme activity and polynucleotides encoding the same

Abstract

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

Description

[0001] This application is a divisional application of the invention patent application filed on May 26, 2014, with the State Intellectual Property Office of the People's Republic of China, entitled "Polypeptide with lysozyme activity and polynucleotide encoding said polypeptide", application number "201280058088.1".

[0002] Reference sequence list

[0003] This application contains a sequence list in a computer-readable form, which is incorporated herein by reference.

[0004] References to atomic coordinates

[0005] This application lists the atomic coordinates of the three-dimensional structure of Acremonium alkalophilum CBS114.92 lysozyme. Figure 6 middle. Background of the Invention

[0006] Invention Field

[0007] This invention relates to polypeptides with lysozyme activity, catalytic domains, and polynucleotides encoding these polypeptides and catalytic domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising these polynucleotides, along with methods for producing and using these polypeptides and catalytic domains.

[0008] Related technical specifications

[0009] Lysozyme is an O-glycosyl hydrolase produced by many organisms as a defense mechanism against bacteria. This enzyme hydrolyzes the bacterial cell wall by breaking the glycosidic bonds of peptidoglycan, an important structural molecule in bacteria. After the bacterial cell wall is weakened by the action of lysozyme, osmotic pressure leads to bacterial cell lysis.

[0010] Lysozyme is found in many organisms, such as 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 the carbon 1 of N-acetylmuramic acid and the carbon 4 of N-acetyl-D-glucosamine. In vivo, these two carbohydrates polymerize to form cell wall polysaccharides.

[0011] There is growing interest in the potential of lysozyme as an antimicrobial agent. For example, lysozyme activity has been shown against pathogens such as Streptococcus pneumoniae, Bacillus anthracis, Enterococcus faecalis, Bacillus stearothermophilus, Clostridium botulinum, Clostridium butyricum, Clostridium perfringens, Clostridium sporogenes, Clostridium butyricum, and Listeria monocytogenes.

[0012] Lysozymes have been classified into five distinct glycoside hydrolases (GH) families (CAZy, www.cazy.org): hen egg white lysozyme (GH22), goose egg white lysozyme (GH23), bacteriophage T4 lysozyme (GH24), sphingomonas flagellin (GH73), and Chalaropsis lysozyme (GH25). Lysozymes from families GH23 and GH24 are primarily known from bacteriophages and have not yet been identified in fungi. Lysozyme family GH25 has been found to be structurally unrelated to other lysozyme families.

[0013] Uses of lysozyme have been proposed in animal feed (see, for example, WO 00 / 21381 and WO 04 / 026334), in cheese production (see, for example, WO 05 / 080559), food preservation (Hughey and Johnson (1987) Appl Environ Microbiol 53:2165), detergents (see, for example, US 5,041,236 and EP 0425016), in oral care (see, for example, US 4,355,022, WO 04 / 017988 and WO 08 / 124764), cosmetic and dermatological, contraceptive, urological, and gynecological (see, for example, WO 08 / 124764).

[0014] A GH25 lysozyme has been reported from the genus Chalaropsis (Felsch JW, Ingagami T, and Hash JH. (1975), “The N,O-Diacetylmuramidase of Chalaropsis species; V The complete amino acid sequence”, J. Biol. Chem. 250(10):3713-3720).

[0015] As the main product available in the commercial market, egg white lysozyme does not cleave N,6-O-diacetylmuraminase in the cell wall of Staphylococcus aureus, and therefore cannot dissolve this important human pathogen in particular (Masschalck B, Deckers D, Michiels CW (2002), “Lytic and nonlytic mechanism of inactivation of gram-positive bacteria by lysozyme under atmospheric and high hydrostatic pressure”, J Food Prot. 65(12):1916-23).

[0016] Different lysozymes have been observed to exhibit varying specificities against different microorganisms. Therefore, it is desirable to make several lysozymes available so that the appropriate enzyme can be selected for each specific application. Thus, novel peptides with lysozyme activity are promising. Invention Overview

[0017] This invention relates to isolated fungal polypeptides belonging to the GH25 family and possessing lysozyme activity.

[0018] The present invention further relates to isolated polypeptides having lysozyme activity, these polypeptides being selected from the group consisting of the following items:

[0019] (a) A polypeptide having at least 80% sequence identity with the mature polypeptide of SEQ ID NO:4 or the mature polypeptide of SEQ ID NO:8;

[0020] (b) A polypeptide encoded by a polynucleotide having at least 80% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:3 or the mature polypeptide coding sequence of SEQ ID NO:7;

[0021] (c) A polypeptide encoded by a polynucleotide hybridized with the coding sequence of a mature polypeptide of SEQ ID NO:3 or SEQ ID NO:7, or its full-length complement, under medium- to high-rigor conditions;

[0022] (d) A variant of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:8, comprising substitution, deletion, and / or insertion at one or more (e.g., several) positions; and

[0023] (e) A fragment of a polypeptide of (a), (b), (c), or (d) that has lysozyme activity.

[0024] The present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs; recombinant expression vectors; recombinant host cells comprising these polynucleotides; and methods for producing these polypeptides.

[0025] Furthermore, the present invention relates to compositions including the polypeptides of the present invention, such as detergent compositions, animal feed compositions, and bacterial genomic DNA extraction compositions.

[0026] The present invention also relates to polypeptides of the present invention having antimicrobial activity and to methods of using these polypeptides of the present invention as inhibitors of biofilm formation in detergents, dental care products, animal feed, and for breaking down bacterial cell walls.

[0027] The present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO:4 or amino acids -23 to -1 of SEQ ID NO:8, wherein the polynucleotide is operatively linked to a gene encoding a protein; to nucleic acid constructs, expression vectors, and recombinant host cells comprising these polynucleotides; and to a method for producing a protein.

[0028] Sequence List Overview

[0029] SEQ ID NO:1 is the DNA sequence of the P244A7GH24 gene isolated from Acremonium alkalophilum CBS114.92.

[0030] SEQ ID NO:2 is the amino acid sequence as deduced from SEQ ID NO:1.

[0031] SEQ ID NO:3 is the DNA sequence of the P242M9 GH25 gene isolated from Acremonium alkalophilum CBS114.92.

[0032] SEQ ID NO:4 is the amino acid sequence as deduced from SEQ ID NO:3.

[0033] SEQ ID NO:5 is the forward primer F-P242M9.

[0034] SEQ ID NO:6 is the reverse primer R-P242M9.

[0035] SEQ ID NO:7 is the synthetically optimized DNA sequence of the GH25 gene.

[0036] SEQ ID NO:8 is the amino acid sequence as deduced from SEQ ID NO:7.

[0037] SEQ ID NO:9 is the forward primer BamHI.

[0038] SEQ ID NO:10 is the reverse primer EcoRI. Brief description of the attached figures

[0039] Figure 1 Radial diffusion assays of Acremonium alcalophilum GH24 lysozyme (EXP03890, SEQ ID NO:2), Acremonium alcalophilum GH25 lysozyme (EXP03864, SEQ ID NO:4), and a reference lysozyme from Aspergillus fumigatus GH25 in Staphylococcus aureus and Escherichia coli are shown.

[0040] Figure 2 The temperature stability of Acremonium alcalophilum GH25 lysozyme P242M9 (SEQ ID NO:4) at 60°C, 76°C, 70°C, 75°C, 80°C, and 85°C after 30 or 60 seconds is shown, as is the decrease in optical density of a solution of resuspended Micrococcus tarda ATTC No. 4698 as measured in a spectrophotometer at 540 nm.

[0041] Figure 3 The thermal stability of GH25 lysozyme P242M9 (SEQ ID NO:4) as determined by differential scanning calorimetry (DSC) is shown in 50 mM sodium acetate (pH 4.5), 50 mM sodium acetate (pH 5.5), and 50 mM MES (2-(N-morpholino)ethanesulfonic acid) (pH 6.5).

[0042] Figure 4 Lysozyme activities of four concentrations of GH25 lysozyme P242M9 (SEQ ID NO:4), synthetic GH25 lysozyme (SEQ ID NO:8), and eleven variants of SEQ ID NO:8 are shown, as determined by the decrease in optical density of a solution of Clostridium perfringens NN01260 resuspended in.

[0043] Figure 5Lysozyme activities of four concentrations of GH25 lysozyme P242M9 (SEQ ID NO:4), synthetic GH25 lysozyme (SEQ ID NO:8), and eleven variants of SEQ ID NO:8 are shown, as determined by the decrease in optical density of a solution of Clostridium perfringens clinical isolates resuspended.

[0044] Figure 6 The atomic coordinates of the three-dimensional structure of Acremonium alkalophilum CBS114.92GH25 lysozyme are listed. These atomic coordinates can help generate a three-dimensional model depicting the structure of Acremonium alkalophilum CBS114.92GH25 lysozyme, as well as three-dimensional models of homologous structures (such as variants of the aforementioned lysozyme).

[0045] definition

[0046] Lysozyme: The term "lysozyme" activity is defined herein as an O-glycosyl hydrolase that catalyzes the hydrolysis of glycosidic bonds between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. Lysozyme cleaves glycosidic bonds between certain residues in the mucopolysaccharides and peptides of the bacterial cell wall, such as 1,4-β bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin, resulting in lysis. Lysozyme belongs to EC 3.2.1.17 enzyme class. For the purposes of this invention, lysozyme activity is determined according to the turbidity determination described in Example 4. In one aspect, the polypeptide of the invention has at least 20%, for example at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the lysozyme activity of the mature polypeptide of SEQ ID NO:4 or SEQ ID NO:8.

[0047] Alleles: The term "allele" refers to any of two or more variant forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally from mutations and can lead to polymorphism within a population. Gene mutations can be silent (encoding a polypeptide with no change) or can encode a polypeptide with a modified amino acid sequence. Alleles of a polypeptide are polypeptides encoded by alleles of a gene.

[0048] Antimicrobial activity: The term "antimicrobial activity" is defined herein as an activity that kills or inhibits the growth of microorganisms, such as algae, archaea, bacteria, fungi, and / or protozoa. Antimicrobial activity can be bactericidal, for example, intended to kill bacteria, or bacteriostatic, intended to prevent bacterial growth. Antimicrobial activity can include catalyzing the hydrolysis of the 1,4-β-bond between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrin. Antimicrobial activity can also include lysozyme binding to the surface of microorganisms and inhibiting their growth. Antimicrobial action can also include activating bacterial autolysins by inhibiting or reducing bacterial toxins and by using the lysozyme of the present invention as an immunostimulant through phage action. For the purposes of this invention, antimicrobial activity is determined according to the antimicrobial assay described in Example 10.

[0049] Altered / Modified Properties: "Altered / Modified properties" are defined herein as characteristics associated with a variant that has been altered or modified relative to the parental lysozyme or the identified reference sequence. Unless otherwise stated, an altered or modified property may be a characteristic associated with a variant that has been improved relative to another reference lysozyme or the parental lysozyme. Examples of properties that can be altered / modified or improved are given below.

[0050] Thermal stability: The term "thermal stability" refers to the lysozyme activity relative to the parent or identified reference sequence after an incubation period at elevated temperatures, in buffer solutions, or under conditions present, such as during product storage / transportation, or similar to those present during industrial use of the variant. The variant may or may not exhibit a changing thermal activity profile relative to the parent. In one aspect, at selected temperatures, the thermal stability of the variant having lysozyme activity is at least 1.0 times that of the parent or reference sequence, for example, at least 1.1 times, at least 1.5 times, at least 1.8 times, at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, and at least 25 times. Preferably, this activity is tested using the lysozyme turbidity activity assay described in the "Materials and Methods" section.

[0051] Temperature profile / temperature stability: The term "temperature profile / temperature stability" refers to the altered temperature profile exhibited by the variant enzyme compared to the parent or identified reference sequence, wherein this temperature profile is defined as lysozyme activity as a function of temperature. Preferably, the activity at each temperature is expressed as a relative activity (in %) normalized to the value at the optimum temperature. The optimum temperature is the temperature at which the activity is highest within the tested temperatures (i.e., those with jumps of 5°C–10°C).

[0052] pH stability:The term "pH stability" refers to the structural stability of a variant enzyme relative to its parent or identified reference sequence after an incubation period at pH levels outside the range in which the enzyme is active (pH activity range). Such a variant may or may not exhibit a changing pH activity profile relative to its parent. For example, the variant may be inactive at increasing or decreasing pH levels but be able to maintain its three-dimensional structure and then regain activity once it is returned to its pH activity range. Alternatively, after incubation at increasing or decreasing pH levels, the variant may exhibit improved renaturation capacity relative to its parent.

[0053] In one aspect, the pH stability profile is altered to give the lysozyme variant improved stability at acidic pH. As used herein, acidic pH means from pH 2 to 5.5, preferably from 2.5 to 5.25, more preferably from 3 to 5, and even more preferably from 3.5 to 4. Preferably, after incubation at a given pH for 1 hour, the variant lysozyme retains 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 compared to a variant that has been maintained at pH 6.5 for the same period. Preferably, the residual activity of the variant lysozyme is at least 1.1 times, at least 1.3 times, at least 1.5 times, preferably at least 2 times, more preferably at least 5 times, most preferably at least 7 times, and even most preferably at least 10 times higher than the residual activity of the parental lysozyme or the identified reference sequence that has been treated under the same conditions. Preferably, the activity is tested using the lysozyme turbidity activity assay described in the "Materials and Methods" section.

[0054] pH activity: Here, the term "pH activity" is defined as the change in the pH-dependent activity profile exhibited by a variant lysozyme when compared to the pH activity profile of the parent lysozyme or an identified reference sequence. The pH activity profile provides a measure of the enzyme's efficiency in inhibiting microbial growth, eliminating microbial cells, and / or catalyzing hydrolysis reactions within a given pH range, under given conditions (e.g., temperature and solvent composition). Lysozymes have specific pH ranges within which the polypeptide is stable and retains its enzymatic activity; outside these ranges, the lysozyme becomes inactive and may also be unstable. An optimal pH value typically exists within this range where the lysozyme exhibits the highest activity.

[0055] Lysozyme variants that have improved activity at alkaline pH (e.g., from pH 7.5 to 12, preferably from 8 to 11, more preferably from 8.5 to 10, and even more preferably from 9 to 9.5) will be able to function in more alkaline environments (e.g., detergents).

[0056] Variants that exhibit improved activity at acidic pH levels (e.g., from pH 2 to 6.5, preferably from 2.5 to 6, more preferably from 3 to 5.5, and even more preferably from 3.5 to 5) will be able to function under more acidic conditions (e.g., preservatives in certain foods).

[0057] Variants exhibiting improved activity at neutral or weakly acidic pH (e.g., from pH 4 to 7.0, preferably from 4.5 to 6.5, more preferably from 5 to 6.5) will be able to function under weakly acidic or neutral conditions, for example, as probiotic molecules in feed, to stabilize the healthy microbial colony of animals or by inhibiting the growth / intestinal colonization of viral, parasitic, or bacterial pathogens in the animal's GI tract.

[0058] In one aspect, the pH activity profile is altered so that the lysozyme variant exhibits improved activity at a more alkaline pH. Preferably, at a pH at least 0.5 units higher, more preferably at least 1.0 pH unit higher, more preferably at least 1.5 pH unit higher, and even more preferably at least 2.0 pH unit higher, the activity of the lysozyme variant is at least 1.1 times, preferably at least 1.5 times, more preferably at least 2 times, even more preferably at least 5 times, and most preferably at least 10 times higher than that of the parent enzyme or the identified reference sequence. Preferably, the lysozyme variant maintains at least 40%, preferably at least 50%, 60%, 70%, or 80%, or 90%, more preferably at least 95%, and even more preferably at least 100% of the activity exhibited by the parent lysozyme or the identified reference sequence at its pH optimum over the same time period. Preferably, this activity is tested using the lysozyme turbidity activity assay described in the "Materials and Methods" section.

[0059] In another aspect, the pH activity profile is altered so that the lysozyme variant exhibits improved activity at more acidic pH levels. Preferably, at a pH at least 0.5 units lower, more preferably at least 1.0 pH units lower, more preferably at least 1.5 pH units lower, and even more preferably at least 2.0 pH units lower, the activity of this lysozyme variant is at least 1.1 times, preferably at least 1.5 times, more preferably at least 2 times, even more preferably at least 5 times, and most preferably at least 10 times higher than that of the parent enzyme or the identified reference sequence. Preferably, the lysozyme variant maintains at least 40%, preferably at least 50%, 60%, 70%, or 80%, or 90%, more preferably at least 95%, and even more preferably at least 100% of the activity exhibited by the parent lysozyme or the identified reference sequence at its pH optimum over the same period of time. Preferably, this activity is tested using the lysozyme turbidity activity assay described in the "Materials and Methods" section.

[0060] Substrate specificity: The term "substrate specificity" refers to the specificity of the lysozyme with respect to the types of bacteria it can kill / inhibit and / or with respect to a model lysozyme substrate (e.g., p-NP-(NAG-NAM)n or p-NP-(NAG)m oligomers). By modifying the substrate specificity of the lysozyme, the types of bacteria that the lysozyme can kill and / or inhibit can be altered. In one aspect, the substrate specificity of the lysozyme is broadened, thereby allowing the killing and / or inhibition of bacterial types other than those that can be killed and / or inhibited by the wild-type lysozyme.

[0061] Glycation sensitivity: Non-enzymatic saccharification is a spontaneous post-translational process in which reducing sugars covalently bind to free amino groups in proteins, primarily on lysine (K) residues. Saccharification can affect lysozyme activity. According to the present invention, the sensitivity of lysozyme to non-enzymatic saccharification can be reduced by specifying amino acid alterations.

[0062] Improved properties may also include thermal properties, such as granulation stability, vapor stability, and a wider temperature activity profile. Further improvements may include protease sensitivity and / or glycosylation mode. Preferably, improvements are evaluated with respect to desired application conditions.

[0063] Catalytic domain: The term "catalytic domain" refers to the region of an enzyme that contains the catalytic mechanism of the enzyme.

[0064] cDNA: The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription of mature, spliced ​​mRNA molecules obtained from eukaryotic or prokaryotic cells. cDNA lacks the intron sequences that can be present in the corresponding genomic DNA. Early initial RNA transcripts are precursors to mRNA, undergoing a series of processing steps, including splicing, before becoming mature, spliced ​​mRNA.

[0065] Coding sequence: The term "coding sequence" refers to a polynucleotide, which directly defines the amino acid sequence of a polypeptide. The boundaries of a coding sequence are typically defined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. Coding sequences can be genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0066] Control Sequence: The term "control sequence" refers to the nucleic acid sequence necessary for the expression of the polynucleotide encoding the mature polypeptide of the present invention. Individual control sequences may be native (i.e., from the same gene) or exogenous (i.e., from different genes) relative to the polynucleotide encoding the polypeptide, or native or exogenous relative to each other. Such control sequences include, but are not limited to, pre-leaders, polyadenylated sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, as well as transcription and translation termination signals. Connectors may be provided for these control sequences to be introduced at specific restriction sites, thereby facilitating the linkage of these control sequences to the polynucleotide coding region encoding the polypeptide.

[0067] Expression: The term “expression” includes any steps involved in the production of a polypeptide, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0068] Expression vector: The term "expression vector" refers to a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operatively linked to a control sequence that provides for its expression.

[0069] Fragment: The term "fragment" means a polypeptide or catalytic domain having one or more (e.g., several) amino acids deleted from the amino and / or carboxyl termini of a mature polypeptide or domain; wherein the fragment has lysozyme activity. In one aspect, the fragment comprises at least 184 amino acid residues (e.g., amino acids 25 to 208 of SEQ ID NO:4) or at least 195 amino acid residues (e.g., amino acids 22 to 216 of SEQ ID NO:4). In another aspect, the fragment comprises at least 184 amino acid residues (e.g., amino acids 10 to 193 of SEQ ID NO:8) or at least 195 amino acid residues (e.g., amino acids 5 to 199 of SEQ ID NO:8).

[0070] Host cell: The term "host cell" refers to any cell type that is sensitive to transformation, transfection, transduction, etc., and has a nucleic acid construct or expression vector containing the polynucleotides of the present invention. The term "host cell" also encompasses any offspring of a parent cell that differs from the parent cell due to mutations occurring during replication.

[0071] Isolated: The term “isolated” refers to a substance in a form or environment that is not naturally occurring. Non-limiting examples of isolated substances include (1) any substance that is not naturally occurring, (2) any substance, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor that has been at least partially removed from one or more of the naturally occurring components associated with it; (3) a substance that has been artificially modified by humans relative to a substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components naturally associated with it (e.g., multiple copies of the gene encoding the substance; using a promoter stronger than the promoter naturally associated with the gene encoding the substance). Isolated substances may be present in fermentation broth samples.

[0072] Mature polypeptide: The term "mature polypeptide" refers to a polypeptide in its final form following translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). In one aspect, based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6) predicting that amino acids 1 to 19 of SEQ ID NO:4 and amino acids -40 to -18 of SEQ ID NO:8 are signal peptides, the mature polypeptide is amino acids 20 to 227 of SEQ ID NO:4 or amino acids 1 to 208 of SEQ ID NO:8. It is known in the art that host cells can produce mixtures of two or more different mature polypeptides expressed by the same polynucleotide (i.e., with different C-terminal and / or N-terminal amino acids).

[0073] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" refers to a polynucleotide encoding a mature polypeptide having lysozyme activity. In one aspect, based on the SignalP program (Nielsen et al., 1997, ibid.) predicting that nucleotides 1 to 57 of SEQ ID NO:3 and nucleotides 1 to 69 of SEQ ID NO:7 encode a signal peptide, the mature polypeptide coding sequence is the binding sequence of nucleotides 58 to 147 and nucleotides 302 to 835 of SEQ ID NO:3 with nucleotides 121 to 744 of SEQ ID NO:7.

[0074] Nucleic acid constructs: The term “nucleic acid construct” refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from naturally occurring genes or modified in a way that does not otherwise occur in nature to contain nucleic acid segments, or that is synthesized and contains one or more control sequences.

[0075] Operable ligation: The term “operable ligation” refers to a configuration in which a control sequence is located at the appropriate position relative to the coding sequence of a polynucleotide, such that the control sequence guides the expression of the coding sequence.

[0076] Sequence consistency: The parameter “sequence consistency” is used to describe the correlation between two amino acid sequences or two nucleotide sequences.

[0077] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) was used to determine sequence consistency between two amino acid sequences. This algorithm was implemented using the Needle program in the EMBOSS software 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 vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 substitution matrix (the EMBOSS version of BLOSUM62). The Needle output (obtained using the -nobrief option) marked as "Longest Consistency" was used as the percentage consistency and calculated as follows:

[0078] (Consistent residues × 100) / (Alignment length - Total number of vacancies in the alignment)

[0079] For the purposes of this invention, the Niedermann-Onsch algorithm (Niedermann and Onsch, 1970, ibid.) was used to determine sequence consistency between two deoxyribonucleotide sequences. This algorithm is implemented by the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output (obtained using the –nobrief option) marked as “Longest Consistency” was used as the percentage consistency and calculated as follows:

[0080] (Consistent DNA × 100) / (Alignment length - Total number of gaps in alignment).

[0081] Rigor conditions: Different rigor conditions are defined as follows.

[0082] The term "very low stringency conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 25% formamide, following standard DNA blotting procedures. Vector material is finally washed three times at 45°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0083] The term "low-rigor conditions" refers to pre-hybridization and hybridization for probes at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 50°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0084] The term "medium-rigor conditions" refers to pre-hybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 35% formamide, for probes at least 100 nucleotides in length, following standard DNA blotting procedures. Vector material is finally washed three times at 55°C for 15 minutes each time with 2X SSPE and 0.2% SDS.

[0085] The term "medium-high stringency conditions" refers to pre-hybridization and hybridization at 42°C for probes at least 100 nucleotides in length, following a standard DNA blotting procedure in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector material is finally washed three times at 60°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0086] The term "high-rigor conditions" refers to the standard Southern blotting procedure followed for probes at least 100 nucleotides in length, involving pre-hybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 65°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0087] The term "very high stringency conditions" refers to pre-hybridization and hybridization at 42°C for probes at least 100 nucleotides in length, following standard DNA blotting procedures, in 5X SSPE, 0.3% SDS, 200 mg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 70°C for 15 minutes each time with 2X SSC and 0.2% SDS.

[0088] Subsequence: The term "subsequence" refers to a polynucleotide having one or more (e.g., several) nucleotides deleted from the 5' and / or 3' end of the coding sequence of a mature polypeptide; wherein the subsequence encodes a fragment having lysozyme activity. In one aspect, a subsequence comprises at least 552 nucleotides (e.g., the linker sequence of nucleotides 73 to 147 and nucleotides 302 to 778 of SEQ ID NO:3), or at least 585 nucleotides (e.g., the linker sequence of nucleotides 64 to 147 and nucleotides 302 to 802 of SEQ ID NO:3). In another aspect, a subsequence comprises at least 552 nucleotides (e.g., the sequence of nucleotides 148 to 699 of SEQ ID NO:7), or at least 585 nucleotides (e.g., the sequence of nucleotides 133 to 717 of SEQ ID NO:7).

[0089] Substantially Pure Polynucleotide: The term "substantially pure polynucleotide" means a polynucleotide formulation free of other foreign or unwanted nucleotides and in a form suitable for use within a genetically engineered peptide production system. Thus, a substantially pure polynucleotide comprises, by weight, up to 10%, up to 8%, up to 6%, up to 5%, up to 4%, up to 3%, up to 2%, up to 1%, and up to 0.5% of other polynucleotide material naturally or recombinantly associated with that polynucleotide. However, a substantially pure polynucleotide may include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators. Preferably, the polynucleotide is at least 90% pure by weight, for example, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%. Preferably, the polynucleotide of the present invention is in a substantially pure form.

[0090] Substantially pure polypeptide: The term "substantially pure polypeptide" means a polypeptide comprising, by weight, at most 10%, at most 8%, at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, and at most 0.5% of other polypeptide material naturally or recombinantly associated with the polypeptide. Preferably, the polypeptide is at least 92% pure by weight of the total polypeptide material present in the formulation, for example, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, and 100% pure. Preferably, the polypeptide of the present invention is present in a substantially pure form. This can be accomplished, for example, by preparing the polypeptide using well-known recombinant methods or classical purification methods.

[0091] Variants: The term "variant" refers to a polypeptide having lysozyme activity by altering (i.e., substituting, inserting, and / or deleting) one or more (e.g., several) amino acid residues at one or more (e.g., several) positions. Substitution means that an amino acid occupying a position is replaced by a different amino acid; deletion means that an amino acid occupying a position is removed; and insertion means that one, two, or three amino acids are added adjacent to and immediately following the amino acid occupying the position. Variants according to the invention may include 1 to 5; 1 to 10; 1 to 15; 1 to 20; 1 to 25; 1 to 30; 1 to 35; 1 to 40; 1 to 45; i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 The numbers 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 can be changed.

[0092] These variants of the invention have at least one alteration / modification selected from the group consisting of the following position numbers: 6, 10, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190, wherein the position corresponds to the position in the mature sequence of SEQ ID NO:8. The variant polypeptide sequence is preferably a polypeptide not found in nature.

[0093] Wild-type lysozyme: The term "wild-type" lysozyme refers to a lysozyme expressed by naturally occurring microorganisms (such as bacteria, yeast, or filamentous fungi found in nature).

[0094] Rules for naming variants

[0095] For the purposes of this invention, the mature polypeptide disclosed in SEQ ID NO:8 is used to determine the corresponding amino acid residues in another lysozyme. The amino acid sequence of the other lysozyme is aligned with the mature polypeptide disclosed in SEQ ID NO:8, and based on this alignment, the Niedermann-Onsch algorithm (Niedermann and Onsch, 1970, *Journal of Molecular Biology* 48:443-453) is used to determine the amino acid position number corresponding to any amino acid residue of the mature polypeptide disclosed in SEQ ID NO:8. This algorithm is implemented using the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, *Trends in Genetics* 16:276-277) (preferably version 5.0.0 or later). The parameters used are a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle, marked "Longest Consistency" (obtained using the –nobrief option), is used as the percentage consistency and calculated as follows: (consistent residues × 100) / (alignment length - total number of gaps in the alignment). The Needle-Onsch algorithm is used for sequence comparison and for calculating sequence consistency.

[0096] Several computer programs can be used to determine the recognition of corresponding amino acid residues in another lysozyme by comparing multiple polypeptide sequences. These computer programs include, but are not limited to: MUSCLE (multiple sequence comparison by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research 32:1792-1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066; Katoh et al., 2005, Nucleic Acids Research 32:1792-1797), and MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids Research 30:3059-3066). Research (Nucleic Acids Research) 33:511-518; Katoh and Toh, 2007, Bioinformatics 23:372-374; Katoh et al., 2009, Methods in Molecular Biology 537:39-64; Katoh and Toh, 2010, Bioinformatics 26:1899-1900), and using ClustalW's EMBOSS EMMA (1.83 or later; Thompson et al., 1994, Nucleic Acids Research) 22:4673-4680), using their respective default parameters.

[0097] When other enzymes diverge from the mature polypeptide of SEQ ID NO:8, making conventional sequence-based comparisons ineffective in detecting their relationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295:613-615), alternative pairwise sequence comparison algorithms can be used. Greater sensitivity in sequence-based searches can be achieved using search procedures that utilize probabilistic representations (characteristic curves) of polypeptide families to search a database. For example, the PSI-BLAST procedure generates multiple profiles through an iterative database search process and is capable of detecting distant homologs (Atschul et al., 1997, Nucleic Acid Research 25:3389-3402). Even greater sensitivity can be achieved if the polypeptide family or superfamily has one or more representatives in a protein structure database. Programs (e.g., GenTHREADER) (Jones, 1999, *Journal of Molecular Biology* 287:797-815; McGuffi and Jones, 2003, *Bioinformatics* 19:874-881) utilize information from multiple sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation probabilities) as input to neural networks that predict the structural folding of query sequences. Similarly, the method of Gough et al., 2000, *Journal of Molecular Biology* 313:903-919 can be used to align sequences of unknown structures with superfamily models existing in the SCOP database. These alignments can then be used to generate homology models of peptides, and the accuracy of such models can be evaluated using various tools developed for this purpose.

[0098] For proteins with known structures, several tools and resources are available for retrieving and generating structural alignments. For example, the SCOP superfamily of proteins has already been structurally aligned, and those alignments are accessible and downloadable. Various algorithms (e.g., distance alignment matrix (Holm and Sander, 1998, Proteins 33:88-96) or combined extensions (Shindyalov and Bourne, 1998, Protein Engineering 11:739-747)) can be used to align two or more protein structures, and implementations of these algorithms can be further utilized to query structural databases along with the structure of interest to discover possible structural homologs (e.g., Helm and Park, 2000, Bioinformatics 16:566-567).

[0099] In the description of variations of the invention, the following nomenclature is used for ease of reference. The accepted IUPAC single-letter and three-letter amino acid abbreviations are adopted.

[0100] replace: For amino acid substitutions, the following nomenclature is used: original amino acid, position, substituted amino acid. Therefore, the substitution of threonine at position 226 with alanine is represented as "Thr226Ala" or "T226A". Multiple mutations are separated by plus signs ("+"), for example, "Gly205Arg+Ser411Phe" or "G205R+S411F" represent the substitution of glycine (G) for arginine (R) and serine (S) for phenylalanine (F) at positions 205 and 411, respectively.

[0101] Missing: For amino acid deletions, the following nomenclature is used: original amino acid, position, *. Therefore, a glycine deletion at position 195 is represented as "Gly195*" or "G195*". Multiple deletions are separated by a plus sign ("+"), for example, "Gly195*+Ser411*" or "G195*+S411*".

[0102] insert: For amino acid insertions, the following nomenclature is used: original amino acid, position, original amino acid, inserted amino acid. Therefore, the insertion of lysine after glycine at position 195 is represented as "Gly195GlyLys" or "G195GK". Insertions of multiple amino acids are represented as [original amino acid, position, original amino acid, inserted amino acid #1, inserted amino acid #2; etc.]. For example, the insertion of lysine and alanine after glycine at position 195 is represented as "Gly195GlyLysAla" or "G195GKA".

[0103] In such cases, the inserted amino acid residues are numbered by adding lowercase letters to the position numbers of the amino acid residues preceding them. In the example above, the sequence would therefore be:

[0104] Parent variants 195 195 195a 195b G GKA

[0105] Multiple variations: Variants containing multiple variations are separated by a plus sign ("+"), such as "Arg170Tyr+Gly195Glu" or "R170Y+G195E", which represent that arginine and glycine at positions 170 and 195 are replaced by tyrosine and glutamic acid, respectively.

[0106] Different changes:When different variations can be introduced at a single position, these variations are separated by a comma. For example, "Arg170Tyr,Glu" means that arginine at position 170 is replaced by either tyrosine or glutamic acid. Therefore, "Tyr167Gly,Ala+Arg170Gly,Ala" represents the following variants: "Tyr167Gly+Arg170Gly", "Tyr167Gly+Arg170Ala", "Tyr167Ala+Arg170Gly", and "Tyr167Ala+Arg170Ala".

[0107] Detailed Description of the Invention

[0108] Polypeptides with lysozyme activity

[0109] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 80% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0110] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 85% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0111] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 90% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0112] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 91% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0113] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 92% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0114] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 93% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0115] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 94% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0116] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 95% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0117] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 96% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0118] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 97% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0119] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 98% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0120] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 99% sequence identity with the mature polypeptide of SEQ ID NO:4.

[0121] In one respect, these peptides differ from the mature peptide of SEQ ID NO:4 by no more than 45 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

[0122] The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:4 or its alleles, or is a fragment thereof having lysozyme activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO:4. In yet another aspect, the polypeptide comprises or consists of amino acids 20 to 227 of SEQ ID NO:4.

[0123] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 80% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0124] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 85% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0125] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 90% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0126] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 91% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0127] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 92% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0128] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 93% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0129] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 94% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0130] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 95% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0131] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 96% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0132] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 97% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0133] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 98% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0134] In one embodiment, the present invention relates to an isolated polypeptide having lysozyme activity having at least 99% sequence identity with the mature polypeptide of SEQ ID NO:8.

[0135] In one respect, these peptides differ from the mature peptide of SEQ ID NO:8 by no more than 45 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44.

[0136] The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO:8 or its alleles, or is a fragment thereof having lysozyme activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO:8. In yet another aspect, the polypeptide comprises or consists of amino acids 20 to 227 of SEQ ID NO:8.

[0137] In another embodiment, the present invention relates to an isolated polypeptide having lysozyme activity, the isolated polypeptide being encoded by a polynucleotide hybridized to (i) SEQ ID NO:3 or SEQ ID NO:7, (ii) its cDNA sequence, or (iii) the full-length complement of (i) or (ii) under medium-strict, medium-high-strict, high-strict, or very high-strict conditions (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York).

[0138] The polynucleotide or subsequence of SEQ ID NO:3 or SEQ ID NO:7, together with the polypeptide or fragment thereof of SEQ ID NO:4 or SEQ ID NO:8, can be used to design nucleic acid probes for identifying and cloning DNA encoding polypeptides with lysozyme activity from strains of different genera or species according to methods well known in the art. Specifically, such probes can be used to hybridize with genomic DNA or cDNA of cells of interest, following standard DNA blotting procedures, to identify and isolate the corresponding gene therein. Such probes can be much shorter than the entire sequence, but should be at least 15 nucleotides long, for example, at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe is at least 100 nucleotides long, for example, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, or at least 900 nucleotides long. Both DNA and RNA probes can be used. Typically, the probes are labeled for detecting the corresponding gene (e.g., using...). 32 P, 3 H, 35 S, biotin, or avidin. This invention covers such probes.

[0139] Genomic DNA or cDNA libraries prepared from other strains of this type can be screened against DNA that hybridizes to the probes described above and encodes polypeptides with lysozyme activity. Genomic DNA or other DNA from these other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the library or separated DNA can be transferred and immobilized on nitrocellulose or other suitable vector materials. To identify clones or DNA that hybridize to SEQ ID NO:3 or SEQ ID NO:7 or their subsequences, the vector material is used for DNA blotting.

[0140] For the purposes of this invention, the hybridization instruction indicates that the polynucleotide hybridizes with a labeled nucleic acid probe corresponding to the following under moderate to very high stringency conditions: (i) SEQ ID NO:3 or SEQ ID NO:7; (ii) the mature polypeptide coding sequence of SEQ ID NO:3 or SEQ ID NO:7; (iii) its cDNA sequence; (iv) its full-length complement; or (v) its daughter sequence. Molecules hybridizing with the nucleic acid probe under these conditions can be detected using, for example, X-ray membranes or any other detection method known in the art.

[0141] In one aspect, the nucleic acid probe is nucleotides 58 to 147 or nucleotides 302 to 835 of SEQ ID NO:3, or nucleotides 121 to 744 of SEQ ID NO:7. In another aspect, the nucleic acid probe is a polypeptide encoding SEQ ID NO:4 or SEQ ID NO:8; its mature polypeptide; or a fragment thereof. In yet another aspect, the nucleic acid probe is SEQ ID NO:3 or SEQ ID NO:7, or its cDNA sequence.

[0142] In another embodiment, the present invention relates to an isolated polypeptide having lysozyme activity, the isolated polypeptide being encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:3 or its cDNA sequence.

[0143] In another embodiment, the present invention relates to an isolated polypeptide having lysozyme activity, the isolated polypeptide being encoded by a polynucleotide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the mature polypeptide coding sequence of SEQ ID NO:7 or its cDNA sequence.

[0144] In a preferred embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO:4 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) sites. In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO:4 does not exceed 45, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

[0145] In a preferred embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO:8 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) sites. In one embodiment, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO:8 does not exceed 45, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

[0146] These amino acid changes can be minor, i.e., conserved amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; small deletions typically of 1–30 amino acids; small extensions to the amino or carboxyl terminus, such as methionine residues at the amino terminus; small linker peptides of up to 20–25 residues; or small extensions that facilitate purification by altering net charge or another function, such as polyhistidine sequences (tracts), antigenic epitopes, or binding domains.

[0147] Examples of conserved substitutions are found in the following group: 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 small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that do not alter specific activity in general are known in the art and, for example, described by H. Neurath and RL Hill in *The Proteins*, Academic Press, New York, 1979. Common substitutes are 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.

[0148] Alternatively, changes in amino acids essentially alter the physicochemical properties of the peptide. For example, changes in amino acids can improve the peptide's thermal stability, modify its substrate specificity, and change its optimal pH value.

[0149] Essential amino acids in peptides 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, a single alanine mutation is introduced at each residue in the molecule, and the lysozyme activity of the resulting mutant molecule is tested to identify amino acid residues critical to the molecule's activity. See also Hilton et al., 1996, J. Biol. Chem., 271:4699-4708. The active site of an enzyme or other biological interactions can also be determined by physical analysis of the structure, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutations in amino acids at presumed contact sites. See, for example, de Vos et al., *Science* 255:306-312; Smith et al., 1992, *Journal of Molecular Biology* 224:899-904; Wlodaver et al., 1992, *FEBS Lett.* 309:59-64. Essential amino acids can also be identified by comparison with related peptides.

[0150] Using known mutagenesis, recombination, and / or tampering methods, followed by an associated screening procedure, one or more amino acid substitutions, deletions, and / or insertions can be made and tested. These associated screening procedures are, for example, those described by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences) 86:2152-2156; WO95 / 17413; or WO 95 / 22625. Other methods that can be used include: error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US Patent No. 5,223,409; WO92 / 06204), and targeted mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0151] The activity of cloned, mutagenic peptides expressed by host cells can be detected by combining mutagenesis / reorganization methods with high-throughput automated screening methods (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenic DNA molecules encoding active peptides can be recovered from 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 the peptide.

[0152] A polypeptide can be a hybrid polypeptide, in which a region of one polypeptide is fused to the N-terminus or C-terminus of a region of another polypeptide.

[0153] The polypeptide can also be a fusion polypeptide or a cleavable fusion polypeptide, wherein another polypeptide is fused to the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are generated by fusing a polynucleotide encoding another polypeptide to the polynucleotide of the present invention. Techniques for generating fusion polypeptides are known in the art and include linking the coding sequences encoding the polypeptides such that they are within a frame and the expression of the fusion polypeptide is under the control of the same one or more promoters and terminators. Fusion polypeptides can also be constructed using integrin technology, wherein the fusion polypeptide is generated post-translational (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0154] Fusion peptides may further include a cleavage site between the two peptides. This site is cleaved upon secretion of the fusion protein, thereby releasing both peptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following: 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., 199... 5. Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9:378-381; Eaton et al., 1986, Biochemistry 25:505-512; Collins-Racie et al., 1995, Biotechnology 13:982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248; and Stevens, 2003, Drug Discovery World 4:35-48.

[0155] exist The crystal structure of Acremonium alkalophilum CBS114.92 lysozyme was determined at a resolution of [resolution value missing]. The atomic coordinates of this structure are shown in [symbol missing]. Figure 6 These atomic coordinates can be used to generate three-dimensional models depicting the structure or homologous structures (e.g., the variants of this invention) of Acremonium alkalophilum CBS114.92 lysozyme.

[0156] Acremonium alkalophilum CBS114.92 lysozyme belongs to the GH25 hydrolase family designated EC: 3.2.1.17. The catalytic mechanism is believed to be the classic Koshland retaining mechanism, where a net retention configuration is confirmed around the anomeric carbon. This is typically achieved via a two-step double displacement mechanism involving a covalent glycosyl-enzyme intermediate. The reaction occurs with acid / base interaction and nucleophilic assistance provided by two amino acid side chains. In the first step (often referred to as the glycosylation step), one amino acid residue (D95) acts as a nucleophile attacking the anomeric center to displace the glycosidic ligand and form the glycosylase intermediate. Simultaneously, the second amino acid residue (E97) acts as an acid catalyst and protonates the glycosidic oxygen upon bond cleavage. In the second step (often referred to as the deglycosylation step), the glycosyl-enzyme intermediate is hydrolyzed by water, where the second amino acid residue (E97) acts as a base catalyst to deprotonate the incoming water molecules. It is believed that the pK ratio of the acid / base groups plays a role in the catalytic process. a The value cycles between high and low values ​​to optimize its effect at each step of catalysis.

[0157] Using X-ray diffraction, amino acid residues D95 and E97 (used for numbering with SEQ ID NO:8) have been identified as catalytic residues. In embodiments of the invention (other than one or more modifications listed herein), the amino acids E97 and D95 ​​(used for numbering with SEQ ID NO:8) corresponding to the lysozyme variants of the invention have not been altered. Mutations in the nucleophile variant are known from several lysozyme molecules to result in an enzyme that retains some catalytic activity because water (perhaps in OH-) - In the first step of the catalytic mechanism, the form of α can act as a nucleophile (Malcolm, BA et al., (1989), “Site-directed mutagenesis of the catalytic residues Asp-52 and Glu-35 of chicken egg white lysozyme”, Proceedings of the National Academy of Sciences 86(1), 133-137).

[0158] Variants with lysozyme activity

[0159] The lysozyme variant of the present invention comprises or consists of alterations at one or more (e.g., several) positions corresponding to the mature polypeptide of SEQ ID NO:8, including positions 6, 10, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190, wherein each alteration is independently a substitution, insertion or deletion and the variant has antimicrobial and / or lysozyme activity.

[0160] In one embodiment, the change is a replacement. In another embodiment, the change is an insertion. In yet another embodiment, the change is a deletion.

[0161] The lysozyme variants of the present invention include or consist of the following polypeptides, the polypeptides comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the mature polypeptide of SEQ ID NO:4.

[0162] The lysozyme variants of the present invention include or consist of the following polypeptides, the polypeptides comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the mature polypeptide of SEQ ID NO:8.

[0163] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 85% identity with the mature polypeptide of SEQ ID NO:8.

[0164] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 90% similarity to the mature polypeptide of SEQ ID NO:8.

[0165] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 93% similarity to the mature polypeptide of SEQ ID NO:8.

[0166] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 95% similarity to the mature polypeptide of SEQ ID NO:8.

[0167] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 96% similarity to the mature polypeptide of SEQ ID NO:8.

[0168] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 97% similarity to the mature polypeptide of SEQ ID NO:8.

[0169] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 98% similarity to the mature polypeptide of SEQ ID NO:8.

[0170] The lysozyme variants of the present invention comprise or consist of the following polypeptides, wherein the polypeptide comprises an amino acid sequence having at least 99% similarity to the mature polypeptide of SEQ ID NO:8.

[0171] In one aspect, the number of variations in the invention is 1-45, for example 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, and 1-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 variations.

[0172] In another aspect, the variant includes alterations, such as substitutions, insertions, or deletions, at one or more (e.g., several) positions of the mature polypeptide corresponding to SEQ ID NO:8, at positions 6, 10, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183, and 190. In another aspect, the variant includes alterations at any two of the following positions corresponding to the mature polypeptide of SEQ ID NO:8: 6, 10, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183, and 190. In another aspect, the variant includes alterations at any of the following three positions corresponding to the mature polypeptide of SEQ ID NO:8: 6, 10, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183, and 190. In another aspect, the variant includes alterations at each of the following positions corresponding to the mature polypeptide of SEQ ID NO:8: 6, 10, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183, and 190.

[0173] One embodiment of the invention involves altering the pH activity profile of the lysozyme while preserving lysozyme activity and / or antimicrobial activity. A preferred embodiment of the invention is that the lysozyme exhibits improved activity at a more alkaline pH; that is, the pH at which peak antimicrobial or lysozyme activity occurs increases / becomes more alkaline.

[0174] In one embodiment, the isolated variant of the invention includes a change or composition thereof at position 10 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or composition thereof for an amino acid at position 10 (used for numbering SEQ ID NO:8), which alters the pH activity profile of the lysozyme. In another embodiment, such a change includes a substitution of or composition thereof for W10H, which increases the pH at which peak antimicrobial activity is achieved.

[0175] In one embodiment, the isolated variant of the invention includes a change or composition thereof at position 39 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or composition thereof for an amino acid at position 39 (used for numbering SEQ ID NO:8), which alters the pH activity profile of the lysozyme. In another embodiment, such a change includes a substitution of or composition thereof for S39D, which increases the pH at which peak antimicrobial activity is achieved.

[0176] In another embodiment, the separate variant of the invention consists of a change at position 10 (numbered using SEQ ID NO:8) together with changes in one or more of the following positions: 6, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190. A preferred embodiment is to replace W10H with changes in one or more of the following positions: 6, 11, 28, 30, 33, 37, 39, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190.

[0177] In one embodiment, a separate variant of the invention consists of a change at position 39 (numbered using SEQ ID NO:8) together with changes at one or more of the following positions: 6, 10, 11, 28, 30, 33, 37, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190. A preferred embodiment is to replace S39D with changes in one or more of the following positions: 6, 10, 11, 28, 30, 33, 37, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190.

[0178] In another embodiment, the separate variant of the invention consists of a change at positions 10 and 39 (using SEQ ID NO:8 for numbering). A preferred embodiment consists of substitutions for W10H and S39D.

[0179] In another embodiment, the separate variants of the invention consist of changes at positions 10 and 39 (numbered using SEQ ID NO:8) together with changes at one or more of the following positions: 6, 11, 28, 30, 33, 37, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190. A preferred embodiment is to replace W10H and S39D with changes in one or more of the following positions: 6, 11, 28, 30, 33, 37, 59, 60, 61, 62, 63, 92, 93, 94, 96, 98, 99, 100, 101, 106, 133, 134, 135, 136, 137, 139, 140, 142, 143, 158, 161, 162, 178, 183 and / or 190.

[0180] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 6 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 6 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0181] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 11 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 11 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0182] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 30 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 30 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0183] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 33 (used for numbering SEQ ID NO:8). In one aspect, such a change includes including or is composed of an amino acid substitution at position 33 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0184] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 37 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 37 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0185] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 101 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 101 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0186] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 139 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 139 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0187] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 161 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 161 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0188] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 162 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 162 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0189] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 183 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 183 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0190] In one embodiment, the isolated variant of the invention includes or is composed of a change at position 190 (used for numbering SEQ ID NO:8). In one aspect, such a change includes a substitution of or is composed of an amino acid at position 190 (used for numbering SEQ ID NO:8), which alters the substrate specificity of the lysozyme.

[0191] Sources of peptides with lysozyme activity

[0192] The polypeptides of the present invention having lysozyme activity can be obtained from microorganisms of any genus. For the purposes of the invention, in conjunction with a given source, the term "obtained from" as used herein shall refer to a polypeptide encoded by a polynucleotide produced by that source or by a strain in which a polynucleotide from that source has been inserted. In one aspect, polypeptides obtained from a given source are secreted extracellularly.

[0193] The polypeptide can be a fungal polypeptide. For example, the polypeptide can be a polypeptide of a filamentous fungus, such as *Apocytogenes*, *Aspergillus*, *Aureospora*, *Fusarium*, *Pythium*, *Penicillium*, *Clostridium*, or *Trichoderma*.

[0194] In another aspect, the polypeptide is *Aquilaria sinensis*, *Aspergillus niger*, *Aspergillus avocado*, *Aspergillus sulphureus*, *Aspergillus fumigatus*, *Aspergillus niger*, *Aspergillus oryzae*, *Chrysosporium inops*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium merdarium*, and *Chrysosporium merdarium*. Queenslandicum, Tropical Golden Spores, Chrysosporium zonatum, Fusarium moniliforme, Fusarium graminearum, Fusarium kuwaiense, Fusarium graminearum, Fusarium graminearum, Fusarium graminearum, Fusarium heterosporum, Fusarium hyacinthum, Fusarium oxysporum, Fusarium oxysporum, Fusarium oxysporum var. rosace ... fimeti, microsporum spore shell, oosporum spore shell, Peruvian spore shell (Thielavia peruviana), hairy spore shell, tumor spore shell, heat-resistant spore shell, terrestrial spore shell, Trichoderma harzianum, Corning spore, long-branched spore, Trichoderma reesei, or green spore polypeptide.

[0195] In another aspect, the polypeptide is an Acremonium alcalophilum polypeptide, such as one obtained from Acremonium alcalophilum CBS 114.92.

[0196] It will be understood that, for the aforementioned categories, this invention covers both the complete and incomplete states, as well as other taxonomic equivalents, such as asexual forms, while ignoring their known species names. Those skilled in the art will readily identify appropriate equivalents.

[0197] These strains are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Microbial Culture Collection (DSMZ), the Centrale des Cultures (CBS), and the Southern Regional Research Center (NRRL) of the Agricultural Research Culture Collection (ARC).

[0198] The polypeptide 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 their natural habitats are well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening a library of genomic DNA or cDNA from another microorganism, or a mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with one or more probes, the polynucleotide can be isolated or cloned using techniques known to those skilled in the art (see, for example, Sarabrook et al., 1989, ibid.).

[0199] Polynucleotides

[0200] The present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention, as described herein.

[0201] Techniques for isolating or cloning polynucleotides are known in the art and include isolation from genomic DNA or cDNA, or combinations thereof. Cloning of polynucleotides from genomic DNA can be achieved, for example, by using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures can be used, such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and polynucleotide-based amplification (NASBA). These polynucleotides can be cloned from Aspergillus or Apocytogenes strains, or related organisms, and therefore, for example, can be allelic or species variants of the polynucleotide's polypeptide coding region.

[0202] Modification of the polynucleotide encoding the polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar to” the polypeptide refers to a non-naturally occurring form of the polypeptide. These polypeptides may differ from polypeptides isolated from their natural sources in some engineering manner, such as variations in specific activity, thermal stability, pH optimum, etc. These variations may be based on polynucleotides present as the coding sequences of mature polypeptides in SEQ ID NO:3, SEQ ID NO:7, or their cDNA sequences (e.g., their subsequences), and / or constructed by introducing nucleotide substitutions that do not alter the amino acid sequence of the polypeptide but correspond to the codon usage intended for the host organism to produce the enzyme, or by introducing nucleotide substitutions that produce different amino acid sequences. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2:95-107.

[0203] Nucleic acid constructs

[0204] The present invention also relates to nucleic acid constructs comprising polynucleotides of the present invention operably linked to one or more control sequences, which, under conditions compatible with these control sequences, guide the expression of coding sequences in suitable host cells.

[0205] Polynucleotides can be manipulated in a variety of ways to provide polypeptide expression. Depending on the expression vector, manipulation of the polynucleotide before its insertion into the vector may be desired or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0206] The control sequence can be a promoter, i.e., a polynucleotide recognized by the host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide exhibiting transcriptional activity in the host cell, including mutated, truncated, and heterozygous promoters, and can be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0207] In bacterial host cells, examples of promoters suitable for guiding the transcription of the nucleic acid constructs of this invention are promoters obtained from the following: Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis α-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus thermophilus malt amylase gene (amyM), Bacillus subtilis fructan sucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular). Microbiology (Molecular Microbiology) 13:97-107), Escherichia coli lac operon, Escherichia coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces agarase gene (dagA), and prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences 75:3727-3731), along with the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences 80:21-25). Other promoters are described in “Useful proteins from recombinant bacteria”, Gilbert et al., 1980, Scientific American, 242:74-94; and Sambrook et al., 1989, ibid. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0208] Examples of suitable promoters for guiding the transcription of the nucleic acid constructs of this invention in filamentous fungal host cells are promoters derived from the following genes: Aspergillus nidulans acetamase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Fusarium variegatum amyglucosidase (WO 00 / 56900), Fusarium variegatum Daria (WO 00 / 56900), Fusarium variegatum Quinn (WO 00 / 56900), etc. 00 / 56900), *Rhizopus oryzae* lipase, *Rhizopus oryzae* aspartic protease, *Trichoderma reesei* β-glucosidase, *Trichoderma reesei* cellobiose hydrolase I, *Trichoderma reesei* cellobiose hydrolase II, *Trichoderma reesei* glucan endonuclease I, *Trichoderma reesei* glucan endonuclease II, *Trichoderma reesei* glucan endonuclease III, *Trichoderma reesei* glucan endonuclease IV, *Trichoderma reesei* glucan endonuclease V, *Trichoderma reesei* xylanase I, *Trichoderma reesei* xylanase II, *Trichoderma reesei* β-xylosidase, along with NA 2tpi promoters (promotes derived from Aspergillus genes encoding neutral α-amylase, wherein an untranslated preamble has been replaced with an untranslated preamble from an Aspergillus gene encoding triose phosphate isomerase; non-restrictive examples include promoters derived from Aspergillus niger genes encoding neutral α-amylase, wherein an untranslated preamble has been replaced with an untranslated preamble from Aspergillus nidulans or Aspergillus oryzae genes encoding triose phosphate isomerase); and mutant, truncated, and heterozygous promoters thereof.

[0209] In yeast hosts, useful promoters are derived from the following genes: *Saccharomyces cerevisiae* enolase (ENO 1), *Saccharomyces cerevisiae* galactokinase (GAL1), *Saccharomyces cerevisiae* alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), *Saccharomyces cerevisiae* triose phosphate isomerase (TPI), *Saccharomyces cerevisiae* metallothionein (CUP1), and *Saccharomyces cerevisiae* 3-phosphate glycerate kinase. Romanos et al., 1992, *Yeast* 8:423-488, describe other useful promoters in yeast host cells.

[0210] The control sequence can also be a transcription terminator, which is recognized by the host cell to terminate transcription. This terminator is operatively linked to the 3' end of the polynucleotide encoding the polypeptide. Any terminator that functions within the host cell can be used in this invention.

[0211] Preferred terminators for bacterial host cells are derived from genes of Bacillus clausti alkaline protease (aprH), Bacillus licheniformis α-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0212] The preferred terminator for filamentous fungal host cells is derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0213] Preferred terminators for yeast host cells are derived from the genes of *Saccharomyces cerevisiae* enolase, *Saccharomyces cerevisiae* cytochrome C (CYC1), and *Saccharomyces cerevisiae* glyceraldehyde-3-phosphate dehydrogenase. Romanos et al., 1992, ibid., describe other useful terminators for yeast host cells.

[0214] The control sequence can also be a stable region of the mRNA downstream of the promoter and upstream of the gene coding sequence, which increases the expression of the gene.

[0215] Examples of suitable mRNA stable regions were obtained from the following: Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0216] The control sequence can also be a pre-translation, i.e., an untranslated region of mRNA important for translation in the host cell. This pre-translation is operatively linked to the 5' end of the polynucleotide encoding the polypeptide. Any pre-translation that functions in the host cell can be used.

[0217] The preferred precursors for filamentous fungal host cells are obtained from the genes of Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0218] Suitable precursors for yeast host cells are derived from the genes of the following: Saccharomyces cerevisiae enolase (ENO 1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphoglycerate dehydrogenase (ADH2 / GAP).

[0219] The control sequence can also be a polyadenylation sequence, operatively linked to the 3' end of the polynucleotide and recognized by the host cell during transcription as a signal to add polyadenylation residues to the transcribed mRNA. Any polyadenylation sequence that functions in the host cell can be used.

[0220] Preferred polyadenylated sequences for use in filamentous fungal host cells are derived from the genes of Aspergillus nidulans anthranilate synthase, Aspergillus niger glucosidase, Aspergillus niger α-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0221] Polyadenylation sequences that can be used in yeast host cells have been described by Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0222] The control sequence can also be a signal peptide coding region encoding a signal peptide linked to the N-terminus of a polypeptide and guiding the polypeptide into the cell's secretion pathway. The 5' end of the polynucleotide's coding sequence may inherently contain a signal peptide coding sequence naturally linked to the region encoding the polypeptide within the translation reading frame. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is exogenous to the coding sequence. An exogenous signal peptide coding sequence may be required when the coding sequence does not naturally contain such a sequence. Alternatively, an exogenous signal peptide coding sequence may simply replace the native signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence in the secretion pathway that guides the expressed polypeptide into the host cell can be used.

[0223] The effective signal peptide coding sequences for bacterial host cells are derived from the following genes: Bacillus NCIB 11837 malt amylase, Bacillus subtilis protease, Bacillus subtilis β-lactamase, Bacillus thermophilus α-amylase, Bacillus thermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are described in Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

[0224] The effective signal peptide coding sequences for filamentous fungal host cells are obtained from the following genes: Aspergillus niger neutral amylase, Aspergillus niger glucosylase, Aspergillus oryzae TAKA amylase, Aspergillus oryzae cellulase, Aspergillus oryzae endoglucanase V, Aspergillus sparseis lipase, and Rhizopus oryzae aspartic protease.

[0225] Useful signal peptides in yeast host cells are obtained from the genes of *Saccharomyces cerevisiae* α-factor and *Saccharomyces cerevisiae* invertase. Romanos et al., 1992, ibid., describe the coding sequences of other useful signal peptides.

[0226] The control sequence can also be a propeptide-coding sequence encoding the propeptide located at the N-terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propeptide progenitor (or, in some cases, a zymogen). The propeptide progenitor is generally inactive and can be converted into an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propeptide progenitor. The propeptide-coding sequence can be obtained from the genes of Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Thermophilus laccase (WO 95 / 33836), Rhizopus oryzae aspartic protease, and Saccharomyces cerevisiae α-factor.

[0227] In the presence of both a signal peptide and a propeptide sequence, the propeptide sequence is located immediately after the N-terminus of the polypeptide, and the signal peptide sequence is located immediately after the N-terminus of the propeptide sequence.

[0228] It may also be desirable to add regulatory sequences that regulate peptide expression in relation to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to turn on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. Regulatory systems in prokaryotes include lac, tac, and trp-operated gene systems. In yeast, the ADH2 or GAL1 system can be used. In filamentous fungi, the *Aspergillus niger* glucosylamylase promoter, the *Aspergillus oryzae* TAKA α-amylase promoter, and the *Aspergillus oryzae* glucosylamylase promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the peptide will be operatively linked to the regulatory sequence.

[0229] expression carrier

[0230] The present invention also relates to recombinant expression vectors comprising the polynucleotide, promoter, and transcription and translation termination signals of the present invention. Different nucleotides and control sequences can be conjugated together to produce a recombinant expression vector, which may include one or more convenient restriction enzyme sites to allow insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide can be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into a suitable vector for expression. In generating the expression vector, the coding sequence is located within the vector such that the coding sequence is operatively linked to the suitable control sequence for expression.

[0231] Recombinant expression vectors can be any vector (e.g., plasmids or viruses) that can readily undergo recombinant DNA procedures and induce polynucleotide expression. The choice of vector will typically depend on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid.

[0232] The vector can be a self-replicating vector, that is, a vector existing as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, mini-chromosome, or artificial chromosome. The vector can contain any tools used to ensure self-replication. Alternatively, the vector can be one that, when introduced into the host cell, is integrated into the genome and replicates along with one or more chromosomes in which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids (which together contain the total DNA of the genome to be introduced into the host cell), or transposons can be used.

[0233] The vector preferably contains one or more selective markers that allow easy selection of transformed, transfected, transduced, or similar cells. The selective marker is a gene whose product provides resistance to biocides or viruses, heavy metals, auxotrophic prototrophs, etc.

[0234] Examples of selective bacterial markers include the dal gene in *Bacillus licheniformis* or *Bacillus subtilis*, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selective markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamipase), argB (ornithine carbamoyltransferase), bar (glufosinate acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotate nucleoside-5'-phosphate decarboxylase), sC (adenosyl sulfate transtransferase), and trpC (o-aminobenzoic acid synthase), along with their equivalents. Preferred markers for use in *Aspergillus* cells are the amdS and pyrG genes from *Aspergillus nidus* or *Aspergillus oryzae*, and the bar gene from *Streptomyces hygroscopicus*.

[0235] The vector preferably contains one or more elements that allow the vector to be integrated into the host cell genome or that allow the vector to replicate autonomously in the cell without relying on the genome.

[0236] For integration into the host cell genome, the vector can rely on a polynucleotide sequence encoding the polypeptide or any other element of the vector integrated into the genome via homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides to guide integration into one or more precise locations on one or more chromosomes within the host cell genome via homologous recombination. To increase the likelihood of integration at precise locations, these integrating elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, that have high sequence identity with the corresponding target sequence to enhance the likelihood of homologous recombination. These integrating elements can be any sequence homologous to the target sequence within the host cell genome. Furthermore, these integrating elements can be non-coding or coding polynucleotides. On the other hand, the vector can be integrated into the host cell genome via non-homologous recombination.

[0237] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously within the host cell in question. The origin of replication can be any plasmid replication factor that mediates autonomous replication and functions within the cell. The terms "origin of replication" or "plasmid replication factor" refer to a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0238] Examples of bacterial origins of replication are plasmids pBR322, pUC19, pACYC177, and pACYC184, which allow replication in Escherichia coli, and pUB110, pE194, pTA1060, and pAMβ1, which allow replication in Bacillus.

[0239] Examples of replication origins used in yeast host cells are 2-micron replication origins, ARS1, ARS4, combinations of ARS1 and CEN3, and combinations of ARS4 and CEN6.

[0240] Examples of useful origins of replication in filamentous fungal cells are AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987, Nucleic Acid Research 15:9163-9175; WO 00 / 24883). The AMA1 gene can be isolated and plasmids or vectors containing this gene can be constructed according to the methods disclosed in WO 00 / 24883.

[0241] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase polypeptide production. An increased number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selective marker gene accompanying the polynucleotide, wherein cells containing amplified selective marker gene copies and thus additional polynucleotide copies can be selected by culturing cells in the presence of appropriate selective reagents.

[0242] The procedures for connecting the elements described above to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, ibid.).

[0243] signal peptide

[0244] The present invention also relates to an isolated polynucleotide encoding a signal peptide comprising the following amino acids: amino acids 1 to 19 of SEQ ID NO:4, or amino acids -40 to -18 of SEQ ID NO:4, or composed thereof. These polynucleotides may additionally contain a gene encoding a protein operatively linked to the signal peptide. The protein is preferably heterologous to the signal peptide. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 57 of SEQ ID NO:3 or nucleotides 1 to 69 of SEQ ID NO:7.

[0245] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells containing these polynucleotides.

[0246] The present invention also relates to a method for producing a protein, comprising (a) culturing a recombinant host cell containing such a polynucleotide; and (b) recovering the protein.

[0247] The protein can be native or heterologous to the host cell. The term "protein" is not intended to refer to a specific length of encoded product and therefore encompasses peptides, oligopeptides, and polypeptides. The term "protein" also encompasses two or more polypeptides that combine to form the encoded product. These proteins also include hybrid polypeptides and fusion polypeptides.

[0248] Preferably, the protein is a hormone, enzyme, receptor or a portion thereof, antibody or a portion thereof, or reporter. For example, the protein can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, such as aminopeptidase, amylase, carbohydrate enzyme, carboxypeptidase, catalase, cellobiase, cellulase, chitosanase, keratinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, invertase, laccase, lipase, mannosidase, allosteric hydrolase, oxidase, pectinase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or β-xylosidase.

[0249] This gene can be obtained from any prokaryotic, eukaryotic, or other source.

[0250] host cells

[0251] This invention also relates to recombinant host cells containing the polynucleotide of the invention, operatively linked to one or more control sequences that direct the production of the polypeptide of the invention. The construct or vector containing the polynucleotide is introduced into the host cell such that the construct or vector is maintained as a chromosomal integrase or as an autonomously replicating extrachromosomal vector, as previously described. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations occurring during replication. The selection of the host cell depends largely on the gene encoding the polypeptide and its origin.

[0252] The host cell can be any cell that has been used to recombine and generate the polypeptide of the present invention, such as a prokaryotic cell or a eukaryotic cell.

[0253] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Bacillus aeruginosa, Lactobacillus, Lactococcus, Marine Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Coptis, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0254] The bacterial host cell can be any Bacillus cell, including (but not limited to) Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus scoagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0255] The bacterial host cell can also be any streptococcal cell, including but not limited to Streptococcus equi, Streptococcus pyogenes, Streptococcus mammae, and Streptococcus equi subsp. veterinaryis.

[0256] The host cell for this bacteria can also be any Streptomyces cell, including but not limited to Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces griseus, and Streptomyces lividans.

[0257] DNA can be introduced into Bacillus cells via protoplast transformation (see, for example, Chang and Cohen, 1979, Mol. Gen. Genet. 168:111-115), or competent cell transformation (see, for example, Young and Spizizen, 1961, J. Bacteriol. 81:823-829; or Dubnau and David...). Off-Abelson, 1971, Journal of Molecular Biology 56:209-221, electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6:742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). DNA can be introduced into E. coli cells via protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166:557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acid Research 16:6127-6145). DNA can be introduced into Streptomyces cells through protoplast transformation, electroporation (see, for example, Gong et al., 2004, Folia Microbiol. (Praha) 49:399-405), conjugation (see, for example, Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, for example, Burke et al., 2001, Proceedings of the National Academy of Sciences 98:6289-6294). DNA can be introduced into Pseudomonas cells by electroporation (see, for example, Choi et al., 2006, J. Microbiol. Methods, 64:391-397) or conjugation (see, for example, Pinedo and Smets, 2005, Appl. Environ. Microbiol., 71:51-57).DNA can be introduced into streptococcal cells via natural induction (see, for example, Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, for example, Catt and Jollick, 1991, Microbios 68:189-207), electroporation (see, for example, Buckley et al., 1999, Appl. Environ. Microbiol. 65:3800-3804), or conjugation (see, for example, Clewell, 1981, Microbiol. Rev. 45:409-436). However, DNA can be introduced into host cells using any method known in the art.

[0258] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0259] The host cell can be a fungal cell. As used herein, “fungus” includes Ascomycota, Basidiomycota, Chytridiomycota, Zygomycota, along with Oomycota and all mitotic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK).

[0260] The host cell of this fungus can be a yeast cell. As used herein, "yeast" includes ascospore-producing yeasts (Endosporales), basidiomycetes, and yeasts belonging to the class Deuteromycetes (budding fungi). Since the classification of yeasts may change in the future, for the purposes of this invention, yeast should be defined as described in *Biology and Activities of Yeast* (edited by Skinner, Passmore, and Davenport, Soc. App. Bacteriol. Symposium, Vol. 9, 1980).

[0261] Yeast host cells can be Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cells, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cells.

[0262] The host cell for fungi can be a filamentous fungal cell. "Filamentous fungi" encompasses all subphyla of filamentous fungi and oomycetes (as defined by Hawksworth et al., 1995, ibid.). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, dextran, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs through hyphal elongation, and carbon metabolism is obligate aerobic. In contrast, the vegetative growth of yeast (e.g., *Saccharomyces cerevisiae*) occurs through budding of single-celled cells, and carbon metabolism can be fermentative.

[0263] The host cells of filamentous fungi can be *Acremonium*, *Aspergillus*, *Aureobasidium*, *Bjerkandera*, *Ceriporiopsis*, *Chrysosporium*, *Coprinus*, *Coriolus*, *Cryptococcus*, *Filibasidium*, *Fusarium*, *Humicola*, *Magnaporthe*, *Mucor*, *Myceliophthora*, and *Neurospora*. Cells of the genera *Neocallimastix*, *Neurospora*, *Paecilomyces*, *Penicillium*, *Phanerochaete*, *Phlebia*, *Piromyces*, *Pleurotus*, *Schizophyllum*, *Talaromyces*, *Thermoascus*, *Thielavia*, *Tolypocladium*, *Trametes*, or *Trichoderma*.

[0264] For example, the host cells of filamentous fungi can be *Aspergillus awamori*, *Aspergillus foetidus*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Bjerkandera adusta*, *Ceriporiopsisaneirina*, *Ceriporiopsis caregiea*, *Ceriporiopsis gilvescens*, *Ceriporiopsis pannocinta*, *Ceriporiopsis rivulosa*, *Ceriporiopsis subrufa*, *Ceriporiopsis subvermispora*, and *Chrysosporium*. *Chrysosporium keratinophilum*, *Chrysosporium lucknowense*, *Chrysosporium merdarium*, *Chrysosporium pannicola*, *Chrysosporium queenslandicum*, *Chrysosporium tropicum*, *Chrysosporium zonatum*, *Coprinus cinereus*, *Coriolus hirsutus*, *Fusarium bactridioides*, *Fusarium cerealis*, *Fusarium crookwellense*, *Fusarium culmorum*, *Fusarium graminearum*, *Fusarium graminum*, *Fusarium heterosporum*, *Fusarium* Fusarium oxysporum, Fusarium reticulatum, Fusarium rosenbergiiFusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii The cells of *Trichoderma eryngii*, *Thielavia terrestris*, *Trametes villosa*, *Trametes versicolor*, *Trichoderma harzianum*, *Trichoderma koningii*, *Trichoderma longibrachiatum*, *Trichoderma reesei*, or *Trichoderma viride*.

[0265] Fungal cells can be transformed through a process involving protoplast formation, protoplast transformation, and cell wall regeneration in a manner known per se. Suitable procedures for transforming Aspergillus and Trichoderma host cells are described in EP238023, Yelton et al., 1984, Proceedings of the National Academy of Sciences 81:1470-1474, and Christensen et al., 1988, Bio / Technology 6:1419-1422. Suitable methods for transforming Fusarium species are described in Malardier et al., 1989, Gene 78:147-156, and WO 96 / 00787. Yeast can be transformed using the procedures described in Becker and Guarente, edited by Abelson, JN and Simon, MI, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 194, pp. 182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences 75:1920.

[0266] Production methods

[0267] The present invention also relates to a method for producing the polypeptide of the invention, comprising (a) culturing cells under conditions favorable for the production of the polypeptide, the cells producing the polypeptide in their wild-type form; and (b) recovering the polypeptide. In a preferred aspect, the cells are Acremonium alcalophilum cells. In a more preferred aspect, the cells are Acremonium alcalophilum cells. In a most preferred aspect, the cells are Acremonium alcalophilum CBS114.92.

[0268] The present invention also relates to a method for producing the polypeptide of the present invention, comprising (a) culturing the recombinant host cell of the present invention under conditions conducive to the production of the polypeptide; and (b) recovering the polypeptide.

[0269] These host cells are cultured in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, the cells can be cultured by shake flask culture or by small-scale or large-scale fermentation (including continuous fermentation, batch fermentation, feed-feed fermentation, or solid-state fermentation) in a suitable medium and under conditions allowing for the expression and / or isolation of the polypeptide in a laboratory or industrial fermenter. This culture occurs using procedures known in the art in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the U.S. Type Culture Collection catalogue). If the polypeptide is secreted into the medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0270] The polypeptide can be detected using methods specific to these polypeptides known in the art, such as the lysozyme spot assay described below. These detection methods include, but are not limited to, the use of specific antibodies, the formation of enzyme products, or the disappearance of enzyme substrates. For example, an enzyme assay can be used to determine the activity of the polypeptide.

[0271] The peptide can be recovered using methods known in the art. For example, the peptide can be recovered from the nutrient medium through conventional procedures, including but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

[0272] The polypeptide can be purified to obtain a substantially pure polypeptide by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatographic focusing, and size exclusion chromatography), electrophoresis procedures (e.g., preparative isoelectric point focusing), differential dissolution (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, edited by Janson and Ryden, VCH Publishers, New York, 1989).

[0273] In an alternative aspect, the polypeptide is not recycled, but rather the host cell of the present invention expressing the polypeptide is used as the source of the polypeptide.

[0274] plant

[0275] The present invention also relates to isolated plants, such as transgenic plants, plant parts, or plant cells, containing the polynucleotides of the present invention for expression in recyclable amounts and to produce polypeptides or domains. The polypeptides or domains can be recovered from the plant or plant part. Alternatively, plants or plant parts containing polypeptides or domains can be used to improve the quality of food or feed, such as improving nutritional value, palatability, and rheological properties, or to neutralize anti-nutritional factors.

[0276] Genetically modified plants can be dicotyledonous (dicotyledonous plants) or monocotyledonous (monocotyledonous plants). Examples of monocotyledonous plants are grasses, such as meadow grass (bluegrass, Kentucky bluegrass); forage grasses such as fescue (Festuca) and ryegrass (Lolium); temperate grasses such as creeping bentgrass (Agrostis); and cereals such as wheat, oats, rye, barley, rice, sorghum, and corn.

[0277] Examples of dicotyledonous plants are tobacco, legumes such as lupins, potatoes, beets, peas, beans, and soybeans, as well as cruciferous plants (Brassicaceae family) such as cauliflower and rapeseed, and the closely related model organism Arabidopsis thaliana.

[0278] Examples of plant parts include stems, callus, leaves, roots, fruits, seeds, and tubers along with individual tissues containing these parts, such as epidermis, mesophyll, parenchyma, vascular tissue, and meristematic tissue. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes, and cytoplasm, are also considered plant parts. Furthermore, any plant cell, regardless of tissue origin, is considered a plant part. Similarly, plant parts, such as specific tissues and cells isolated to facilitate the use of this invention, are also considered plant parts, such as embryos, endosperm, aleurones, and seed coats.

[0279] Also included within the scope of this invention are such plants, plant parts, and the offspring of plant cells.

[0280] Transgenic plants or plant cells expressing the polypeptide or domain can be constructed using methods known in the art. In short, plants or plant cells are constructed by incorporating one or more expression constructs encoding the polypeptide or domain into a plant host genome or chloroplast genome and propagating the resulting modified plants or plant cells into transgenic plants or plant cells.

[0281] The expression construct is conveniently a nucleic acid construct containing a polynucleotide encoding a polypeptide or domain, which is operatively linked to an appropriate regulatory sequence required for expression of the polynucleotide in a selected plant or plant part. Furthermore, the expression construct may include a selective marker suitable for identifying plant cells in which the expression construct has been integrated, and the DNA sequence necessary for introducing the construct into the plant in question (the latter depending on the DNA introduction method to be used).

[0282] The selection of regulatory sequences (such as promoter and terminator sequences, and optionally signaling or transport sequences) is determined, for example, based on when, where, and how the desired polypeptide or domain is expressed. For instance, the expression of genes encoding polypeptides or domains can be constitutive or inducible, or developmentally, stage-, or tissue-specific, and the gene product can be targeted to specific tissues or plant parts, such as seeds or leaves. Regulatory sequences, for example, are described by Tague et al., 1988, Plant Physiology 86:506.

[0283] For constitutive expression, 35S-CaMV, maize ubiquitin-1, or rice actin-1 promoters can be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18:675-689; Zhang et al., 1991, Plant Cell 3:1155-1165). Organ-specific promoters can be promoters from the following sources: those from storage pool tissues (e.g., seeds, potato tubers, and fruits) (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303); those from metabolic pool tissues (e.g., meristems) (Ito et al., 1994, Plant Mol. Biol. 24:863-878); seed-specific promoters, such as gluten, prolysin, globulin, or albumin promoters from rice (Wu et al., 1998, Plant Cell Physiol. 39:885-889); broad bean promoters from ginsenoside B4; and unknown seed protein genes from broad beans (Conrad et al., 1998, J. Plant...). Physiol. (Journal of Plant Physiology) 152:708-711), promoters from seed oil body proteins (Chen et al., 1998, Plant Cell Physiol. 39:935-941), promoters from the napA storage protein from rapeseed, or any other seed-specific promoters known in the art, for example, as described in WO 91 / 14772.In addition, promoters can be leaf-specific promoters, such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol., 102:991-1000), the Chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol., 26:85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet., 248:668-674), or wound-inducible promoters, such as the potato pin2 promoter (Xu et al., 1993, Plant... Mol. Biol. (Plant Molecular Biology) 22:573-588. Similarly, promoters can be induced by abiotic treatments such as temperature, drought, or salinity changes, or by exogenous application of promoter-activating substances such as ethanol, estrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, as well as heavy metals.

[0284] Promoter enhancer elements can also be used to achieve higher expression of peptides or domains in plants. For example, a promoter enhancer element can be an intron located between the promoter and the multinucleotide sequence encoding the peptide or domain. For example, Xu et al., 1993, ibid., disclosed the use of the first intron of the rice actin 1 gene to enhance expression.

[0285] The selective marker gene and any other part of the expression construct may be selected from those available in the art.

[0286] Nucleic acid constructs can be incorporated into plant genomes using conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, bioprojectile transformation, and electroporation (Gasser et al., 1990, Science 244:1293; Potrykus, 1990, Bio / Technology 8:535; Shimamoto et al., 1989, Nature 338:274).

[0287] Agrobacterium tumefaciens-mediated gene transfer is a method for producing transgenic dicotyledonous plants (see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19:15-38 for commentary) and for transforming monocotyledonous plants, although other transformation methods can also be used for these plants. One method for producing transgenic monocotyledonous plants is particle bombardment of embryogenic callus or developing embryos (with microscopic gold or tungsten particles coated with transforming DNA) (Christou, 1992, Plant J. 2:275-281; ​​Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio / Technology 10:667-674). As described in Omirulleh et al., 1993, Plant Molecular Biology 21:415-428, alternative methods for monocotyledonous transformation are based on protoplast transformation. Other transformation methods include those described in U.S. Patent Nos. 6,395,966 and 7,151,204 (both incorporated herein by reference in their entirety).

[0288] Following transformation, transformants incorporating the expression construct are selected using methods well-known in the art and regenerated into complete plantations. Transformation procedures are typically designed to selectively remove the selected gene during regeneration or in progeny by using, for example, co-transformation with two separate T-DNA constructs or site-specific excision of the selected gene via a specific recombinase.

[0289] In addition to directly transforming a specific plant genotype using the constructs of the present invention, transgenic plants can be produced by hybridizing a plant with the construct with a second plant lacking the construct. For example, a construct encoding a polypeptide or domain can be introduced into a specific plant variety through hybridization without requiring direct transformation of a given variety. Therefore, the present invention covers not only plants directly regenerated from cells transformed according to the present invention, but also the offspring of such plants. As used herein, offspring can refer to the offspring of any generation of the parent plant prepared according to the present invention. Such offspring may contain the DNA construct prepared according to the present invention. Hybridization results in the introduction of transgenes into a plant line through cross-pollination between the donor plant line and the starting line. Non-limiting examples of such steps are described in U.S. Patent No. 7,151,204.

[0290] Plants can be produced through backcrossing. For example, plants include genotypes, lines, inbreds, or hybrids that are referred to as backcrosses.

[0291] Genetic markers can be used to assist in the introgression of one or more transgenic elements of the present invention from one genetic background to another. Marker-assisted selection offers numerous advantages over conventional breeding, including the ability to avoid errors caused by phenotypic variation. Furthermore, genetic markers can provide data on the relative prevalence of elite germplasm in the offspring of a specific hybrid. For example, when a plant with the desired trait (or otherwise a non-agronomically desirable genetic background) is crossed with an elite parent, genetic markers can be used to select offspring that not only possess the trait of interest but also have a larger proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a specific genetic background is minimized.

[0292] The present invention also relates to methods for generating the polypeptide or domain of the present invention, the methods comprising (a) culturing a transgenic plant or plant cell containing a polynucleotide encoding the polypeptide or domain under conditions conducive to the generation of the polypeptide or domain; and (b) recovering the polypeptide or domain.

[0293] application

[0294] Examples of preferred applications of the lysozyme or its compositions of the present invention are given below. The dosage of the lysozyme and other conditions for using the lysozyme can be determined based on methods known in the art.

[0295] These polypeptides of the present invention typically have sites for use with bacteria, fungi, zymogens, or algae. Typically, these sites are in aqueous systems such as cooling water systems, laundry rinse water, or oil systems such as cutting oils, lubricants, and oil fields, where it is desirable to kill or at least control the growth of microorganisms. However, the present invention can also be used in all applications where lysozyme compositions are known to be useful, such as wood protection, rubber, adhesives, glues, paper, cardboard, fabrics, leather, plastics, creases, and feed.

[0296] The lysozyme or its composition of the present invention can be used in several applications to degrade materials comprising peptidoglycan or chitodextrin by treating them with the lysozyme or its composition (see, for example, Proctor and Cunningham, (1988) Critical Reviews in Food Science and Nutrition 26:359-395; Carini et al. (1985) Microbiol. Alimen. Nutr. 3:299-320; Huey and Johnson (1987) Applied Environmental Microbiology 53:2165-2170; Cunningham et al. (1991) World's Poultry Science Journal 47:141-163).

[0297] The lysozyme of the present invention is used in cleaning and / or detergent applications.

[0298] Preferably, the lysozyme of the present invention is incorporated into and / or used with the detergent composition described below. When repeated washing is performed at temperatures below 60°C, the risk of foul odors increases in the washing machine (for washing clothes and dishes) and on textiles or articles washed in the machine. This foul odor appears to be caused by the growth of microorganisms (e.g., bacteria, fungi, algae, or other single-celled organisms) in the washing machine.

[0299] Furthermore, the present invention relates to a process for washing fabrics, the process comprising treating the fabric with a washing solution comprising a detergent composition and a lysozyme or lysozyme composition of the present invention. The washing treatment can be carried out, for example, by machine washing or by manual washing. The washing solution can be, for example, an aqueous washing solution comprising the detergent composition and having a pH between 3 and 12.

[0300] Fabrics subjected to the methods of the present invention can be conventional washable garments, such as homewear. Preferably, most garments are ready-made garments and fabrics, including woven fabrics, textiles, twill work pants, yarns, and terry cloths made from cotton, cotton blends, or natural or synthetic cellulose (e.g., derived from wood pulp) or blends thereof. Examples of blends are mixtures of cotton or synthetic fibers / viscose with one or more accompanying materials, such as wool, synthetic fibers (e.g., polyamide fibers, acrylic fibers, polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers, polyurethane fibers, polyurea fibers, aramid fibers), and cellulose-containing fibers (e.g., synthetic fibers / viscose, ramie, flax / linen, jute, cellulose acetate fibers, lyocell fibers).

[0301] This invention provides a method for reducing microbial contamination on surfaces such as textile garments or hard surfaces such as metal, plastic, or rubber parts in washing machines or dishwashers, bathroom tiles, floors, countertops, drains, sinks, and basins by treating surfaces contaminated with microorganisms using the lysozyme or lysozyme composition of this invention. Such a treatment is also contemplated to reduce odors on textiles and on hard surfaces containing microbial contamination.

[0302] The reduction in microbial contamination can be assessed in several ways, such as by having a panel assess whether the taste as a result of the treatment is reduced compared to a treatment without lysozyme; alternatively, a sample can be taken from the surface and cultured to assess whether the count of microorganisms as a result of the treatment is reduced.

[0303] Application of the lysozyme of the present invention in animal feed

[0304] The lysozyme of the present invention can also be used in animal feed. In one embodiment, the present invention provides a method for preparing an animal feed composition, the method comprising adding the lysozyme of the present invention to one or more animal feed ingredients.

[0305] The lysozyme of this invention stabilizes the healthy microbial colonies 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, piglets or pigs (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, but also healthy microbial communities in fish (including but not limited to salmon, trout, tilapia, catfish, and carp); and crustaceans (including but not limited to small shrimp and prawns) including: viral pathogens (e.g., coronaviruses, porcine reproductive and respiratory syndrome virus (PRRSV), Persivirus causing bovine viral diarrhea virus, etc.); parasitic pathogens (coccidia protozoa, Eimeria maxima, Eimeria stenosis). (mitis); or bacterial pathogens, such as Clostridium perfringens, Escherichia coli, Campylobacter coli, Campylobacter hyointestinalis, and Campylobacter jejuni, Yersinia spp., Treponema suis, Brachyspira hyodysenteriae, Lawsonia intracellularis, and Salmonella, such as Salmonella enterica, Salmonella Typhimurium, and Salmonella Mbandaka. In a preferred embodiment, a lysozyme is applied to chickens and has antimicrobial activity against Clostridium perfringens. In another embodiment, the lysozyme of the present invention is used as a feed additive, wherein it can have a positive effect on the microbial balance of the chicken's digestive tract and thereby improve animal production performance.

[0306] According to WO 00 / 21381 and WO 04 / 026334, the lysozyme of the present invention can also be used in animal feed as an enhancing enzyme to improve feed digestibility and increase its utilization efficiency.

[0307] In another embodiment, the lysozyme of the present invention can be used as a feed additive, wherein it can have a positive effect on the animal's digestive tract and thereby improve animal production performance, or improve animal health, such as reduced mortality, based on weight gain, feed conversion ratio (FCR). FCR is calculated as feed intake per animal relative to weight gain per animal.

[0308] Application of the lysozyme of the present invention as an antimicrobial agent

[0309] The lysozyme of the present invention can be used as an antimicrobial agent. One aspect of the present invention is a method for reducing microbial contamination, comprising treating a microbially contaminated surface with a lysozyme of the present invention.

[0310] To evaluate whether the lysozyme of the present invention can function as an antimicrobial agent, it can be tested in a turbidity assay. In this assay, its ability to degrade microbial cells, such as dried substrates of *Exiguobacterium undae* cells (isolated from stinky socks) or *Micrococcus luteus* cells dissolved in buffer or detergent, and thereby reduce, for example, the optical density (OD) at 540 nm, is tested compared to a microbial suspension treated only with buffer.

[0311] The lysozyme of the present invention is used for disinfection or as a disinfectant.

[0312] According to U.S. Patent No. 6,777,223, the lysozyme of the present invention can be used as a disinfectant or for disinfection, such as for treating infections in the eyes or mouth, or for cleaning and disinfecting contact lenses, and for preventing or removing biofilms on surfaces.

[0313] The lysozyme of this invention can also be used in oral care. For example, it can be used alone or in combination with other enzymes or even antimicrobial peptides in toothpaste or other oral care products. The polypeptide can be introduced into the oral cavity or applied to articles to be introduced into the oral cavity. See, for example, WO08 / 124764.

[0314] The polypeptides of the present invention are generally considered to be suitable for cleaning, disinfecting, or inhibiting microbial growth on any surface. Examples of surfaces that can advantageously contact the polypeptides of the present invention include, for example, the surfaces of processing equipment used in dairy plants, chemical or pharmaceutical processing plants, water sanitation systems, oil treatment plants, pulp processing plants, water treatment plants, and cooling towers. The polypeptides of the present invention should be used in amounts that are effective in cleaning, disinfecting, or inhibiting microbial growth on the surface in question.

[0315] Additionally, the polypeptides of the present invention can be used in food processing plants and in any area where food is prepared or served, such as in hospitals, nursing homes, and restaurants, for cleaning surfaces and cooking utensils.

[0316] Uses of the lysozyme of the present invention in food applications

[0317] The lysozyme of this invention can also be used to selectively inhibit the uncontrolled growth of Clostridium butyricum during cheese ripening, especially those made from pressed curd, such as Swiss cheese, Parmesan, Edam, Gouda, Cheddar, and many other cheeses.

[0318] The lysozyme of this invention can also be used in brewing to control or inhibit microbial contamination.

[0319] Application of the lysozyme of the present invention in treatment

[0320] The lysozyme of this invention can also be used for the topical treatment of malnutrition and inflammatory lesions of the skin and soft tissues. See, for example, Palmieri and Boraldi (1977) Arch. Sci. Med. (Torino) 134:481-485.

[0321] The lysozyme of this invention can also be used in skin care. For example, the polypeptide can be applied to the skin of a patient suffering from a skin infection (such as acne). The lysozyme can also be used in wound dressings applied to injured skin to assist in wound healing. See, for example, U.S. Application No. 20080254079.

[0322] The lysozyme of this invention can also be used in lipsticks, lip balms, lip glosses, or lip stains. For example, such products can be used to treat localized lip infections, such as cold sores. See, for example, U.S. Application No. 20080254079.

[0323] The lysozyme of this invention can also be used to treat bronchopulmonary diseases.

[0324] The lysozyme of the present invention can also be used as a digestive enzyme or digestive aid. The lysozyme of the present invention can also be used to improve the use of dead / live bacteria as a food source, for example, by controlling unwanted microbial contamination.

[0325] The lysozyme of the present invention can also be used as a therapeutic agent in humans or other animals, for example, to control or inhibit the overgrowth of bacteria in the intestines of people with diseases (e.g., pancreatic diseases) or immunocompromised patients.

[0326] Application of the lysozyme of the present invention for extracting bacterial genomic DNA

[0327] The lysozyme of this invention can also be used to assist in the extraction of bacterial genomic DNA from pure cultures and environmental samples containing multiple bacterial species. To sequence bacterial DNA, the bacterial cell wall needs to be broken down to isolate the DNA. Egg white lysozyme is a standard enzyme for isolating DNA from Gram-positive bacteria and works by hydrolyzing peptidoglycan chains present in the cell wall, thereby assisting in the degradation of the cell wall. However, some Gram-positive cell walls are not degraded by egg white lysozyme. For example, as described by Pitcher and Saunders (1989), Applied Environmental Microbiology 56(3):782-787, it is suggested that cells from, for example, Staphylococcus aureus be lysed with lysin. However, these methods do not work for all types of Gram-positive bacteria, and therefore novel lysozymes potentially offer the acquisition of novel genomes that cannot be isolated using commercially available lysozyme solutions.

[0328] Adding one or more of the lysozymes of the present invention, optionally together with lysococcal lysozyme or egg white lysozyme, breaks down cell walls from bacteria (preferably Gram-positive bacteria), which is not possible using currently available commercial solutions. In one embodiment, the lysozyme is a GH25 lysozyme or a variant thereof having SEQ ID NO:4, SEQ ID NO:8. In another embodiment, the lysozyme effectively breaks down cell walls from bacteria such as Bacillus, Micrococcus, Zobellia, Cellulophaga, and Streptomyces. Another embodiment includes bacteria such as Bacillus subtilis, Micrococcus luteus, Zobellia uliginosa, Cellulophaga lytica, and Streptomyces mobaraensis. In another embodiment, the lysozyme combined with egg white lysozyme effectively breaks down the cell walls of bacteria such as Bacillus, Micrococcus, Zhou's bacteria, Fibrophagium, and Streptomyces, including Bacillus subtilis, Micrococcus luteus, Zobilella auriculi, Fibrophagium lysate, and Streptomyces mollissima. A specific embodiment involves breaking down the cell walls of bacteria derived from Streptomyces mollissima.

[0329] The lysozyme of the present invention can be used in compositions or kits for breaking down cell walls from bacteria, optionally together with lysozyme or egg white lysozyme. The lysozyme component can be a GH25 lysozyme of the present invention or a GH25 lysozyme having SEQ ID NO:4, SEQ ID NO:8, or a variant thereof.

[0330] Other applications of the lysozyme of the present invention

[0331] The lysozyme of the present invention can also be used to control microbial growth during fermentation processes, such as in the production of ethanol or other products from biomass. See, for example, WO 2007 / 109750. Therefore, this lysozyme can be used, for example, in a process for producing a fermentation product, the process comprising (a) liquefying and / or saccharifying a carbohydrate material and (b) using a fermentation bio-fermentation, wherein a lysozyme of the present invention is applied at a concentration sufficient to kill and / or inhibit the growth of bacterial cells before, during, and / or after fermentation.

[0332] The lysozyme of this invention can also be used to control the growth of microorganisms in fish or shrimp farms.

[0333] Other uses include preservation of food, beverages, and cosmetics, such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, enzyme formulations, or food ingredients.

[0334] Composition

[0335] In one yet another aspect, the present invention relates to compositions comprising polypeptides of the invention having antimicrobial and / or lysozyme activity.

[0336] The composition may include the polypeptide of the present invention as the main enzyme component, for example, a single-component composition. Alternatively, the composition may contain a variety of enzyme activities, such as aminopeptidase, amylase, carbohydrate enzyme, carboxypeptidase, catalase, cellulase, chitosanase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, α-galactosidase, β-galactosidase, glucosylamylase, α-glucosidase, β-glucosidase, halogen peroxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinase, peptidase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0337] 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 particles or microparticles. The polypeptide included in the composition can be stabilized according to methods known in the art.

[0338] Examples of preferred applications of the polypeptide compositions of the present invention are given below. The dosage of the polypeptide compositions of the present invention and other conditions for using the compositions can be determined based on methods known in the art.

[0339] Bacterial genomic DNA extraction composition

[0340] The lysozyme of the present invention can be added to and thus become a component of a composition for the extraction of genomic DNA from bacteria. Alternatively, the lysozyme can be used in combination with one or more other lysozymes, such as, but not limited to, lysozyme, mutalysin, or egg white lysozyme. The composition can be formed as part of a kit, which can be mixed together according to a set of instructions for the extraction of genomic DNA from bacteria. The kit may contain a buffer, one or more metal ion binders (e.g., EDTA), a protease (e.g., proteinase K), a detergent (e.g., SDS or Triton X), and one or more lysozymes (e.g., the GH25 lysozyme, lysozyme, mutalysin, or egg white lysozyme of the present invention). Bacterial genomic DNA can be extracted from pure cultures and environmental samples containing multiple bacterial species. A preferred embodiment is a GH25 lysozyme or a variant thereof having SEQ ID NO:4, SEQ ID NO:8.

[0341] Animal feed composition

[0342] The present invention also relates to methods for using the polypeptides of the present invention having lysozyme activity in animal feed, as well as feed compositions and feed additives including the lysozyme of the present invention.

[0343] The term "animal" includes all animals, including humans. Examples of animals are non-ruminants and ruminants. Ruminants include, for example, sheep, goats, and cattle, such as beef cattle and dairy cattle. In one specific embodiment, the animal is a non-ruminant. Non-ruminants include monogastric animals, such as piglets or pigs (including but not limited to piglets, growing pigs, and sows); poultry, such as turkeys, geese, ducks, and chickens (including but not limited to broilers, chicks, and laying hens); horses (including but not limited to hot-blooded, cold-blooded, and warm-blooded horses), calves; and fish (including but not limited to salmon, trout, tilapia, catfish, and carp); and crustaceans (including but not limited to shrimp and prawns).

[0344] The term feed or feed composition means any compound, preparation, mixture, or composition suitable for or intended for ingestion by animals. In the use according to the invention, the lysozyme can be fed to animals before, after, or simultaneously with their diet. The latter is preferred. Such lysozyme compositions can, of course, be mixed with other enzymes.

[0345] Lysozyme can be added to feed in any form, such as as relatively pure lysozyme, or as a mixture with other components intended to be added to animal feed, i.e., in the form of animal feed additives, such as so-called animal feed premixes. In another aspect, the present invention relates to compositions for use in animal feed, such as animal feed and animal feed additives, such as premixes.

[0346] In addition to the lysozyme of the present invention, the animal feed additive of the present 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 macro mineral.

[0347] Furthermore, optional feed additives include colorants such as carotenoids (e.g., β-carotene), astaxanthin, and lutein; stabilizers; growth enhancers and aroma / flavoring compounds such as methoxycresol, anethole, decadecyl lactone, undecyl lactone and / or dodecadecyl lactone, ionone, irisone, gingerol, piperidine, propylene phthalide, butylene phthalide, capsaicin and / or tannins; polyunsaturated fatty acids (PUFAs); and reactive oxygen species. Types; and the following supports may also be used, which may contain, for example, 40%-50% by weight of wood fiber, 8%-10% by weight of stearic acid, 4%-5% by weight of turmeric powder, 4%-58% by weight of rosemary powder, 22%-28% by weight of limestone, 1%-3% by weight of gum (such as gum arabic), 5%-50% by weight of sugar and / or starch, and 5%-15% by weight of water.

[0348] The feed or feed additive of the present invention may also include at least one other enzyme selected from the following: phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); α-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 α-amylase (EC 3.2.1.1); and / or β-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

[0349] Examples of polyunsaturated fatty acids are C18, C20, and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid, and γ-linolenic acid.

[0350] Examples of substances that produce reactive oxygen species include chemicals such as perborate, persulfate, or sodium percarbonate; and enzymes such as oxidases, oxygenases, or synthases.

[0351] Typically, fat- and water-soluble vitamins, as well as trace minerals, form part of a so-called premix intended to be added to feed, while macro-minerals are usually added to feed separately. Each type of these compositions is an animal feed additive of the present invention when enriched with the proteases of the invention.

[0352] In one specific embodiment, the animal feed additive of the present invention is intended to be included (or specified to be included) in animal diets or animal feeds at a level of 0.1 ppm to 1000 ppm, preferably 0.5 ppm to 200 ppm, and more preferably 1 ppm to 100 ppm. The above-mentioned levels can also be used for premixes.

[0353] The animal feed compositions of the present invention may contain at least one plant protein, such as plant protein derived from or produced by a plant, including modified proteins and protein derivatives. Plant proteins may be derived from plant protein sources such as legumes and cereals, for example, materials from plants of the Leguminosae, Brassicaceae, Chenopodiaceae, and Poaceae families, such as soybean meal, lupin meal, and rapeseed meal. Alternatively, plant protein sources may be materials from one or more plants of the Chenopodiaceae family, such as beets, sugar beets, spinach, or quinoa. Other examples of plant protein sources are rapeseed, sunflower seeds, cottonseed, and cabbage, as well as cereals such as barley, wheat, rye, oats, corn, rice, triticale, and sorghum.

[0354] The animal feed compositions 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%-25%. The animal feed compositions of the present invention may also contain corn distillers grains with solubles (DDGS), typically in an amount of 0%-30%.

[0355] In a further specific embodiment, the animal feed composition of the present invention comprises 0%-80% corn; and / or 0%-80% sorghum; and / or 0%-70% wheat; and / or 0%-70% barley; and / or 0%-30% oats; and / or 0%-40% soybean meal; and / or 0%-25% fish meal; and / or 0%-25% meat and bone meal; and / or 0%-20% whey.

[0356] Animal diets can be prepared, for example, into pastes (non-granulated) or pelleted feeds. Typically, the milled feed is mixed and sufficient amounts of essential vitamins and minerals are added according to the instructions for the type discussed. Enzymes are added in the form of solid or liquid enzyme formulations. For example, for pastes, solid or liquid enzyme formulations can be added before or during the mixing step. For pelleted feeds, the (liquid or solid) lysozyme / enzyme preparation can also be added before or during the feed formulation step. Typically, liquid lysozyme / enzyme preparations are added after the pelleting step. The enzyme can also be incorporated into feed additives or premixes.

[0357] The final enzyme concentration in the diet is in the range of 0.01-200 mg enzyme protein / kg diet, for example, 0.5-25 mg enzyme protein / kg animal diet.

[0358] Cleaning or detergent composition

[0359] The lysozyme of the present invention can be added to a detergent composition and thus become a component thereto, particularly in liquid detergents having a pH of 7 or lower.

[0360] For example, the detergent compositions of the present invention can be formulated into hand or machine wash cleaning compositions, including laundry additive compositions suitable for pretreating stained fabrics and fabric softener compositions added during rinsing, or into detergent compositions for use in general household hard surface cleaning operations, or into detergent compositions for use in hand or machine dishwashing operations.

[0361] In one specific aspect, the present invention provides a detergent additive comprising the lysozyme of the present invention. The detergent additive and the detergent composition may include one or more other enzymes, such as a protease, lipase, keratinase, amylase, carbohydrate-degrading enzyme, cellulase, pectinase, mannanase, arabinase, galactanase, xylanase, oxidases such as laccase, and / or peroxidase.

[0362] Generally speaking, the characteristics of the selected one or more enzymes should be compatible with the selected detergent (i.e., optimal pH value, compatibility with other enzymatic and non-enzymatic components, etc.), and the one or more enzymes should be present in an effective amount.

[0363] In one embodiment, the invention relates to cleaning or detergent compositions comprising the enzymes of the invention in combination with one or more additional cleaning components. The selection of additional cleaning components is within the skill of those skilled in the art and includes conventional ingredients, including exemplary, non-limiting components listed below.

[0364] The selection of components (for textile care) may include considerations such as the type of textile to be cleaned, the type and / or extent of soiling, the temperature at which cleaning is performed, and the formulation of the detergent product. Although the components mentioned below are categorized according to a specific function, this should not be construed as limiting, as the component may have one or more additional functions that a skilled professional would understand.

[0365] This cleaning or detergent composition can be suitable for washing textiles, such as fabrics, clothes or linens, or for cleaning hard surfaces, such as floors, tables, or dishes.

[0366] The present invention also relates to polynucleotides encoding these polypeptides, nucleic acid constructs, vectors, and host cells comprising these polynucleotides, along with methods for producing and using these polypeptides.

[0367] surfactants

[0368] Detergent compositions may include one or more surfactants, which may be anionic and / or cationic and / or nonionic and / or semi-polar and / or zwitterionic, or mixtures thereof. In one specific embodiment, the detergent composition includes a mixture of one or more nonionic surfactants and one or more anionic surfactants. Such surfactants are typically present at levels ranging from about 0.1% to 60% by weight, for example, from about 1% to about 40%, or from about 3% to about 20%, or from about 3% to about 10%. The surfactants are selected based on the desired cleaning application, and include any one or more conventional surfactants known in the art. Any surfactant known in the art for use in detergents may be utilized.

[0369] When included therein, the detergent will typically contain from about 1% to about 40% by weight, for example from about 5% to about 30% (including from about 5% to about 15%), or from about 20% to about 25% of anionic surfactants. Non-limiting examples of anionic surfactants include sulfates and sulfonates, specifically, linear alkylbenzene sulfonates (LAS), isomers of LAS, branched alkylbenzene sulfonates (BABS), phenyl alkyl sulfonates, α-olefin sulfonates (AOS), olefin sulfonates, chain olefin sulfonates, alkane-2,3-dimethylbis(sulfate), hydroxyalkyl sulfonates and disulfonates, alkyl sulfates (AS) (e.g., sodium dodecyl sulfate (SDS)), fatty alcohol sulfates (FAS), and primary alcohol sulfates (PAS). Alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxy sulfates or fatty alcohol ether sulfates), secondary alkyl sulfonates (SAS), paraffinic sulfonates (PS), ester sulfonates, sulfonated fatty acid glycerides, α-sulfonic acid fatty acid methyl esters (α-SFMe or SES) (including methyl ester sulfonates (MES)), alkyl succinic acids or alkenyl succinic acids, dodecenyl / tetradecenyl succinic acids (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfonic acid succinic acids or soaps, and combinations thereof.

[0370] When included therein, the detergent will typically contain from about 0% to about 10% by weight of a cationic surfactant. Non-limiting examples of cationic surfactants include alkyl dimethyl ethanol quaternary ammonium (ADMEAQ), hexadecyl trimethyl ammonium bromide (CTAB), dimethyl distearate ammonium chloride (DSDMAC), and alkyl benzyl dimethyl ammonium, alkyl quaternary ammonium compounds, alkoxylated quaternary ammonium (AQA) compounds, and combinations thereof.

[0371] When included therein, the detergent will typically contain from about 0.2% to about 40% by weight of a nonionic surfactant, for example from about 0.5% to about 30%, particularly from about 1% to about 20%, from about 3% to about 10%, for example from about 3% to about 5%, or from about 8% to about 12%. Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters (e.g., ethoxylated and / or propoxylated fatty acid alkyl esters), alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polysaccharides (APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamides (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucosamide (GA), or fatty acid glucosamide (FAGA)), together with products available under the trade names SPAN and TWEEN, and combinations thereof.

[0372] When included therein, the detergent will typically contain from about 0% to about 10% by weight of a semi-polarized surfactant. Non-limiting examples of semi-polarized surfactants include amine oxides (AOs), such as alkyl dimethylamine oxides, N-(cocoylalkyl)-N,N-dimethylamine oxides and N-(butter-alkyl)-N,N-bis(2-hydroxyethyl)amine oxides, fatty acid alkanolamides and ethoxylated fatty acid alkanolamides, and combinations thereof.

[0373] When included therein, the detergent will typically contain from about 0% to about 10% by weight of a zwitterionic surfactant. Non-limiting examples of zwitterionic surfactants include betaine, alkyldimethylbetaine, sulfobetaine, and combinations thereof.

[0374] Water-soluble

[0375] A co-hydrosolvent is a compound that dissolves hydrophobic compounds (or conversely, polar substances in a nonpolar environment) in aqueous solution. Typically, co-hydrosolvents possess both hydrophilic and hydrophobic characteristics (as known from surfactants as so-called amphiphilic properties); however, the molecular structure of co-hydrosolvents generally does not favor spontaneous self-aggregation, see, for example, the review by Hodgdon and Kaler (2007), Current Opinion in Colloid & Interface Science, 12:121-128. Co-hydrosolvents do not exhibit a critical concentration above which self-aggregation, as observed with the formation of micelles, thin layers, or other well-defined intermediate phases by surfactants and lipids, occurs. Many co-hydrosolvents instead exhibit a continuous aggregation process, where the size of the aggregates increases with increasing concentration. However, many co-hydrosolvents alter the phase state, stability, and colloidal properties of systems containing both polar and nonpolar substances (including mixtures of water, oils, surfactants, and polymers). Water-soluble solvents are used across a wide range of industries, from pharmaceuticals and personal care to food and technological applications. The use of water-soluble solvents in detergent compositions allows for, for example, more concentrated surfactant formulations (such as in the process of compressing liquid detergents by removing water) without causing undesirable phenomena such as phase separation or high viscosity.

[0376] Detergents may contain 0% to 5% by weight, for example, about 0.5% to about 5%, or about 3% to about 5%, of a water-soluble solvent. Any water-soluble solvent known in the art for use in detergents may be used. Non-limiting examples of water-soluble solvents include sodium benzenesulfonate, sodium p-toluenesulfonate (STS), sodium xylenesulfonate (SXS), sodium cumenesulfonate (SCS), sodium cymene sulfonate, amine oxides, alcohols and polyethylene glycol ethers, sodium hydroxynaphthenate, sodium hydroxynaphthenate, sodium ethylhexylsulfonate, and combinations thereof.

[0377] Builders and co-builders

[0378] Detergent compositions may contain, by weight, about 0% to 65%, for example about 5% to about 45%, of detergent builders or co-builders, or mixtures thereof. In dishwashing detergents, the level of builders is typically 40% to 65%, particularly 50% to 65%. Builders and / or co-builders may specifically be chelating agents that form water-soluble complexes having Ca and Mg. Any builders and / or co-builders known in the art for use in laundry detergents may be utilized. Non-limiting examples of detergent builders include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), carbonates such as sodium carbonate, soluble silicates such as sodium silicate, layered silicates (e.g., SKS-6 from Hoechst), ethanolamines such as 2-aminoethanol (MEA), diethanolamine (DEA, also known as iminodiethanol), triethanolamine (TEA, also known as 2,2',2'-meta-aminotriethanol), and carboxymethyl inulin (CMI), and combinations thereof.

[0379] The detergent composition may also contain 0%-20%, for example, about 5% to about 10% by weight, a detergent co-agent, or a mixture thereof. The detergent composition may include a co-agent alone or in combination with a builder, such as a zeolite builder. Non-limiting examples of co-agents include homopolymers of polyacrylates or copolymers thereof, such as poly(acrylic acid) (PAA) or copolymers of (acrylic acid / maleic acid) (PAA / PMA). Other non-limiting examples include citrates, chelating agents such as aminocarboxylates, aminopolycarboxylates, and phosphonates, and alkyl- or alkenyl succinic acids. Other specific examples include 2,2',2”-N-aminotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diphosphonic acid (HEDP), ethylenediaminetetra-(methylenephosphonic acid) (EDTMPA), diethylenetriaminepenta-(methylenephosphonic acid) (DTPMPA or DTMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic-N-monoacetic acid (ASMA), aspartic-N,N-diacetic acid (ASDA), aspartic-N-monopropionic acid (ASMP), and iminodisuccinic acid. N-(2-sulfomethyl)-aspartic acid (IDA), N-(2-sulfoethyl)-aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(2-sulfomethyl)-glutamic acid (SMGL), N-(2-sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MIDA), α-alanine-N,N-diacetic acid (α-ALDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), o-amino acid N,N-diacetic acid (ANDA), sulfanilamide-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA), sulfonyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)-ethylenediamine-N,N',N'-triacetic acid (HEDTA), diethanolglycine (DEG), diethylenetriaminepenta (DTPMP), aminotris(methylenephosphonic acid) (ATMP), and combinations thereof and their salts. Further exemplary builders and / or co-builders are described, for example, in WO 09 / 102854 and US 5977053.

[0380] bleaching system

[0381] The detergent may contain 0%-50% by weight, for example, about 0.1% to about 25% of a bleaching system. Any bleaching system known in the art for use in laundry detergents can be utilized. Suitable bleaching system components include bleaching catalysts, photobleaching agents, bleaching activators, hydrogen peroxide sources (e.g., sodium percarbonate and sodium perborate), preformed peracids, and mixtures thereof. Suitable preformed peracids include, but are not limited to: peroxycarboxylic acids and their salts, percarbonate and their salts, and perimidic acids. Acids and their salts, peroxymonosulfate and its salts (e.g., potassium persulfate (Oxone(R))), and mixtures thereof. Non-limiting examples of bleaching systems include peroxide-based bleaching systems that may include, for example, an inorganic salt that forms a bleaching activator combination with a peracid, including alkali metal salts such as perborates (typically monohydrates or tetrahydrates), percarbonates, persulfates, superphosphates, and sodium salts of persilicates. The term bleaching activator herein refers to a compound that reacts with a peroxide bleaching agent (like hydrogen peroxide) to form a peracid. The peracid formed in this way... Acids constitute activated bleaching agents. Suitable bleaching activators to be used herein include those belonging to the ester, amide, imide, or acid anhydride classes. Suitable examples are tetraacetylethylenediamine (TAED), sodium 4-[(3,5,5-trimethylhexanoyl)oxy]benzenesulfonate (ISONOBS), diperoxylauric acid, 4-(dodecyloxy)benzenesulfonate (LOBS), 4-(decyloxy)benzenesulfonate, 4-(decyloxy)benzoate (DOBS), 4-(nonanoyloxy)benzenesulfonate (NOBS), and / or those disclosed in WO Those in 98 / 17767. Specific families of bleaching activators of interest are disclosed in EP 624154, and in that family, triethyl acetylacetic acid (ATC) is particularly preferred. ATC, or short-chain triglycerides (like triacetin), has the advantage of being environmentally friendly because it eventually degrades into citric acid and alcohol. Furthermore, triethyl acetylacetic acid and triacetin exhibit good hydrolytic stability in the product during storage, and it is an effective bleaching activator. Finally, ATC provides good washing ability for laundry additives. Alternatively, the bleaching system may include, for example, amides, imines, or sulfone-type peroxy acids. The bleaching system may also include peracids, such as 6-(phthalimide)percapanoic acid (PAP). The bleaching system may also include a bleaching catalyst. In some embodiments, the bleaching component may be an organic catalyst selected from the group consisting of: organic catalysts having the following formula:

[0382]

[0383] (iii) and its mixtures; wherein each R 1Independently, it is a branched alkyl group containing 9 to 24 carbons or a straight-chain alkyl group containing 11 to 24 carbons, preferably, each R 1 Independently, it is a branched alkyl group containing 9 to 18 carbons or a straight-chain alkyl group containing 11 to 18 carbons; more preferably, each R 1 Independently selected from the group consisting of: 2-propylheptyl, 2-butyloctyl, 2-pentylnonyl, 2-hexyldecyl, n-dodecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, isonyl, isodecyl, iso-tridecyl, and iso-pentadecanyl. Other exemplary bleaching systems are described, for example, in WO 2007 / 087258, WO 2007 / 087244, WO 2007 / 087259, and WO 2007 / 087242. Suitable photobleaching agents may be, for example, sulfonated zinc phthalocyanine.

[0384] polymer

[0385] The detergent may contain 0%-10% by weight, for example 0.5%-5%, 2%-5%, 0.5%-2%, or 0.2%-1% of a polymer. Any polymer known in the art for use in detergents may be used. The polymer may function as a co-adjuvant as mentioned above, or may provide anti-redeposition, fiber protection, dirt release, dye transfer inhibition, oil stain removal, and / or anti-foaming properties. Some polymers may have more than one of the above-mentioned properties and / or more than one of the following motifs. Exemplary polymers include (carboxymethyl) cellulose (CMC), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) or poly(ethylene oxide) (PEG), ethoxylated poly(ethyleneimine), carboxymethyl inulin (CMI), and polycarboxylates such as PAA, PAA / PMA, poly-aspartic acid, and lauryl methacrylate / acrylic acid copolymers, hydrophobically modified CMC (HM-CMC) and silicones, copolymers of terephthalic acid and oligomeric polyethylene glycol, copolymers of poly(ethylene terephthalate) and poly(ethylene oxyterephthalate) (PET-POET), PVP, poly(vinylimidazolium) (PVI), poly(vinylpyridine-N-oxide) (PVPO or PVPNO), and polyvinylpyrrolidone-vinylimidazolium (PVPVI). Other exemplary polymers include sulfonated polycarboxylate esters, polyethylene oxide and polypropylene oxide (PEO-PPO), and diquaternary ammonium ethoxysulfonate. Other exemplary polymers are disclosed, for example, in WO 2006 / 130575. Salts of the aforementioned polymers are also considered.

[0386] Fabric colorant

[0387] The detergent compositions of the present invention may further include fabric colorants, such as dyes or pigments, which, when formulated in the detergent composition, can deposit on the fabric when the fabric comes into contact with a washing liquid comprising the detergent composition, and thus alter the color of the fabric by absorption / reflection of visible light. Fluorescent whitening agents emit at least some visible light. In contrast, fabric colorants alter the color of a surface because they absorb at least a portion of the visible light spectrum. Suitable fabric colorants include dyes and dye-clay conjugates, and may also include pigments. Suitable dyes include small molecule dyes and polymer dyes. Suitable small molecule dyes include those selected from the group consisting of dyes falling under the Colour Index (CI) classification: Direct Blue, Direct Red, Direct Violet, Acid Blue, Acid Red, Acid Violet, Basic Blue, Basic Violet, and Basic Red, or mixtures thereof, as described, for example, in WO 2005 / 03274, WO 2005 / 03275, WO 2005 / 03276 and EP 1876226 (which are incorporated herein by reference). The detergent composition preferably includes from about 0.00003 wt% to about 0.2 wt%, from about 0.00008 wt% to about 0.05 wt%, or even from about 0.0001 wt% to about 0.04 wt% of a fabric toner. The composition may include from 0.0001 wt% to 0.2 wt% of a fabric toner, which may be particularly preferred when the composition is in the form of a unit-dose packet. Suitable toners are also disclosed, for example, in WO 2007 / 087257 and WO 2007 / 087243.

[0388] Other enzymes

[0389] In one aspect, the present invention provides a detergent additive comprising the lysozyme of the present invention. The detergent additive, together with the detergent composition, may include one or more [other] enzymes, such as proteases, lipases, keratins, amylases, carbohydrate-degrading enzymes, cellulases, pectins, mannanases, arabinases, galactanases, xylanases, oxidases such as laccase, and / or peroxidases.

[0390] Generally speaking, the properties of the one or more enzymes selected should be compatible with the selected detergent (i.e., optimal pH, compatibility with other enzymatic and non-enzymatic components, etc.), and the one or more enzymes should be present in an effective amount.

[0391] Cellulase:Suitable cellulases include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Suitable cellulases include those from the genera *Bacillus*, *Pseudomonas*, *Pyrophyllus*, *Fusarium*, *Clostridium*, and *Apocytozoa*, such as fungal cellulases produced by *Pyrophyllus*, *Thermophyllus*, and *Fusarium* as disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757, and WO 89 / 09259.

[0392] Particularly suitable cellulases are alkaline or neutral cellulases that offer color protection benefits. Examples of such cellulases are those described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, and WO 98 / 08940. Other examples are cellulase variants, such as those described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307, and PCT / DK 98 / 00299.

[0393] Commercially available cellulases include Celluzyme and Carezyme (Novozymes A / S), Clazinase and Puradax HA (Genencor International Inc.), as well as KAC-500(B). TM (Kao Corporation).

[0394] Protease: Suitable proteases include those of animal, plant, or microbial origin. Microbial origin is preferred. This includes chemically modified or protein-engineered mutants. The protease can be a serine protease or a metalloproteinase, preferably an alkaline microbial protease or a trypsin-like protease. Examples of alkaline proteases are subtilisin proteases, especially those derived from Bacillus, such as subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (described in WO 89 / 06279). Examples of trypsin-like proteases are trypsins (e.g., of porcine or bovine origin) and Fusarium proteases described in WO 89 / 06270 and WO 94 / 25583.

[0395] Examples of useful proteases are variants as described in WO 92 / 19729, WO 98 / 20115, WO 98 / 20116 and WO 98 / 34946, particularly variants in which substitution occurs at one or more of the following positions: 27, 36, 57, 76, 87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274.

[0396] Preferred commercially available proteases include Alcalase TM Savinase TM Prime TM Duralase TM Esperase TM , and Kannase TM (Novozymes A / S), Maxatase TM Maxacal TM Maxapem TM Property TM Purafect TM Purafect OxP TM FN2 TM and FN3 TM (Genencor International Inc.)

[0397] Lipase and keratinase:Suitable lipases and keratins include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutant enzymes. Examples include lipases from the genus *Thermophilic*, such as those from *Thermophilic Hypotherium latifolium* (formerly named *Pythium latifolium*) as described in EP258068 and EP305216; cutinases from the genus *Pythium*, such as *Pythium specificum* (WO 96 / 13580); lipases from strains of the genus *Pseudomonas* (some of which are now renamed *Burkholderia*), such as *Alcaligenes* or *Alcaligenes-like* (EP 218272), *Pseudomonas cepacia* (EP 331376), *Pseudomonas* strain SD705 (WO 95 / 06720 & WO 96 / 27002), *P. wisconsinensis* (WO 96 / 12012), and GDSL-type *Streptomyces* lipase (WO 10 / 065455); cutinases from *Oryza sativa* (WO... 10 / 107560); cutinase from Pseudomonas mendoza (US 5,389,536); lipase from Thermobifida fusca (WO 11 / 084412); lipase from Bacillus stearothermophilus (WO11 / 084417); lipase from Bacillus subtilis (WO 11 / 084599); and lipase from Streptomyces griseus (WO11 / 150157) and S. pristinaespiralis (WO 12 / 137147).

[0398] Other examples are lipase variants, such as those described in EP 407225, WO 92 / 05249, WO 94 / 01541, WO94 / 25578, WO 95 / 14783, WO 95 / 30744, WO 95 / 35381, WO 95 / 22615, WO 96 / 00292, WO 97 / 04079, WO 97 / 07202, WO 00 / 34450, WO 00 / 60063, WO 01 / 92502, WO 07 / 87508, and WO 09 / 109500.

[0399] Preferred commercially available lipase products include Lipolase TM Lipex TM Lipolex TM and Lipoclean TM (Novozymes), Lumafast (from Genencor), and Lipomax (from Gist-Brocades).

[0400] Other examples include lipases sometimes referred to as acyltransferases or perhydrolases, such as an acyltransferase homologous to Candida antarcticis lipase A (WO 10 / 111143), an acyltransferase from Mycobacterium smegmatis (WO 05 / 56782), a perhydrolase from the CE 7 family (WO 09 / 67279), and variants of Mycobacterium smegmatis perhydrolase, particularly the S54V variant (WO10 / 100028) used in Gentle Power Bleach, a commercial product from Huntsman TextileEffects Pte Ltd.

[0401] amylase: Suitable amylases (α and / or β) include those of bacterial or fungal origin. This includes chemically modified or protein-engineered mutants. Amylases include, for example, amylases obtained from specific strains of Bacillus, such as Bacillus licheniformis, described in more detail in GB 1,296,839.

[0402] Examples of useful amylases are the variants described in WO 94 / 02597, WO 94 / 18314, WO 96 / 23837, and WO97 / 43424, especially variants with substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444.

[0403] Commercially available amylase is Duramyl TM Termonyl TM Fungaly TM Stainzyme TM Natalase TM and BAN TM (Novozymes), Rapidase TM and Purastar TM (From Genentech International).

[0404] Peroxidase / oxidase:Suitable peroxidases / oxidases include those of plant, bacterial, or fungal origin. This includes chemically modified or protein-engineered mutants. Examples of useful peroxidases include peroxidases from *Coprinus comatus*, such as those from *Coprinus gracilistylus* and their variants, as described in WO 93 / 24618, WO 95 / 10602, and WO 98 / 15257. Commercially available peroxidases include Guardzyme. TM (Novozymes A / S).

[0405] These one or more detergent enzymes can be incorporated into the detergent composition by adding individual additives containing one or more enzymes, or by adding a combination additive containing all of these enzymes. The detergent additives of the present invention, i.e., individual additives or combination additives, can be formulated as, for example, granules, liquids, slurries, etc. Preferred detergent additive formulations are granules, especially non-dusting granules; liquids, particularly stable liquids; or slurries.

[0406] Non-dust-forming particles can be produced, such as those disclosed in US 4,106,991 and 4,661,452, and can optionally be coated using methods known in the art. Examples of waxy coating materials are poly(ethylene oxide) products having an average molecular weight of 1,000 to 20,000; ethoxylated nonylphenol having 16 to 50 ethylene oxide units; ethoxylated fatty alcohols, wherein the alcohol contains 12 to 20 carbon atoms and wherein 15 to 80 ethylene oxide units are present; fatty alcohols; fatty acids; and mono-, di-, and triglycerides of fatty acids. Examples of film-forming coating materials suitable for application via fluidized bed technology are given in GB 1483591. For example, liquid enzyme preparations can be stabilized according to established methods by adding polyols such as propylene glycol, sugars or sugar alcohols, propionic acid, or boric acid. Protected enzymes can be prepared according to the methods disclosed in EP 238,216.

[0407] auxiliary materials

[0408] Any detergent components known in the art for use in laundry detergents may also be used. Other optional detergent components include preservatives, shrinkage inhibitors, anti-redeposition agents, wrinkle inhibitors, bactericides, binders, corrosion inhibitors, disintegrants / disintegration agents, dyes, enzyme stabilizers (including boric acid, borates, CMC, and / or polyols such as propylene glycol), fabric finishing agents including clays, fillers / processing aids, fluorescent whitening agents / brightening agents, foam promoters, foam (foam) conditioners, fragrances, soil suspending agents, softeners, defoamers, dulling inhibitors, and wicking agents, used alone or in combination. Any ingredients known in the art for use in laundry detergents may be used. The selection of such ingredients is well within the skill of those skilled in the art.

[0409] Dispersant: The detergent compositions of the present invention may further comprise dispersants. Specifically, powdered detergents may comprise dispersants. Suitable water-soluble organic materials include homopolymerized or copolymerized acids or salts thereof, wherein the polycarboxylic acid comprises at least two carboxyl groups separated from each other by no more than two carbon atoms. Suitable dispersants, for example, are described in Powdered Detergents, Surfactant Science Series, Volume 71, Marcel Dekker, Inc.

[0410] Dye transfer inhibitors: The detergent compositions of the present invention may further include one or more dye transfer inhibitors. Suitable polymeric dye transfer inhibitors include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidinone and polyvinylimidazole, or mixtures thereof. When present in the subject composition, the dye transfer inhibitor may be present at levels from about 0.0001% to about 10%, from about 0.01% to about 5%, or even from about 0.1% to about 3% by weight of the composition.

[0411] Fluorescent whitening agentThe detergent compositions of the present invention will preferably further comprise additional components that can color the items being cleaned, such as optical brighteners or light-enhancing agents. The light-enhancing agent is preferably present at a level of about 0.01% to about 0.5%. Any optical brightener suitable for use in laundry detergent compositions can be used in the compositions of the present invention. The most commonly used optical brighteners are those belonging to the following categories: diaminostilbene-sulfonic acid derivatives, diarylpyrazoline derivatives, and diphenyl-bistyryl derivatives. Examples of diaminostilbene-sulfonic acid derivatives of fluorescent whitening agents include sodium salts of the following: 4,4'-bis-(2-diethanolamino-4-anilino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2,4-diphenylamino-s-triazine-6-ylamino)stilbene-2,2'-disulfonate; 4,4'-bis-(2-anilino-4(N-methyl-N-2-hydroxy-ethylamino)-s-triazine 4,4'-bis-(4-phenyl-2,1,3-triazol-2-yl)stilbene-2,2'-disulfonate, 4,4'-bis-(2-anilino-4(1-methyl-2-hydroxy-ethylamino)-s-triazine-6-ylamino)stilbene-2,2'-disulfonate, and 2-(stilbene-4"-naphthalene-1.,2':4,5)-1,2,3-triazine-2"-sulfonate. Preferred fluorescent whitening agents are Tinopal DMS and Tinopal CBS, available from Ciba-Geigy AG, Basel, Switzerland. Tinopal DMS is the disodium salt of 4,4'-bis-(2-morpholino-4-anilino-s-triazine-6-ylamino)stilbene disulfonate. Tinopal CBS is the disodium salt of 2,2'-bis-(phenyl-styrene)disulfonate. Also preferred is the commercially available Parawhite KX fluorescent whitening agent, supplied by Paramount Minerals and Chemicals, Mumbai, India. Other fluorescent agents suitable for use in this invention include 1,3-diarylpyrazoline and 7-aminoalkylcoumarin. Suitable fluorescent whitening agent levels range from a lower level of about 0.01 wt%, from 0.05 wt%, from about 0.1 wt%, or even from a lower level of about 0.2 wt% to a higher level of 0.5 wt% or even 0.75 wt%.

[0412] Fouling release polymersThe detergent compositions of the present invention may also include one or more soil-releasing polymers that facilitate the removal of soil from fabrics, such as cotton or polyester-based fabrics, particularly hydrophobic soil from polyester-based fabrics. For example, soil-releasing polymers may be nonionic or anionic terephthalate-based polymers, polyvinylcaprolactam and related copolymers, vinyl graft copolymers, polyester polyamides, see, for example, Powdered Detergents, Surfactant Science Series, Volume 71, Chapter 7, Marcel Dekker, Inc. Another type of soil-releasing polymer is an amphiphilic alkoxylated oil-removing polymer comprising a core structure and a plurality of alkoxylated groups attached to the core structure. The core structure may comprise a polyalkylimide structure or a polyalkanolamine structure, as detailed in WO 2009 / 087523 (which is incorporated herein by reference). Furthermore, random graft copolymers are suitable soil-releasing polymers. Suitable graft copolymers are described in more detail in WO 2007 / 138054, WO 2006 / 108856 and WO 2006 / 113314 (which are incorporated herein by reference). Other waste-releasing polymers are substituted polysaccharide structures, especially substituted cellulose structures, such as modified cellulose derivatives, for example those described in EP 1867808 or WO 2003 / 040279 (both of which are incorporated herein by reference). Suitable cellulose polymers include cellulose, cellulose ethers, cellulose esters, cellulose amides and mixtures thereof. Suitable cellulose polymers include anionic modified cellulose, nonionic modified cellulose, cationic modified cellulose, zwitterionic modified cellulose and mixtures thereof. Suitable cellulose polymers include methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, ester carboxymethylcellulose and mixtures thereof.

[0413] Anti-redeposition agent: The detergent compositions of the present invention may further include one or more anti-redeposition agents, such as carboxymethyl cellulose (CMC), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene oxide and / or polyethylene glycol (PEG), homopolymers of acrylic acid, copolymers of acrylic acid and maleic acid, and ethoxylated polyethyleneimine. The cellulose-based polymers described above under the category of dirt-releasing polymers may also function as anti-redeposition agents.

[0414] Other suitable excipientsIncluding, but not limited to, shrink-proof agents, wrinkle-resistant agents, bactericides, adhesives, carriers, dyes, enzyme stabilizers, fabric softeners, fillers, foam modifiers, water-soluble solvents, fragrances, pigments, defoamers, solvents, and structural agents and / or structural elastic agents for liquid detergents.

[0415] biofilm

[0416] Microorganisms grown in biofilms become less sensitive to all types of antimicrobial agents compared to the same microorganisms grown in conventional suspension culture.

[0417] It is well known that starved bacteria are much less sensitive to a variety of antimicrobial challenges. For example, many classic antibiotics (such as penicillin) are ineffective in slow-growing or non-dividing bacteria. Lysozyme remains effective because it attacks and destroys the peptidoglycan layer regardless of the bacterial growth state.

[0418] Biofilm control; example: dental waterline:

[0419] To name just a few, biofilms forming within the dental waterline can comprise biofilms of Pseudomonas aeruginosa, Proteus mirabilis, and Legionella. Species commonly found in the oral cavity may also colonize this system as a result of anti-retraction valve failure. The risk of cross-infection becomes even more significant when immunocompromised patients are involved, and the number of patients in this category continues to grow steadily in this era. There is a need for effective control of bacterial biofilm accumulation at the dental waterline. Commentary on biofilms can be found in: Watnick P and Kolter R (2000), “Biofilm, city of microbes,” J Bacteriol.; 182(10):2675-9.

[0420] A typical example of a commercially available throat lozenge product is Lysopaine, manufactured by Boehringer Ingelheim France.

[0421] Active ingredient:

[0422] Bacitracin 200 U.I.

[0423] (Up to 65 iu / mg)

[0424] Papain 2mg

[0425] Up to 30 NK / mg

[0426] Lysozyme hydrochloride 5mg

[0427] Up to 26000 U FIP / mg: Units determined by measuring the OD kinetics of bacterial lysis in buffer solution. Units determined by measuring the change in turbidity induced by the lysis of bacterial cultures suspended in buffer solution.

[0428] Inactive ingredients:

[0429] Saccharin excipients

[0430] magnesium stearate excipient

[0431] Menthol flavoring agent (aromatisant)

[0432] Sorbitol excipients

[0433] For local treatment of punctate infections, limit the infection to the buccal membrane of the oropharynx. Caution: If clinical indications for a general bacterial infection are clear, antibiotic treatment is recommended.

[0434] toothpaste:

[0435] Lysozyme can be used alone or in combination with other enzymes or even antimicrobial peptides. Examples of other enzymes are glucose oxidase and lactoperoxidase.

[0436] A typical toothpaste composition that includes lysozyme is Biotene, Inc., 2030 East University Drive, Rancho Domiguez, CA 90220, USA.

[0437] Active ingredients

[0438] Contains: lactoperoxidase (100gm)

[0439] Inactive ingredients

[0440] Glucose oxidase, lysozyme, sodium monofluorophosphate, sorbitol, glycerol, calcium pyrophosphate, hydrated silica, xylitol, cellulose gum, flavoring agent, sodium benzoate, β-d-glucose, potassium thiocyanate

[0441] The following examples further illustrate the invention, but these examples should not be construed as limiting the scope of the invention.

[0442] Example

[0443] strain

[0444] Aspergillus oryzae strain MT3568 was used for the expression of the Acremonium alkalophilum gene encoding the GH25 enzyme. Aspergillus oryzae MT3568 is a gene derivative of Aspergillus oryzae JaL355 that disrupts the amdS (acetamase) gene (WO 2002 / 40694), in which the pyrG auxotroph is repaired by disrupting the Aspergillus oryzae acetamase (amdS) gene with the pyrG gene. Acremonium alkalophilum CBS 114.92 was isolated by A. Yoneda in 1984 from sludge from pig manure compost near Lake Tsukui, Japan, according to the Central Bureau of Schnimmelkulture. Aspergillus oryzae Toc1512 was used for the expression of the Acremonium alkalophilum gene encoding the GH25 enzyme (SEQ ID:7) and a variant thereof. Aspergillus oryzae is a pyrG-deficient strain that can be transformed with the pyrG gene, and the transformant strain is selected based on its ability to grow in the absence of uridine.

[0445] Culture media and solutions

[0446] YP culture medium It consists of 10g of yeast extract, 20g of bacterial peptone, and 1 liter of deionized water.

[0447] LB medium It consists of 10g of tryptone, 5g of yeast extract, 5g of sodium chloride, and 1 liter of deionized water.

[0448] MDU-2 Bp medium It consists of 45g maltose-1H2O, 7g yeast extract, 12g KH2PO4, 1g MgSO4-7H2O, 2g K2SO4, 5g urea, 1g NaCl, and 0.5ml AMG trace metal solution (pH 5.0) per liter.

[0449] G2-Gly medium It consists of 18g of yeast extract, 24g of glycerin (86%-88%), 1ml of Dowfax 63N10, and 1 liter of deionized water.

[0450] Horikoshi agar mediumIt consists of the following components: 1% (w / v) dextrose, 1% soluble starch, 0.5% (w / v) peptone, 0.5% (w / v) yeast extract, 0.02% (w / v) MgSO4·7H2O, 0.1% (w / v) K2HPO4, and 15 g (w / v) bacterial agar. 1% (w / v) Na2CO3 is added separately after sterilization.

[0451] Bacteria using agar plates It consists of LB medium and 15g of bacterial agar per liter.

[0452] PDA agar plates The culture medium consisted of potato extract (prepared by boiling 300g of sliced ​​(washed but unpeeled) potatoes in water for 30 minutes, followed by decanting or filtering the culture through a coarse filter cloth). Distilled water was then added until the total volume of the suspension was one liter, followed by the addition of 20g (w / v) dextrose and 20g (w / v) agar powder. The medium was sterilized by autoclaving at 15 psi for 15 minutes (Handbook of Bacteriological Analysis, 8th Edition, Revision A, 1998).

[0453] COVE Sugar Board The medium consisted of 342 g sucrose, 20 g agar powder, 20 ml COVE salt solution, and 1 liter of deionized water. The medium was sterilized by autoclaving at 15 psi for 15 minutes (Handbook of Bacteriological Analysis, 8th Edition, Revision A, 1998). The medium was then cooled to 60°C and 10 mM acetamide, 15 mM CsCl, and Triton X-100 (50 μl / 500 ml) were added.

[0454] NaNO3 sugarcane board The medium consisted of 20 ml of COVE salt solution, 20 g of agar powder, 342 g of sucrose, and 1 liter of deionized water. The medium was sterilized by autoclaving at 15 psi for 15 minutes (Handbook of Bacteriological Analysis, 8th Edition, Revision A, 1998). The medium was then cooled to 60°C and 10 mM NaNO3 and Triton X-100 (50 μl / 500 ml) were added.

[0455] LB agar plates It consists of 37g of agar and 1 liter of deionized water.

[0456] COVE salt solution It consists of 26g of MgSO4·7H2O, 26g of KCl, 26g of KH2PO4, 50ml of COVE trace metal solution, and 1 liter of deionized water.

[0457] COVE Trace Metal SolutionIt consists of 0.04g of Na₂B₄O₇·10H₂O, 0.4g of CuSO₄·5H₂O, 1.2g of FeSO₄·7H₂O, 0.7g of MnSO₄·H₂O, 0.8g of Na₂MoO₄·2H₂O, 10g of ZnSO₄·7H₂O, and 1 liter of deionized water.

[0458] AMG Trace Metal It consists of 14.3g ZnSO4-7H2O, 2.5g CuSO4-5H2O, 0.5g NiCl2, 13.8g FeSO4, 8.5g MnSO4, and 3.0g citric acid per liter.

[0459] COVE N-gly Bevel It consists of 10g of 100% glycerol, 50ml of COVE salt solution, 218g of sorbitol, 2.02g of KNO3, 25g of agar, and 1 liter of deionized water.

[0460] Example 1: Lysozyme Assay

[0461] Xanthan gum is the gene-producing organism for all xanthan gum production. Isolation of Xanthan gum cells from highly viscous xanthan gum solutions is a cost-intensive process in industrial production (Homma et al., EP 690072; Murofushi et al., EP 718311; US ​​5702927). Currently, a preferred method for recovering xanthan gum from fermentation broth is precipitation with alcohol, primarily isopropanol, after pasteurization to destroy bacterial cells and enzymes (Cottrell, WI; Kang, SKDev, (1978), “Xanthan gum: A unique bacterial polysaccharide for food applications,” Ind. Microbiol, 19:177). Subsequently, the xanthan gum / cell debris precipitate is spray-dried and ground into powder. The alcohol is recovered by distillation. Because significant amounts of Xanthan gum cell wall fragments are present in some commercially available xanthan gum formulations, and these fragments are peptidoglycan-rich Xanthan gum cell wall material, the gum can be used as a convenient assay for peptidoglycan degradation activity.

[0462] Solid plate measurement:

[0463] Commercially prepared xanthan gum (Sigma #G-1253) was dissolved in buffer or bacterial growth medium in the presence of 0.7% agarose to a final concentration of 0.5% w / v and then autoclaved. The enzyme preparation, supernatant, or whole organism was placed in wells cut from agar plates or directly on the surface of the medium. These preparations resulted in clearing zones on the plates. These clearing zones indicate the degradation of bacterial cell wall material.

[0464] Liquid cleaning test:

[0465] Commercially prepared xanthan gum is dissolved in a buffer solution, with or without sodium chloride. The solution is autoclaved and used for xanthan gum cleanup studies. An enzyme preparation, supernatant, or whole biological medium is added to the assay medium and incubated. The resulting treatment is measured in a spectrophotometer to determine the OD of the solution. Typically, a wavelength of 600 nm is used.

[0466] Example 2: Cloning and characterization of the lysozyme-encoding gene (SEQ ID NO:4) from Acremonioum alkalophilum

[0467] The genome sequence information was generated by the Joint Genome Institute (JGI) of the U.S. Department of Energy. *Acremonium alkalophilum* CBS 114.92 was isolated in 1984 by A. Yoneda from sludge from pig manure compost near Lake Tsukui, Japan, according to the Central Bureau of Physics and Genomics. A preliminary genome assembly was downloaded from JGI and analyzed using the Pedant-Pro™ Sequence Analysis Suite (Biomax Informatics AG, Martin Redd, Germany). A gene model constructed by the software was used as a starting point for detecting GH25 homologues in the genome. A more accurate gene model was constructed using several known GH25 protein sequences as guides.

[0468] Acremonium alkalophilum CBS 114.92 was propagated for 7 days on Horikoshi agar (pH 9) at 30°C. Mycelia were harvested directly from the plates and DNA was isolated using the FastDNA SPIN Kit for Soil (www.mpbio.com). The DNA was eluted in 100 μL of 10 mM TRIS buffer and 0.1 mM EDTA (pH 7.5) and stored at 4°C until use.

[0469] The synthetic oligonucleotide primer pairs shown in Table 1 below were designed for PCR amplification of the *A. alkalophilum* CBS114.92 P242M9 GH25 gene derived from *A. alkalophilum* genomic DNA. IN-FUSION was used. TM The cloning kit (Clontech, Mountain View, California, USA) directly cloned the fragment into the expression vector pDau109 (WO2005 / 042735).

[0470] Table 1: Primers used for GH25 PCR amplification

[0471]

[0472] Bold letters represent coding sequences. The underlined sequence is homologous to the insertion site of pDau109.

[0473] The PCR reaction (25 μl) consisted of 12.5 μl of 2X IPROOF. TM The mixture consisted of HF Master Mix, 0.5 μl of primer F-P242M9 (100 μM), 0.5 μl of primer R-P242M9 (100 μM), 0.5 μl of genomic DNA (100 ng / μl), and 11 μl of deionized water.

[0474] The PCR reaction (25 μl) consisted of Phusion High-Fidelity DNA Polymerase (catalog number F-530S, Thermos Scientific, USA), 5 μl of 5X Pfusion buffer, 0.5 μl of 10 mM dNTPs, 0.5 μl of primer F-P242M9 (100 μM), 0.5 μl of primer R-P242M9 (100 μM), 0.5 μl of genomic DNA (100 ng / μl), and 18 μl of deionized water. The PCR reaction was carried out in... Dual-Block thermal circulator ( The samples were incubated in a Dual-Block Thermal Cycler (MJ Research Inc., Walsham, Massachusetts, USA) and programmed to run at 95°C for 2 minutes for one cycle; 98°C for 10 seconds, 72°C for 2 minutes, and 72°C for 30 seconds for 35 cycles; and 72°C for 10 minutes for one cycle. The samples were cooled to 12°C and further processed before removal.

[0475] The PCR reaction of 5 μl was analyzed by 1% agarose gel electrophoresis using TAE buffer, and a product band of approximately 874 bp was observed. ILLUSTRA was used according to the manufacturer's instructions. TM GFX TM PCR DNA and Gel Strip Purification Kit (ILLUSTRA) TM GFX TM The remaining PCR reaction was purified using the PCR DNA and Gel Band Purification Kit.

[0476] Then use IN-FUSION TM The cloning kit cloned the fragment into pDau109 digested with Hind III and Bam HI, generating plasmid pP242M9. Cloning the P242M9 gene into Hind III-Bam HI digested pDau109 resulted in transcription of the *Aspergillus echinococcosis* P242M9 gene under the control of the NA2-tpi dual promoter. NA2-tpi is a modified promoter from the gene encoding neutral α-amylase in *Aspergillus niger*, in which the untranslated precursor has been replaced with an untranslated precursor from the gene encoding triose phosphate isomerase in *Aspergillus nidulans*.

[0477] Based on the IN-FUSION that produced the P242M9 GH25 construct TM Follow the cloning protocol outlined in the cloning kit instructions. Transform the treated plasmid and insert fragment into Fusion Blue according to the manufacturer's protocol. TM *E. coli* cells (Crotec, Mountain View, California, USA) were plated on LB agar supplemented with 50 μg ampicillin / ml. After incubation overnight at 37°C, colonies were observed to grow on the LB amblycilin-supplemented plates under selection. Following the manufacturer's instructions, ten colonies transformed with the P242M9 GH25 construct were cultured in LB medium supplemented with 50 μg ampicillin / ml and cultured using 5Prime (5 PRIME GmbH, Kingstreet). The FAST Plasmid Mini Kit (4a, 22767 Hamburg, Germany) is used to isolate plasmids.

[0478] The isolated plasmids were sequenced using vector primers to identify representative plasmid expression clones free from PCR errors. This was done using an Applied Biosystems Model 3730xl Automated DNA Sequencer, version 3.1 BIG-DYE. TM Termination chemistry (Applied Biosystems, Inc., Foster City, California, USA) and primer walking strategies were used for DNA sequencing of the Acremonium alkalophilum CBS114.92GH25 genome clone. The quality of the nucleotide sequence data was carefully examined, and all sequences were compared against each other using PHRED / PHRAP software (University of Washington, Seattle, WA, USA). The obtained sequences were identical to those from JGI.

[0479] The nucleotide sequence and deduced amino acid sequence of the *Acremonium alkalophilum* P242M9 GH25 gene are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The coding sequence (including the stop codon) is 838 bp and is interrupted by a 154 bp (nucleotides 148 to 301) intron. The predicted protein encoded is 227 amino acids. A 19-residue signal peptide was predicted using the SignalP program (Nielsen et al., 1997, *Protein Engineering*, 10:1-6). The predicted mature protein contains 207 amino acids.

[0480] Aspergillus oryzae strain MT3568 was used in all experiments. MT3568 is a derivative of Aspergillus oryzae JaL355 with disrupted amdS (acetamidinase) (WO 2002 / 40694), in which the pyrG auxotrophic symptom is repaired during the knockout of the amdS gene. Protoplasts of Aspergillus oryzae MT3568 were prepared according to the method described on pages 14-15 of European Patent EP 0238023. Fresh protoplasts of Aspergillus oryzae MT3568 were prepared and transformed with the pP242M9 GH25 plasmid. Plasmid DNA from the mini prep procedure described above was used to transform Aspergillus oryzae MT3568.

[0481] Six μL of a solution containing approximately 3.0 μg of total DNA was used for transformation. The DNA was gently added to 100 μL of *Aspergillus oryzae* MT3568 protoplasts and 250 μL of 60% PEG 4000 (Sigma-Aldrich catalog number 95904). The 60% (w / v) PEG 4000 was prepared as follows: PEG 4000 powder was dissolved in redistilled H₂O and then heated in a microwave oven at 800 watts for 10–20 seconds until dissolved. The dissolved solution was cooled to room temperature and then adjusted with CaCl₂ solution and Tris-HCl solution (pH 7.5) to a final concentration of 10 mM for each solution. After adding the 60% PEG 4000 solution, the tubes were gently mixed and incubated at 37°C for 30 minutes. Add the mixture to 6 ml of top agar containing 10 mM acetamide and plate it onto a COVE-sorbitol plate containing 10 mM acetamide.

[0482] These plates were incubated at 37°C for 3 days or more and then moved to 26°C for two days. The inoculation was performed by first soaking 10 μl of a white inoculation needle (Nunc A / S, Denmark) in 0.1%... In an 80% solution, spore-forming colonies were brought into contact with a selection plate, and spores were picked from four individual colonies by re-scratching a fresh COVE sorbitol plate containing 10 mM acetamide. After 5 days at 26°C, spores from the re-scratched colonies were inoculated into 96-well deep-dish plates (NUNC, catalog 260251, Thermo Scientific, USA). These wells of the deep-dish plates contained 500 μL of YP + 2% glucose or YP + 2% maltodextrin medium. The inoculated plates were sealed with gas-permeable tape (89009-656, VWR.com). The plates were incubated at 30°C for 5 days. Expression was confirmed by SDS-PAGE analysis of 20 μL of harvested culture medium on 10% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and Coomassie blue staining. A transformant was selected for further work and designated Aspergillus oryzae EXP03864.

[0483] Spores of EXP03864 were inoculated into 100 ml of DAP-4C-1 medium (in a 500 ml Erlenmeyer flask with baffles). These cultures were incubated at 26°C and 150 rpm for 3 days and, if necessary, for 4 days. SDS gels were run as shown above to determine protein levels.

[0484] Plate test for lysozyme activity

[0485] As described in the lysozyme plate assay section, spot assays were performed using xanthan gum at pH 5, 7, and 8.

[0486] Prepare a 1.5% agarose solution (Ingenieur catalog 15510-027, electrophoresis grade) in the following buffer:

[0487] pH ~5 in water

[0488] pH ~ 7 in 0.02M potassium phosphate (pH 7)

[0489] pH ~ 8 - in 0.02M potassium phosphate (pH 8)

[0490] Autoclave the agarose at 121°C for 20 minutes. Dissolve 0.5% xanthan gum (Sigma G1253) in the melted 1.5% agarose and pour the mixture into Petri plates. Once these plates are in place, prepare sample application wells using the end of a P-1000 pipette attached to the vacuum line (cut to 3 mm in diameter).

[0491] 20 μL of EXP03899 culture medium was placed in the application well and incubated overnight at 37°C. Samples exhibiting lysozyme activity were observed through the clear zone, where cell debris was observed in xanthan gum. Culture medium from EXP03864 showed such a clear zone, while the untransformed Aspergillus oryzae host MT3568 did not produce a clearly visible clear zone. The remaining EXP03899 culture medium was filtered through a Fast PES Bottle top filter with a 0.22 μm cutoff and stored in equal portions at -20°C until further use.

[0492] Example 3: RDA (Radial Diffusion Measurement)

[0493] Initially, as described earlier by Lehrer et al. (Lehrer RI, Rosenman M, Harwig SS et al. (1991), “Ultrasensitive assays for endogenous antimicrobial polypeptides”, J Immunol Methods, 137:167–73), the antimicrobial activity of culture supernatants and purified fractions containing recombinantly expressed lysozyme was confirmed using RDAs with several modifications. In short, cool 30 mL of melted 1 / 10 Mueller-Hinton broth (MHB) with 1% agarose to 42°C (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989), and inoculate with Staphylococcus aureus ATCC 51365 or Escherichia coli DSM682. ATCC 10536 (Add to 5.0×10) 5 CFU / mL was poured into a single-well omnitray (Nunc) plate. The omnitray plate was then covered with a TSP (Nunc) plate and allowed to cure. After 1 hour, the TSP plate was removed, leaving 96 1-mm wells in which 10 μL of the compound of interest could be tested.

[0494] 10 μl of test solution was spotted onto each pair of wells and these plates were incubated at 37°C O / N. The next day, the clear areas indicated no growth of the test bacteria and thus indicated antimicrobial activity. The clear areas were visualized by staining with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazolium), which is reduced to purple formazan in living cells (Mosmann, Tim (1983), “Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays”, Journal of Immunological Methods 65(1–2):55–63). This staining provides black staining for living cells and no staining for clear areas without living cells.

[0495] Aspergillus fumigatus GH25 lysozyme (prepared as disclosed in Korczynska et al., Acta Cryst. (2010) F66, 973-977) was included as a reference in this test. The purified samples shown in Table 2 below have been tested in RDA assays.

[0496] Table 2: Radial diffusion determination of GH24 and GH25 lysozymes

[0497]

[0498] Measurement of transparent areas

[0499] Three copies conduct In this experiment, all samples produced the same zone of inhibition (transparency / inhibition). See [link / reference]. Figure 1 Table 3 below shows the transparent area in mm.

[0500] Table 3: Antimicrobial clear zones of GH25 lysozyme against Staphylococcus aureus and Escherichia coli.

[0501]

[0502]

[0503] The purified lysozyme A. alcalophilum GH24 (SEQ ID NO:4) showed antimicrobial activity against live cells of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli.

[0504] No antimicrobial activity was found in the culture supernatant from the unconverted Aspergillus production host (results not shown).

[0505] A large, indistinctly defined transparent area was observed around the application zone of *A. alcalophilum* GH24 (SEQ ID NO:2). This experiment indicates that *A. alcalophilum* lysozyme (SEQ ID NO:2) and *Aspergillus fumigatus* GH25 reference lysozyme have different activities and specificities against the two bacteria tested in this example.

[0506] Example 4: Turbidity Measurement

[0507] Lysozyme activity was determined by measuring the decrease (decrease) in absorbance / density at 540 nm in a solution of resuspended Micrococcus lysodeikticus ATTC No. 4698 (Sigma-Aldrich M3770) or Exiguobacterium undea in a spectrophotometer.

[0508] Preparation of substrates for lysosomal micrococci

[0509] Before use, the cells were resuspended in citrate-phosphate buffer (pH 6.5) to a concentration of 0.5 mg cells / mL and the optical density (OD) was measured at 540 nm. The cell suspension was then adjusted so that the cell concentration was equivalent to OD540 = 1.0. The adjusted cell suspension was then frozen for storage before use. The resuspended cells were used within 4 hours.

[0510] Preparation of citrate-phosphate buffer (pH 6.5)

[0511] Mix 29 mL of 0.1 M citric acid with 61 mL of 0.2 M Na2HPO4, and adjust the pH to 6.5 with HCl or NaOH.

[0512] Preparation of stem cells from *Microbacterium mirifica* (substrate)

[0513] Cultures of *Microbacterium mirifica* (DSM14481) were grown overnight in 500 mL shake flasks at 30 °C and 250 rpm in 100 mL LB medium (Fluka 51208, 25 g / L). The overnight culture was then centrifuged at 20 °C and 5000 g for 10 min, and the pellet was washed twice with sterile milli-Q water and resuspended in milli-Q water. The washed cells were centrifuged at 13000 rpm for 1 min, and the supernatant was decanted as much as possible. The washed cells were then dried in a vacuum centrifuge for 1 hour. The cell pellet was resuspended in citrate-phosphate buffer (pH 6.5) to achieve an optical density (OD) of 1 at 540 nm.

[0514] Measuring the antimicrobial activity of lysozyme in turbidity determination

[0515] Dilute the lysozyme sample to be tested in citrate-phosphate buffer (pH 6.5) to a concentration of 100-200 mg enzyme protein / L and keep it on ice until use. In a 96-well microtiter plate (Nunc), add 200 μL of substrate to each well and incubate the plate at 25°C or 37°C for 5 minutes using a VERSAmax microplate reader (Molecular Devices). After incubation, measure the absorbance of each well at 540 nm (initial value). To begin measuring activity, add 20 μL of diluted lysozyme sample to each substrate (200 μL) and begin kinetic measurements of absorbance at 540 nm at 25°C or 37°C for at least 30 minutes and up to 24 hours. Monitor the absorbance measured at 540 nm for each well; if the lysozyme is lysozyme active, observe the absorbance decrease over time.

[0516] Aspergillus fumigatus GH25 lysozyme (Kausinski et al. (2010) ibid.) was included as a reference in this test and the results are shown in Table 4 below.

[0517] Table 4: GH25 lysozyme targeting Micrococcus lysinus and Microbacterium simulans as measured by optical density decrease. Lysozyme activity

[0518]

[0519] NT means not tested

[0520] -Indicates no effect

[0521] +Indicates a minor impact

[0522] ++ indicates a moderate impact

[0523] +++ indicates a significant impact.

[0524] Example 5: Purification of P242M9 GH25 protein from Aspergillus oryzae

[0525] The fermentation supernatant containing lysozyme was filtered through a Fast PES bottle top filter with a 0.22 μm cutoff. The pH was adjusted to 4.5 with 10% acetic acid. After pH adjustment, the solution became slightly cloudy, and this cloudiness was removed by filtration through a Fast PES bottle top filter with a 0.22 μm cutoff.

[0526] Following pretreatment, approximately 650 mL of a solution containing the lysozyme was purified by chromatography on approximately 50 mL of SP agarose (SP 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. The fraction from this column was pooled based on chromatograms (absorption at 280 nm and 254 nm) and SDS-PAGE analysis. The pooled fraction was buffer-exchanged in 50 mM sodium acetate (pH 5.5) and concentrated using an Amicon spin filter with a 10 kDa cutoff.

[0527] The molecular weight estimated from SDS-PAGE is approximately 22 kDa, and the purity is >95%.

[0528] Example 6: Temperature stability of P242M9 GH25

[0529] The temperature stability of Acremonium alcalophilum P242M9 GH25 lysozyme (SEQ ID NO:4) was measured. The aim of this study was to determine the residual activity of Acremonium alcalophilum GH25 lysozyme after heat treatment at 60°C–85°C.

[0530] Samples were incubated at different temperatures for very short periods (30 s and 60 s), and then residual activity was measured at 37 °C for 1 h using *Micrococcus luteus*, a Gram-positive bacterium, as a substrate in citrate-phosphate buffer. Lysozyme activity was determined using an OD decrease turbidity assay.

[0531] Heat treatment

[0532] Heat treatment was performed at different temperatures in a preheated PCR thermal cycler (GeneAmp PCR System 9700) with a buffer of cremonium alcalophilum GH25 lysozyme (SEQ ID NO:4) for 30 / 60 seconds. The tubes were then subjected to 30 or 60 seconds at 60°C, 65°C, 70°C, 75°C, 80°C, and 85°C, followed by flash cooling on an ice bath.

[0533] Turbidity measurement

[0534] Lysozyme activity was determined by measuring the decrease (drop) in absorbance / density of a solution of resuspended Micrococcus luteus (also known as lysozyme) ATTC No. 4698 (Sigma-Aldrich M3770) at 540 nm using a spectrophotometer.

[0535] Preparation of substrates for *Micrococcus ferruginosa*

[0536] Before use, the cells were resuspended in citrate-phosphate buffer (prepared by mixing 29 mL of 0.1 M citrate with 61 mL of 0.2 M Na₂HPO₄ and adjusting the pH to 6.5 with HCl or NaOH) to a concentration of 0.5 mg cells / mL, and the optical density (OD) was measured at 540 nm. The cell suspension was then adjusted so that the cell concentration was equivalent to OD₅₄₀ = 1.0. The adjusted cell suspension was then frozen for storage before use. The suspended cells were used within 4 hours.

[0537] Measuring the antimicrobial activity of lysozyme in turbidity determination

[0538] Dilute the lysozyme sample in citrate-phosphate buffer to a concentration of 100-200 mg enzyme protein / L and keep it on ice until use. In a 96-well microtiter plate (Nunc), add 200 μL of *Micrococcus luteus* substrate to each well and incubate the plate at 37°C for 5 minutes using a VERSAmax microplate reader (Molecular Equipment Corporation). After incubation, measure the absorbance of each well (initial value) at 540 nm. To begin activity measurements, add 20 μL of diluted lysozyme sample to each well of pH-adjusted diluted substrate (200 μL) and begin kinetic measurements of absorbance at 540 nm at 37°C for at least 30 minutes and up to 24 hours.

[0539] The absorbance of each well was monitored at 540 nm, and if the lysozyme exhibited antimicrobial activity, the absorbance was observed to decrease over time. The difference in absorbance between T=0 and time points was calculated as ΔOD and compared between the tested substrates (Table 5 and...). Figure 2 The percentage of residual activity was calculated by comparing the ΔOD of the heat-treated sample with that of the untreated sample. The experiment was performed in triplicate.

[0540] Table 5: Temperature stability of P242M9 GH25 lysozyme as measured by OD decrease

[0541]

[0542] Table 6: Percentage of Remaining Activity of P242M9 GH25 Lysozyme

[0543]

[0544] The results showed that P242M9 GH25 lysozyme was stable even after 30 seconds at 85°C, and retained more than 80% of its activity even after 60 seconds at 85°C.

[0545] Example 7: Thermal stability of P242M9 GH25 determined using DSC

[0546] Using a pre-packaged PD-10 column, aliquots of the protein sample of lysozyme (P242M9 GH25) purified as in Example 7 were buffer-exchanged (see buffers in the table below). The sample was filtered through a 0.45 μm filter and diluted with buffer to approximately 2 A280 units. This buffer was used as a reference solution. The thermal stability of the lysozyme at different pH values ​​was determined by differential scanning calorimetry (DSC) using a VP-capillary DSC instrument equipped with an automated sampler (MicroCal, Piscataway, NJ, USA). The thermal denaturation peak (main endothermic peak) in the autocorrelation curve (Cp vs. T) obtained after heating the lysozyme solution in buffer at a constant programmed heating rate was taken as the thermal denaturation temperature, Td (°C).

[0547] The sample and reference solution (approximately 0.5 ml) were pre-equilibrated at 20 °C for 10 min using a thermal method, and DSC scans were performed from 20 °C to 110 °C at a scan rate of 200 K / h. Data were processed using MicroCal Origin software (version 7.0383). The denaturation temperature was determined with an accuracy of approximately + / - 0.5 °C. The results of the DSC measurements are summarized in Table 7 and... Figure 3 middle.

[0548] Table 7: Denaturation temperature of P242M9 GH25 lysozyme

[0549]

[0550] Example 8: Cloning and expression of the lysozyme-encoding gene (SEQ ID NO:8) from Acremonioum alkalophilum

[0551] Based on the nucleotide sequence identified as SEQ ID NO:3, a synthetic gene with SEQ ID NO:7 was synthesized by GeneArt (GENEART AG BioPark, 11 Josef-Engert-Str., 93053, Regensburg, Germany). The synthesized GH25 gene was amplified by PCR using the PCR primer set listed in Table 8 below. For cloning purposes, restriction enzyme sites BamHI and EcoRI were introduced into the ends of the PCR fragments (restriction enzyme sites are underlined and bold letters represent coding sequences in the primer sequences listed below).

[0552] Table 8: Primers used for PCR amplification of GH25

[0553]

[0554] The PCR fragments were purified by rotation, digested with BamHI and EcoRI (New England Biolabs), and ligated into the Aspergillus oryzae / Escherichia coli plasmid expression vector pENI 1898 using a Rapid DNA Ligation kit. This expression vector had been digested with BamHI and EcoRI beforehand. pENI 1898 was modified from the vector pENI 1861 (WO 03 / 070956) because the AMA sequence had been removed from the vector after digestion with HindIII and subsequent re-ligation into an agarose gel purified vector (Clutterbuck, AJ et al. (1991) Gene 98(1):61-67). Furthermore, pENI1861 carries the pyrG gene and is capable of complementing pyrG-deficient Aspergillus strains.

[0555] After incubation at room temperature for 5 minutes, the ligation reaction was transformed into competent *E. coli* TOP10 cells (Ingenie Biotech) and plated on LB agar plates containing 150 μg / ml ampicillin. The plates were incubated at 37°C for 16 hours. Plasmid DNA was purified from selected transformants and sequenced to confirm the cloning procedure.

[0556] The encoded predicted protein is 248 amino acids, including a 23-residue signal peptide and a 207-amino acid mature protein. The mature sequence of SEQ ID NO:4 is identical to the mature sequence of SEQ ID NO:8.

[0557] Example 9: Cloning and expression of lysozyme GH25 variant

[0558] Variants of the GH25 gene

[0559] Ten variants of the synthesized GH25 lysozyme gene containing a single amino acid alteration were cloned and expressed. The GH25 lysozyme variants consist of a single substitution of the following: W10H, Y28M, S39D, G92P, A93M, E97A, V133M, T142N, F178I, or D190A.

[0560] Cloning of GH25 variants

[0561] To generate the GH25 variant as described above, site-directed mutagenesis based on PCR was performed using mutagenic primers that introduced the desired sequence alteration (substitution). Primers were designed such that the mutation was located in the middle of an oligonucleotide with sufficient flanking nucleotides (15-25). Plasmid DNA containing GH25 was used as a template, and PCR was performed using a corrected DNA polymerase (Phusion DNA polymerase (New England Biolabs)). The PCR product was used to transform competent *E. coli* TOP10 cells (Ingenieur) according to the manufacturer's instructions. Plasmid DNA was isolated from the monoclonal transformed *E. coli* strain and sequenced to confirm the presence of the desired substitution.

[0562] Conversion and Expression of GH25 and Variants

[0563] The plasmid DNA pENI1898 encoding the lysozyme gene was transformed into Aspergillus oryzae ToC1512 protoplasts (WO 2005 / 070962) as described in Example 2 and plated on NaNO3 sucrose plates without uridine to select transformants carrying the correct construct. The plates were incubated at 37°C for 72 hours.

[0564] Single colonies were isolated by scratching NaNO3 sucrose plates at 37°C for 72 hours and then growing them in YP medium at 34°C for 72 hours. Colonies expressing lysozyme were selected after examining the fermentation broth by turbidity measurement in 50 mM 3,3-dimethylglutaric acid at pH 6.4, using the cell wall of *Micrococcus lysodeoxycholica* as a substrate. Lysozyme expression was further confirmed by visualizing a strip of approximately 23 kDa using SDS-PAGE analysis.

[0565] The lysozyme-expressing transformant was scratched again and allowed to regrow at 37°C on a COVE N-gly slant for 5 days, then inoculated into 200 ml G2-Gly shake flasks. After culturing at 30°C with vigorous stirring for 1 day, 3 ml of each culture was transferred into 200 ml MDU-2Bp in the shake flask and incubated at 30°C for 3 days. The culture was purified in a manner similar to that used in the purification of P242M9 GH25 lysozyme in Example 6 above.

[0566] Example 10: Antimicrobial activity

[0567] The antimicrobial activity of P242M9 GH25 lysozyme, synthetic GH25 lysozyme, and 11 variants of synthetic GH25 lysozyme was tested against two Clostridium perfringens strains.

[0568] Methods and Materials

[0569] The activity was determined by measuring the optical density and number of colony-forming units in Clostridium perfringens cultures before and after exposure to lysozyme. Exposure of Clostridium perfringens to active lysozyme resulted in bacterial death and a corresponding decrease in optical density (OD) and the number of colony-forming units.

[0570] In short, dilute the lysozyme stock solution to a concentration of 50 μg / ml in PBS (pH 6). Perform a two-fold dilution series of these 50 μg / ml solutions in PBS (pH 6) to a final concentration of 0.4 μg / ml.

[0571] The overnight culture of *Clostridium perfringens* was then resuspended in PBS (pH 6). OD was measured. 546 The concentration was adjusted to 1. The culture (75 μl) was mixed 1:1 with the prepared lysozyme solution (75 μl) in a microtiter plate (Nunc), which resulted in *Clostridium perfringens* being exposed to lysozyme at final concentrations ranging from 0.4–25 μg / ml (7 concentrations in total lysozyme). The culture was exposed to lysozyme for 4 hours at 42 °C under anaerobic conditions. OD was measured at time point 0 and 4 hours after exposure. 546 and CFU / ml.

[0572] result

[0573] The results of the activity tests are shown in Tables 9, 10, and 11. Figure 4 and 5 Table 9 shows the reduction in colony-forming units (CFU / mL) per mL for two strains of Clostridium perfringens with GH25 lysozyme or its variant. Exposure to active lysozyme typically results in a 1–2 log reduction in CFU / mL. A reduction of ≥1 log CFU / mL is considered significant for lysozyme activity.

[0574] Table 10 shows the decrease in optical density (δOD) of *Clostridium perfringens* NN01260 upon exposure to GH25 lysozyme P242M9 (SEQ ID NO:4), synthetic GH25 lysozyme (SEQ ID NO:8), or 11 variants of SEQ ID NO:8. Table 11 shows the decrease in optical density (δOD) of *Clostridium perfringens* clinical isolates upon exposure to GH25 lysozyme P242M9 (SEQ ID NO:4), synthetic GH25 lysozyme (SEQ ID NO:8), or 11 variants of SEQ ID NO:8.

[0575] Table 9: Decrease in CFU / mL of Clostridium perfringens when treated with wild-type and variant GH25 lysozyme.

[0576]

[0577] ++ Significant activity across the entire range of tested lysozyme concentrations.

[0578] + Significant activity against reduced lysozyme concentrations in the test.

[0579] - Lysozyme showed no significant activity.

[0580] Table 10: Decrease in OD- of Clostridium perfringens NN011260 when treated with wild-type and variant GH25 lysozyme.

[0581]

[0582]

[0583] * The average of three biological replicates

[0584] Table 11: Decrease in OD- of clinical isolates of Clostridium perfringens when treated with wild-type and variant GH25 lysozyme.

[0585]

[0586]

[0587] * The average of three biological replicates

[0588] These results show that mutations at nine positions do not lead to a loss of lysozyme activity. However, a mutation at position 97 results in a loss of activity, indicating that this position should not be mutated.

[0589] Example 11: pH activity curve

[0590] For a range of pH values, the pH activity profile of the lysozyme of the present invention is determined by measuring the decrease (drop) of the absorbance / density at 540 nm in a spectrophotometer of the solution of resuspended Micrococcus luteus ATTC No. 4698 (Sigma-Aldrich M3770) in a spectrophotometer in order to determine at which pH the GH25 lysozyme or variant has peak lysozyme activity.

[0591] Prepare the *Micrococcus luteus* substrate as described in Example 6. As described in Example 6, measure the lysozyme antimicrobial activity, except that the citrate-phosphate buffer was adjusted to one of the following pH values ​​using HCl or NaOH before starting the experiment: 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. Perform the experiments described above in duplicate or triplicate and present the results in Table 12 below.

[0592] Table 12: pH activity curves of wild-type and variant GH25 lysozyme

[0593]

[0594]

[0595] The results showed that, compared with wild-type lysozyme, the GH25 lysozyme variants of the present invention having substitutions for Y28M, G92P, A93M, V133M, T142N, or F178I retained antimicrobial activity without significant changes in the pH activity profile. The GH25 lysozyme variants of the present invention having substitutions for W10H or S39D resulted in a modified pH profile, thus increasing the pH profile by 0.5 pH units while retaining antimicrobial activity.

[0596] Example 12: Gastric stability

[0597] After incubation for up to 1 hour in artificial gastric fluid containing pepsin (pH 2), the activities of lysozyme from hen's egg white, lysozyme from *A. alcalophilum* GH25 (SEQ ID NO: 8), and two variants (W10H and S39D) were measured by turbidity assay. This activity was then compared with standard curves for each lysozyme obtained under the same or equivalent conditions, but without incubation in gastric fluid containing pepsin.

[0598] Different lysozymes were incubated in simulated gastric fluid for time spans of 0, 15, 30, or 60 minutes. After incubation, a stop buffer was added to raise the pH and thereby prevent low pH and pepsin degradation of the lysozyme's peptide bonds. After pepsin inactivation, activity turbidity was determined along with standard curves of different concentrations of lysozyme discussed for comparison.

[0599] Artificial gastric juice: (HCl pH 2, 1 mg / ml pepsin, 0.1 M NaCl).

[0600] Solution A: 0.01M HCl, 0.1M NaCl (prepared by mixing 275μl of 1M HCl with 2.5ml of 1M NaCl and adding 22.23ml of MQ water to a total volume of 25ml).

[0601] Solution B: 0.01M HCl, 0.1M NaCl, 10mg / ml pepsin (prepared by weighing out 50mg of pepsin and adding 5ml of solution A).

[0602] Preparation of standard curve

[0603] Add 160 μl of artificial gastric fluid to each microtiter well, followed by 20 μl of citrate-phosphate buffer (pH 7 for egg white lysozyme, pH 4 for A. alcalophilum GH25 lysozyme, and pH 4.5 for variants W10H and S39D). Add 20 μl of lysozyme solution, then add to each well. Micrococcus tarda Substrate (20 μl, prepared as described in Example 6) (standard curve and time series sample) and OD 540 nm was measured at 37 °C for one hour.

[0604] The antimicrobial activity of lysozyme was measured as described in Example 6, except that the citrate-phosphate buffer was adjusted to the optimal pH for the different lysozymes (as given in the preceding paragraphs) by adding HCl or NaOH. The experiments described above were performed in duplicate, and the results are given in Table 13 below.

[0605] Table 13: Residual activity of lysozyme after incubation with artificial gastric juice

[0606]

[0607] Even after 15 minutes of incubation in artificial gastric fluid, the residual lysozyme activity of the yolk egg white lysozyme was significantly reduced. In contrast, the activity of GH25 lysozyme from Acremonium alcalophilum did not show a reduction. Furthermore, two GH25 variants with substitutions for W10H or S39D also retained lysozyme activity, exhibiting similar residual activity to the GH25 lysozyme from Acremonium alcalophilum.

[0608] Example 13: Isolation of genomic DNA

[0609] The yield of bacterial genomic DNA purification with the addition of Acremonium alcalophilum GH25 lysozyme (SEQ ID NO.8) and a combination of egg white lysozyme and Acremonium alcalophilum GH25 lysozyme (SEQ ID NO.8) was compared with DNA purification without lysozyme and with egg white lysozyme alone. Genomic DNA was isolated from five different bacteria.

[0610] method

[0611] Bacterial quality was obtained by scraping cells from an agar plate with an inoculation loop or by centrifuging the liquid culture (see Table 14). DNA was isolated using a modified version of the QIAamp DNA Blood Mini Kit (catalog number 51106) and purified using the Qiagen QIAcube or a manual process.

[0612] Table 14: Overview of the bacterial strains tested, bacterial starting materials, and purification methods.

[0613]

[0614] DNA isolation using QIAcube:

[0615] Resuspend colony scrapings suitable for a 10 μL inoculation loop in 3 mL of M9 buffer (prepared by dissolving 8.77 g Na₂HPO₄·2H₂O, 3 g KH₂PO₄, 4 g NaCl, and 0.2 g MgSO₄·7H₂O in 1000 mL of H₂O and adjusting the pH to 7 with NaOH or HCl). Centrifuge the solution at 3000 RPM for 5 min and resuspend the precipitate in 3 mL of P1 buffer (included in Qiagen kit #51106). Add 200 μL aliquots to 12 2 mL test tubes and add 15 μL each of water, *A. alcalophilum* GH₂5 lysozyme (10 mg / mL), egg white lysozyme (10 mg / mL), or a 1:1 mixture of *A. alcalophilum* GH₂5 lysozyme (10 mg / mL) and egg white (10 mg / mL) to the test tubes in triplicate. Place the test tubes in the QIAcube and process them according to Qiagen's recommendations.

[0616] Manual DNA isolation:

[0617] Resuspend the liquid culture pellet and colony scrapings in 3 mL of M9 buffer. Centrifuge the bacterial M9 buffer suspension at 3000 RPM for 5 min and resuspend the pellet in 3 mL of P1 buffer. Add 200 μL aliquots to 12 2 mL test tubes and add 15 μL of water, *A. alcalophilum* GH25 lysozyme (10 mg / mL), egg white lysozyme (10 mg / mL), or a 1:1 mixture of *A. alcalophilum* GH25 lysozyme (10 mg / mL) and egg white (10 mg / mL) to each tube in triplicate. Incubate the tubes at 37 °C for 30 min, then add 25 μL of Qiagen protease and 200 μL of Qiagen buffer AL. Incubate the tubes at 56 °C for 20 min. Finally, add 210 μL of 96% ethanol to each tube. Vortex these tubes and transfer the liquid to a Qiagen rotating column and centrifuge at 8000 rpm for 1 min. Wash these columns twice with 500 μl of AW1 buffer (included in Qiagen Kit #51106). After the first wash, centrifuge these columns at 8000 RPM for 1 min, and after the second wash, centrifuge these columns at 15000 RPM for 3 min. Elute genomic DNA from each column by adding 100 μL of milliQ water (heated to 70°C), incubate at room temperature for 1 min, and centrifuge at 8000 RPM for 1 min into Eppendorf tubes. Repeat all incubations in triplicate.

[0618] result

[0619] Genomic DNA yield was estimated by running the isolated DNA on an agarose gel and visually examining the intensity of the DNA bands (Table 15). Relative DNA yields between four different treatments for each bacterial strain were estimated visually using four different scores.

[0620] Table 15: Lysozyme reactions of Acremonium alcalophilum GH25 (SEQ ID NO. 8) and lysozyme of breast egg white Comparison of genomic DNA yields among different combinations of enzymes

[0621]

[0622] The yield was scored visually using the following measures:

[0623] - No DNA bands were seen.

[0624] +weak band

[0625] ++Medium

[0626] +++ Stronger banding.

[0627] Compared to lysozyme made from mother's egg white, in 3 out of 6 cases, using A. alcalophilum GH25 lysozyme yielded equally good or better genomic DNA yields. Compared to either lysozyme alone, in 3 out of 6 cases, using a combination of A. alcalophilum GH25 lysozyme and mother's egg white lysozyme yielded equally good or better genomic DNA yields. sequence list <110> Novozymes A / S <120> A polypeptide with lysozyme activity and a polynucleotide encoding the polypeptide. <130> 12166-EP-EPA <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 782 <212> DNA <213> Acremonium alcalophilum <220> <221> CDS <222> (1)..(133) <220> <221> signal peptide <222> (1)..(57) <220> <221> CDS <222> (215) (345) <220> <221> CDS <222> (516) (779) <400> 1 atg gtc tct ttc aag cag ctc gcc ctc ctg gca ctg ggc gcc gtc caa 48 Met Val Ser Phe Lys Gln Leu Ala Leu Leu Ala Leu Gly Ala Val Gln 1 5 10 15 gta cag gcg cag tgc gtc ggc ccg gct atc aat tcc gcg gct ctt aac 96 Val Gln Only Gln Cys Val Gly Pro Only Ile Asn Ser Only Only Leu Asn 20 25 30 ctc atc aag gag ttt gag gga tgg agg ccc aac att t gtgcgttccc 143 Leu Ile Lys Glu Phe Glu Gly Trp Arg Pro Asn Ile 35 40 ttctacgtta catcacccag ttccctgtt attcagacat tatttctata ttcctggcta 203 acactgtaaa g ac cgc gac ccc gtc ggc ctc ccc acc gtc gga tac ggc 252 Tyr Arg Asp Pro Val Gly Leu Pro Thr Val Gly Tyr Gly 45 50 55 cac ctc tgc cgc gac tcg agc tgc tct gac gtc cct tac cca att ccc 300 His Leu Cys Arg Asp Ser Ser Cys Ser Asp Val Pro Tyr Pro Ile Pro 60 65 70 ctg tcc gtt gcc aac ggc gag cgt ctt ctt cgg agc gac cta gcg 345 Leu Ser Val Ala Asn Gly Glu Arg Leu Leu Arg Ser Asp Leu Ala 75 80 85 gtgagtctat cccctttgca cttcataaaa cgtcgccttc tctgttgtca tttacctgg 405 acagcctccc cctatttctc tcttctatct cccgttctgc aagcttgacc 465 cctgaccaac catatccacc cagacctacc agaactgcat cacgatgcag acg gcc 521 Thr Ala 90 tcg tcc gtc gtc ctg aat gcg aac cag tac ggc gcc ctg gtc agc tgg 569 Ser Ser Val Val Leu Asn Ala Asn Gln Tyr Gly Ala Leu Val Ser Trp 95 100 105 gcc ttc aac gtc ggc tgc ggc gcc acc agc acg tcg act ctg atc cgc 617 Ala Phe Asn Val Gly Cys Gly Ala Thr Ser Thr Ser Thr Leu Ile Arg 110 115 120 cgc ctc aac gcc gga gag agc ccc aac acc gtc gct gcc cag gag ctg 665 Arg Leu Asn Ala Gly Glu Ser Pro Asn Thr Val Ala Ala Gln Glu Leu 125 130 135 cct cgc tgg aac aag gct ggc ggc cag gtc ctg ccc ggc ctg gtg cgc 713 Pro Arg Trp Asn Lys Ala Gly Gly Gln Val Leu Pro Gly Leu Val Arg 140 145 150 cgc cgt gct gcc gag gta gag ctg cat cgt act tcc acc agt gtc cgt 761 Arg Arg Ala Ala Glu Val Glu Leu His Arg Thr Ser Thr Ser Val Arg 155 160 165 170 gct ctg cct gct tgc tct tag 782 Ala Leu Pro Ala Cys Ser 175 <210> 2 <211> 176 <212> PRT <213> Acremonium alcalophilum <400> 2 Met Val Ser Phe Lys Gln Leu Ala Leu Leu Ala Leu Gly Ala Val Gln 1 5 10 15 Val Gln Ala Gln Cys Val Gly Pro Ala Ile Asn Ser Ala Ala Leu Asn 20 25 30 Leu Ile Lys Glu Phe Glu Gly Trp Arg Pro Asn Ile Tyr Arg Asp Pro 35 40 45 Val Gly Leu Pro Thr Val Gly Tyr Gly His Leu Cys Arg Asp Ser Ser 50 55 60 Cys Ser Asp Val Pro Tyr Pro Ile Pro Leu Ser Val Ala Asn Gly Glu 65 70 75 80 Arg Leu Leu Arg Ser Asp Leu Ala Thr Ala Ser Ser Val Val Leu Asn 85 90 95 Ala Asn Gln Tyr Gly Ala Leu Val Ser Trp Ala Phe Asn Val Gly Cys 100 105 110 Gly Ala Thr Ser Thr Ser Thr Leu Ile Arg Arg Leu Asn Ala Gly Glu 115 120 125 Ser Pro Asn Thr Val Ala Ala Gln Glu Leu Pro Arg Trp Asn Lys Ala 130 135 140 Gly Gly Gln Val Leu Pro Gly Leu Val Arg Arg Arg Ala Ala Glu Val 145 150 155 160 Glu Leu His Arg Thr Ser Thr Ser Val Arg Ala Leu Pro Ala Cys Ser 165 170 175 <210> 3 <211> 838 <212> DNA <213> Acremonium alcalophilum <220> <221> CDS <222> (1)..(147) <220> <221> signal peptide <222> (1)..(57) <220> <221> CDS <222> (302)..(835) <400> 3 atg aag ctt ctt ccc tcc ttg to ggc ctg gcc agt ctg gcg tcc ctc 48 Met Lys Leu Leu Pro Ser Leu Ile Gly Leu Ala Ser Leu Ala Ser Leu 1 5 10 15 gcc gtc gcc cgg atc ccc ggc ttt gac att tcg ggc tgg caa ccg acc 96 Ala Val Ala Arg Ile Pro Gly Phe Asp Ile Ser Gly Trp Gln Pro Thr 20 25 30 acc gac ttt gca agg gcg tat gct aat gga gat cgt ttc gtc tac atc 144 Thr Asp Phe Ala Arg Ala Tyr Ala Asn Gly Asp Arg Phe Val Tyr Ile 35 40 45 aag gtacgttca ccttgccacc aagttgcgaa cccgagacaca gactgtgacc 197 Lys gcctcctttg ccctggggca gctcacgcac ccagcat cccaccccc ggcccccc 257 gtaccaccgg aaagctaca tcaccccct accactgcta ccag gcc acc gag ggc 313 Only Thr Glu Gly 50 acc aca ttc aag agc tcc gca ttc agc cgc cag tac acc ggc gca acg 361 Thr Thr Phe Lys Ser Ala Phe Ser Arg Gln Tyr Thr Gly Ala Thr 55 60 65 caa aac gc ttc atc cgc ggc gcc tac cac ttc gcc cag ccc gcc gcg 409 Gln Asn Gly Phe Ile Arg Gly Ala Tyr His Phe Ala Gln Pro Ala Ala 70 75 80 85 tcc tcg ggc gcc gcg cag gcg aga tac ttc gcc agc aac ggc ggc ggc 457 Ser Ser Gly Ala Ala Gln Ala Arg Tyr Phe Ala Ser Asn Gly Gly Gly 90 95 100 tgg tcc aag gac ggc atc acc ctg ccc ggg gcg ctg gac atc gag tac 505 Trp Ser Lys Asp Gly Ile Thr Leu Pro Gly Ala Leu Asp Ile Glu Tyr 105 110 115 aac ccc aac ggc gcc acc tgc tac ggc ctc tcg caa tcg gcc atg gtg 553 Asn Pro Asn Gly Ala Thr Cys Tyr Gly Leu Ser Gln Ser Ala Met Val 120 125 130 aac tgg atc gag gac ttt gtc acc acc tac cac ggc atc acc tcc cgc 601 Asn Trp Ile Glu Asp Phe Val Thr Thr Tyr His Gly Ile Thr Ser Arg 135 140 145 tgg ccc gtc atc tac acc acc acc gac tgg tgg acc cag tgc acc ggc 649 Trp Pro Val Ile Tyr Thr Thr Thr Asp Trp Trp Thr Gln Cys Thr Gly 150 155 160 165 aac tcc aac cgc ttc gcg aac cgc tgc ccg ctg tgg atc gcc cgc tac 697 Asn Ser Asn Arg Phe Ala Asn Arg Cys Pro Leu Trp Ile Ala Arg Tyr 170 175 180 gcc agc tcc gtc ggc act ctg ccc aat ggc tgg ggc ttt tac acc ttc 745 Ala Ser Ser Val Gly Thr Leu Pro Asn Gly Trp Gly Phe Tyr Thr Phe 185 190 195 tgg cag tac aac gac aag tat cct cag ggc ggt gat tcg aac tgg ttc 793 Trp Gln Tyr Asn Asp Lys Tyr Pro Gln Gly Gly Asp Ser Asn Trp Phe 200 205 210 aac ggc gat gcg tcg cgt ctc agg gct ctc gct aac gga gac taa 838 Asn Gly Asp Ala Ser Arg Leu Arg Ala Leu Ala Asn Gly Asp 215 220 225 <210> 4 <211> 227 <212> PRT <213> Acremonium alcalophilum <400> 4 Met Lys Leu Leu Pro Ser Leu Ile Gly Leu Ala Ser Leu Ala Ser Leu 1 5 10 15 Ala Val Ala Arg Ile Pro Gly Phe Asp Ile Ser Gly Trp Gln Pro Thr 20 25 30 Thr Asp Phe Ala Arg Ala Tyr Ala Asn Gly Asp Arg Phe Val Tyr Ile 35 40 45 Lys Ala Thr Glu Gly Thr Thr Phe Lys Ser Ser Ala Phe Ser Arg Gln 50 55 60 Tyr Thr Gly Ala Thr Gln Asn Gly Phe Ile Arg Gly Ala Tyr His Phe 65 70 75 80 Ala Gln Pro Ala Ala Ser Ser Gly Ala Ala Gln Ala Arg Tyr Phe Ala 85 90 95 Ser Asn Gly Gly Gly Trp Ser Lys Asp Gly Ile Thr Leu Pro Gly Ala 100 105 110 Leu Asp Ile Glu Tyr Asn Pro Asn Gly Ala Thr Cys Tyr Gly Leu Ser 115 120 125 Gln Ser Ala Met Val Asn Trp Ile Glu Asp Phe Val Thr Thr Tyr His 130 135 140 Gly Ile Thr Ser Arg Trp Pro Val Ile Tyr Thr Thr Thr Asp Trp Trp 145 150 155 160 Thr Gln Cys Thr Gly Asn Ser Asn Arg Phe Ala Asn Arg Cys Pro Leu 165 170 175 Trp Ile Ala Arg Tyr Ala Ser Ser Val Gly Thr Leu Pro Asn Gly Trp 180 185 190 Gly Phe Tyr Thr Phe Trp Gln Tyr Asn Asp Lys Tyr Pro Gln Gly Gly 195 200 205 Asp Ser Asn Trp Phe Asn Gly Asp Ala Ser Arg Leu Arg Ala Leu Ala 210 215 220 Asn Gly Asp 225 <210> 5 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Forward primer F-P242M9 <400> 5 acacaactgg ggatccacca tgaagcttct tccctccttg a 41 <210> 6 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Reverse primer R-P242M9 <400> 6 agatctcgag aagcttatta gtctccgtta gcgagagc 38 <210> 7 <211> 747 <212> DNA <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> CDS <222> (1)..(744) <220> <221> signal peptide <222> (1)..(69) <220> <221> Mature peptides <222> (121)..(744) <400> 7 atg aag ttc ttc acc acc atc ctc agc acc gcc agc ctt gtt gct gct 48 Met Lys Phe Phe Thr Thr Ile Leu Ser Thr Ala Ser Leu Val Ala Ala ‑40 ‑35 ‑30 ‑25 ctc ccc gcc gct gtt gac tcg aac cat acc ccg gcc gct cct gaa ctt 96 Leu Pro Ala Ala Val Asp Ser Asn His Thr Pro Ala Ala Pro Glu Leu ‑20 ‑15 ‑10 gtt gcc cgg agt cct att cgt cga cgc att ccc gga ttc gat atc tcg 144 Val Ala Arg Ser Pro Ile Arg Arg Arg Ile Pro Gly Phe Asp Ile Ser ‑5 ‑1 1 5 gga tgg cag ccg acg acg gac ttc gca agg gcg tac gca aac gga gac 192 Gly Trp Gln Pro Thr Thr Asp Phe Ala Arg Ala Tyr Ala Asn Gly Asp 10 15 20 cga ttc gtg tac atc aag gca aca gag gga aca aca ttc aaa tcg tcg 240 Arg Phe Val Tyr Ile Lys Ala Thr Glu Gly Thr Thr Phe Lys Ser Ser 25 30 35 40 gca ttc tcc agg cag tac acc gga gca acc cag aac ggc ttc atc cga 288 Ala Phe Ser Arg Gln Tyr Thr Gly Ala Thr Gln Asn Gly Phe Ile Arg 45 50 55 gga gcc tac cac ttc gcc cag cct gca gcc tcc tcg gga gca gcc cag 336 Gly Ala Tyr His Phe Ala Gln Pro Ala Ala Ser Ser Gly Ala Ala Gln 60 65 70 gca agg tac ttc gca tcg aac ggt ggc ggt tgg tcc aag gac ggt atc 384 Ala Arg Tyr Phe Ala Ser Asn Gly Gly Gly Trp Ser Lys Asp Gly Ile 75 80 85 acc ctc cct ggt gcc ttg gat atc gag tac aac ccc aac gga gca aca 432 Thr Leu Pro Gly Ala Leu Asp Ile Glu Tyr Asn Pro Asn Gly Ala Thr 90 95 100 tgt tat ggt ctc tcg cag tcg gcg atg gtg aac tgg att gag gac ttc 480 Cys Tyr Gly Leu Ser Gln Ser Ala Met Val Asn Trp Ile Glu Asp Phe 105 110 115 120 gtg aca acc tac cac ggc atc acc tcg agg tgg cct gtg atc tac acc 528 Val Thr Thr Tyr His Gly Ile Thr Ser Arg Trp Pro Val Ile Tyr Thr 125 130 135 aca acc gac tgg tgg acg cag tgt acc ggc aac tcc aac cga ttc gcg 576 Thr Thr Asp Trp Trp Thr Gln Cys Thr Gly Asn Ser Asn Arg Phe Ala 140 145 150 aac agg tgt ccg ctc tgg atc gcg agg tat gcc tcc tcc gtc ggc acc 624 Asn Arg Cys Pro Leu Trp Ile Ala Arg Tyr Ala Ser Ser Val Gly Thr 155 160 165 ctc ccg aac gga tgg ggc ttc tat acc ttc tgg cag tac aac gat aag 672 Leu Pro Asn Gly Trp Gly Phe Tyr Thr Phe Trp Gln Tyr Asn Asp Lys 170 175 180 tac ccc cag gga gga gat tcc aac tgg ttc aac ggt gat gca tcg agg 720 Tyr Pro Gln Gly Gly Asp Ser Asn Trp Phe Asn Gly Asp Ala Ser Arg 185 190 195 200 ctc agg gca ttg gcg aac ggc gat tag 747 Leu Arg Ala Leu Ala Asn Gly Asp 205 <210> 8 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 8 Met Lys Phe Phe Thr Thr Ile Leu Ser Thr Ala Ser Leu Val Ala Ala ‑40 ‑35 ‑30 ‑25 Leu Pro Ala Ala Val Asp Ser Asn His Thr Pro Ala Ala Pro Glu Leu ‑20 ‑15 ‑10 Val Ala Arg Ser Pro Ile Arg Arg Arg Ile Pro Gly Phe Asp Ile Ser ‑5 ‑1 1 5 Gly Trp Gln Pro Thr Thr Asp Phe Ala Arg Ala Tyr Ala Asn Gly Asp 10 15 20 Arg Phe Val Tyr Ile Lys Ala Thr Glu Gly Thr Thr Phe Lys Ser Ser 25 30 35 40 Ala Phe Ser Arg Gln Tyr Thr Gly Ala Thr Gln Asn Gly Phe Ile Arg 45 50 55 Gly Ala Tyr His Phe Ala Gln Pro Ala Ala Ser Ser Gly Ala Ala Gln 60 65 70 Ala Arg Tyr Phe Ala Ser Asn Gly Gly Gly Trp Ser Lys Asp Gly Ile 75 80 85 Thr Leu Pro Gly Ala Leu Asp Ile Glu Tyr Asn Pro Asn Gly Ala Thr 90 95 100 Cys Tyr Gly Leu Ser Gln Ser Ala Met Val Asn Trp Ile Glu Asp Phe 105 110 115 120 Val Thr Thr Tyr His Gly Ile Thr Ser Arg Trp Pro Val Ile Tyr Thr 125 130 135 Thr Thr Asp Trp Trp Thr Gln Cys Thr Gly Asn Ser Asn Arg Phe Ala 140 145 150 Asn Arg Cys Pro Leu Trp Ile Ala Arg Tyr Ala Ser Ser Val Gly Thr 155 160 165 Leu Pro Asn Gly Trp Gly Phe Tyr Thr Phe Trp Gln Tyr Asn Asp Lys 170 175 180 Tyr Pro Gln Gly Gly Asp Ser Asn Trp Phe Asn Gly Asp Ala Ser Arg 185 190 195 200 Leu Arg Ala Leu Ala Asn Gly Asp 205 <210> 9 <211> 46 <212> DNA <213> Artificial sequence <220> <223> Forward primer BamHI <400> 9 tacacaactg gggatccagc ggccgcacca tgaagttctt caccac 46 <210> 10 <211> 37 <212> DNA <213> Artificial sequence <220> <223> Reverse primer EcoRI <400> 10 atacttgtcc gaattcctaa tcgccgttcg ccaatgc 37

Claims

1. An isolated polypeptide having lysozyme activity, the polypeptide being selected from the group consisting of the following items: (a) A polypeptide having at least 99% or 100% sequence identity with the mature polypeptide of SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 8, wherein the polypeptide is obtained from Acremonium alcalophilum ;as well as (b) A polypeptide having 100% sequence identity with the mature polypeptide of SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO:

8. The mature polypeptide of SEQ ID NO: 4 is amino acids 20 to 227 of SEQ ID NO: 4, and the mature polypeptide of SEQ ID NO: 8 is amino acids 1 to 208 of SEQ ID NO:

8.

2. The polypeptide of claim 1, wherein the polypeptide having lysozyme activity is obtained from... Acremonium alcalophilum .

3. The polypeptide of claim 1, wherein the polypeptide having lysozyme activity decomposes the cell wall of Gram-positive bacteria.

4. The polypeptide of claim 3, wherein the Gram-positive bacteria is selected from Staphylococcus aureus (…). Staphylococcus carnosus ), Microbacterium laurentii ( Exiguobacterium undae Clostridium perfringens (Clostridium perfringens) and Micrococcus tarda ( Micrococcus luteus) .

5. The polypeptide of claim 1, wherein the polypeptide having lysozyme activity exhibits activity against the Gram-negative bacterium *Escherichia coli*.

6. The polypeptide of claim 1, wherein the polypeptide is selected from mature polypeptides including SEQ ID NO: 4 or SEQ ID NO:

8.

7. The polypeptide of claim 1, wherein the polypeptide is selected from the mature polypeptides of SEQ ID NO: 4 or SEQ ID NO:

8.

8. The polypeptide of any one of claims 1-7, wherein the polypeptide is a variant of the mature polypeptide of SEQ ID NO: 4 or SEQ ID NO: 8, the variant comprising substitution, deletion, and / or insertion at one or two positions.

9. The polypeptide of claim 8, wherein the mature polypeptide of SEQ ID NO: 8 is selected from variants of V133M, W10H, S39D, F178I, Y28M, D95A, G92P, D190A, A93M or T142N.

10. A composition comprising the polypeptide as described in any one of claims 1-9.

11. The composition of claim 10, wherein it is a detergent composition.

12. The composition of claim 11, used on clothing, tableware, or for cleaning hard surfaces.

13. The composition of any one of claims 10 to 12, wherein the composition comprises one or more additional enzymes selected from the group consisting of proteases, amylases, lipases, keratins, cellulases, endoglucans, xyloglucans, pectins, pectin lyases, xanthan gums, peroxidases, halogenated peroxidases, catalases, and mannanases, or any mixture thereof.

14. The composition of any one of claims 10 to 12, comprising one or more components selected from the group consisting of surfactants, detergent builders, water-soluble solvents, bleaching systems, polymers, fabric toners, accessories, dispersants, dye transfer inhibitors, optical brighteners, soil-releasing polymers, and anti-redeposition agents.

15. The composition of claim 10 is a bacterial genomic DNA extraction composition.

16. The composition of claim 15, wherein the composition further comprises one or more additional lysozymes.

17. An animal feed composition comprising a polypeptide as described in any one of claims 1-9.

18. The animal feed composition of claim 17, further comprising one or more of the following: amylase; phytase; xylanase; galactanase; α-galactosidase; protease, phospholipase, β-glucanase, or any mixture thereof.

19. An animal feed additive, comprising At least one polypeptide as described in any one of claims 1-9; and At least one fat-soluble vitamin, and / or At least one water-soluble vitamin, and / or At least one trace mineral.

20. The animal feed additive of claim 19, further comprising one or more of the following: amylase; phytase; xylanase; galactanase; α-galactosidase; protease, phospholipase, β-glucanase, or any mixture thereof.

21. Use of the polypeptide according to any one of claims 1-9 in the preparation of a composition for inhibiting biofilm formation.

22. Use of the polypeptide according to any one of claims 1-9 in the preparation of dental compositions for oral care.

23. Use of the polypeptide according to any one of claims 1-9 in the preparation of detergent compositions.

24. Use of the polypeptide according to any one of claims 1-9 in the preparation of animal feed.

25. Use of the polypeptide according to any one of claims 1-9 in the preparation of a composition for decomposing bacterial cell walls.

26. The use as described in claim 25, wherein the polypeptide is used in a process for isolating DNA from bacteria.

27. A method for isolating DNA from bacteria, comprising: (a) Treating the isolated bacteria with the polypeptide as described in any one of claims 1-9; and (b) Recover the bacterial DNA.

28. An isolated polynucleotide encoding a polypeptide as described in any one of claims 1-9.

29. A nucleic acid construct or expression vector comprising a polynucleotide of claim 28 operably linked to one or more control sequences, the one or more control sequences directing the production of a polypeptide encoded by the polynucleotide in an expression host.

30. A recombinant host cell comprising a polynucleotide of claim 28 operably linked to one or more control sequences, the one or more control sequences directing the production of a polypeptide encoded by the polynucleotide.

31. A method for producing a polypeptide as described in any one of claims 1-9, the method comprising: (a) Culturing a cell type that produces the polypeptide in its wild-type form under conditions favorable for its production, wherein said cell type is... Acremonium alcalophilum Cells; and (b) The polypeptide was recovered.

32. A method for producing a polypeptide with lysozyme activity, the method comprising: (a) Culturing the host cells as described in claim 30 under conditions conducive to the production of the polypeptide; and (b) The polypeptide was recovered.

33. A transgenic plant part or plant cell transformed with a polynucleotide encoding a polypeptide as described in any one of claims 1-9, wherein the transgenic plant part or plant cell does not belong to a plant variety.

34. A method for producing a polypeptide with lysozyme activity, the method comprising: (a) Culturing the transgenic plant or plant cells as described in claim 33 under conditions conducive to the production of the polypeptide; and (b) The polypeptide was recovered.

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