Ruminant feed composition comprising muramidase

文档序号：1077587 发布日期：2020-10-16 浏览：14次中文

阅读说明：本技术 包含胞壁质酶的反刍动物饲料组合物 (Ruminant feed composition comprising muramidase ) 是由 A·C·斯托姆 L·V·柯福德于 2019-03-05 设计创作，主要内容包括：本发明涉及使用包含具有胞壁质酶活性的多肽的反刍动物饲料来改善反刍动物消化率的方法。(The present invention relates to methods of improving ruminant digestibility using ruminant feed comprising polypeptides having muramidase activity.)

1. A ruminant feed composition, such as a ruminant feed, a ruminant feed supplement, or a ruminant feed supplement, comprising one or more muramidase enzymes, wherein the muramidase enzyme is in an amount sufficient to be administered at a level of 1 to 200mg enzyme protein per kg of ruminant feed.

2. The ruminant feed composition of claim 1, wherein the ruminant is selected from the group consisting of: cattle, dairy cows and beef cattle.

3. The ruminant feed composition of any of claims 1-2, wherein the muramidase comprises one or more domains from a Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and a novel MUR polypeptide having muramidase activity.

4. A method of improving Energy Corrected Milk (ECM) production in a ruminant, the method comprising administering to the ruminant feed composition of any of claims 1-3.

5. A method of increasing dry matter digestibility (DMd) of a ruminant feed, a ruminant feed supplement, or a ruminant feed additive, the method comprising the steps of: a) providing at least one muramidase; b) providing a ruminant feed, a ruminant feed supplement, or a ruminant feed supplement suitable for a ruminant; c) applying a muramidase to a ruminant feed, a ruminant feed supplement, or a ruminant feed additive to form a ruminant feed composition; and d) feeding the ruminant feed composition, thereby achieving an increase in digestibility of the dry matter.

6. The method of claim 5, wherein the production of Volatile Fatty Acids (VFA) in the rumen is increased as compared to the production of VFA in the rumen of a ruminant that is not fed muramidase.

7. The method of any one of claims 5 to 6, wherein propionate production in the rumen is increased as compared to propionate production in the rumen of a ruminant that is not fed muramidase.

8. The method of any one of claims 5-7, wherein acetate production in the rumen is increased as compared to acetate production in the rumen of a ruminant that is not fed muramidase.

9. A method according to any one of claims 5 to 8 wherein muramidase is administered at a level of from 1 to 200mg enzyme protein/kg dry matter of ruminant feed.

10. The method of any one of claims 5 to 9, wherein Energy Corrected Milk (ECM) production by ruminants is improved by at least 1.0%, preferably at least 1.5%, more preferably at least 2.0% after administration compared to a control.

11. The method of any one of claims 5 to 10, wherein the muramidase is of microbial origin.

12. The method of any one of claims 5 to 11, wherein the muramidase comprises one or more domains from a Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and a novel MUR polypeptide having muramidase activity.

13. The method of any one of claims 5 to 12, wherein the control is a ruminant feed composition that does not comprise muramidase.

14. The method of any one of claims 5 to 12, wherein the control is a ruminant feed composition that does not comprise GH24 muramidase, GH25 muramidase, or a novel MUR polypeptide having muramidase activity.

15. The method of any one of claims 5-14, wherein the control is monensin.

Technical Field

The present invention relates to methods of improving ruminant digestibility using ruminant feed comprising polypeptides having muramidase activity.

Background

Muramidase is lysozyme, also known as N-acetylmuramyl glycan hydrolase, an O-glycosyl hydrolase enzyme produced as a defense mechanism against bacteria by many organisms. The enzyme causes hydrolysis of the bacterial cell wall by cleaving the glycosidic bond of the peptidoglycan; peptidoglycan is an important structural molecule in bacteria. Bacterial cell lysis is caused by unbalanced osmotic pressure after the bacterial cell wall is weakened by muramidase action.

Muramidase enzymes occur naturally in many organisms, such as viruses, plants, insects, birds, reptiles, and mammals. In mammals, muramidase has been isolated from nasal secretions, saliva, tears, intestinal contents, urine, and milk. This enzyme cleaves the glycosidic bond between carbon number 1 of N-acetylmuramic acid and carbon number 4 of N-acetyl-D-glucosamine. In vivo, these two carbohydrates polymerize to form the cell wall polysaccharides of many microorganisms.

Muramidases have been classified into seven different Glycoside Hydrolases (GH) families (CAZy, www.cazy.org): GH18, GH19, egg white lysozyme (GH22), goose egg white lysozyme (GH23), bacteriophage T4 muramidase (GH24), Sphingomonas (Sphingomonas) flagellin (GH73), and coleoptera (Chalaropsis) muramidase (GH 25). Muramidases from families GH23 and GH24 are known mainly from bacteriophages and have only recently been identified in fungi. Muramidase family GH25 has been found to be structurally unrelated to other muramidase families. Furthermore, another class of polypeptides having muramidase activity has been identified in PCT/CN2017/084074, such muramidase being herein referred to as novel MUR polypeptides having muramidase activity, wherein muramidase activity is defined in the definitions section and representative muramidase is listed in the sequence listing.

Muramidase has traditionally been extracted from egg white due to its natural abundance and is called egg white muramidase. Until recently, egg white lysozyme was the only muramidase that was studied for animal feed. Muramidase extracted from egg white is the major product available in the commercial market, but does not cleave N, 6-O-diacetylmuramic acid in the cell wall of e.g. Staphylococcus aureus and is therefore especially unable to lyse this important human pathogen (Masschalck B, Deckers D, Michiels CW (2002), "Lytic and non-Lytic mechanism of inactivation of gram-positive bacteria by muramidase under atmospheric and high hydrostatic pressures", J Food Prot. [ J. Proc. J. Proc. 65(12): 1916-23).

WO 2000/21381 discloses a composition comprising at least two antimicrobial enzymes and a polyunsaturated fatty acid, wherein one of these antimicrobial enzymes is GH22 muramidase from egg white. GB 2379166 discloses a composition comprising a compound that disrupts the peptidoglycan layer of bacteria and a compound that disrupts the phospholipid layer of bacteria, wherein the peptidoglycan disrupting compound is GH22 muramidase from egg white.

WO 2004/026334 discloses an antimicrobial composition for inhibiting the growth of intestinal pathogens in the gut of livestock comprising (a) a cell wall lysing substance or salt thereof, (b) an antimicrobial substance, (c) a chelating agent and (d) an antibiotic, wherein the cell wall lysing substance or salt thereof is GH22 muramidase from egg white.

The demand for products from ruminants, such as dairy products and meat, is increasing, which results in an increased demand for ruminant feed. The invention aims to improve the efficiency of nutrient utilization in feed so as to reduce the influence of dairy products and beef production on the environment.

Disclosure of Invention

The present invention provides a ruminant feed composition, such as a ruminant feed, a ruminant feed supplement, or a ruminant feed supplement, comprising one or more muramidase enzymes in an amount sufficient to be administered at a level of 1 to 200mg enzyme protein per kg ruminant feed.

Further provided is a method of increasing the dry matter digestibility of a ruminant feed, a ruminant feed supplement or a ruminant feed additive, the method comprising the steps of: a) providing at least one muramidase; b) providing a ruminant feed, a ruminant feed supplement, or a ruminant feed supplement suitable for a ruminant; c) applying a muramidase to a ruminant feed, a ruminant feed supplement, or a ruminant feed additive to form a ruminant feed composition; and d) administering the ruminant feed composition to a ruminant, thereby achieving an increase in digestibility of the dry matter.

In one embodiment of the invention, the production of Volatile Fatty Acids (VFAs) in the rumen is increased compared to the production of VFAs in the rumen of a ruminant that is not fed muramidase. In another embodiment, the production of propionate in the rumen is increased as compared to the production of propionate in the rumen of a ruminant that is not fed muramidase, and/or the production of acetate in the rumen is increased as compared to the production of acetate in the rumen of a ruminant that is not fed muramidase.

Muramidase enzymes for use in the present invention may be of microbial origin. In one embodiment, the muramidase comprises one or more domains from a Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and novel MUR polypeptides having muramidase activity.

Drawings

FIG. 1 shows the effect of 9 muramidases (A: SEQ ID NO:3, B: SEQ ID NO:6, C: SEQ ID NO:9, D: SEQ ID NO:12, E: SEQ ID NO:15, F: SEQ ID NO:18, G: SEQ ID NO:21, H: SEQ ID NO:24 and I: SEQ ID NO:27) from 3 different Glycoside Hydrolase (GH) families (GH24, GH25 and novel MUR polypeptides with muramidase activity) and a positive control (PC, containing monensin) on the relative improvement of dry matter digestibility expressed as percentage improvement over the control after 48H of fermentation in rumen fluid and buffer solution.

FIG. 2 shows the relative difference in the rate of dry matter digestion in the rumen after 12h fermentation compared to the negative control due to the effect of increasing doses of muramidase (NC is negative control, PC is positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:18, E is SEQ ID NO: 9).

FIG. 3: the relative difference in the amount of acetate produced by the rumen after 12h of fermentation compared to the negative control due to the effect of increasing doses of muramidase is shown (NC is negative control, PC is positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:18, E is SEQ ID NO: 9).

FIG. 4: the relative difference in the production of rumen propionate after 12h fermentation due to the effect of increasing doses of muramidase compared to the negative control is shown (PC is a positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:18, E is SEQ ID NO: 9).

FIG. 5: the relative difference in the production of ruminal butyrate after 12h of fermentation compared to the negative control due to the effect of increasing doses of muramidase is shown (PC is the positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:18, E is SEQ ID NO: 9).

FIG. 6: the relative difference in total ruminal VFA production after 12h fermentation compared to the negative control due to the effect of increasing doses of muramidase is shown (PC is a positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:18, E is SEQ ID NO: 9).

FIG. 7: the relative difference in carbon in total ruminal VFA production after 12h fermentation compared to the negative control due to the effect of increasing doses of muramidase is shown (PC is a positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:18, E is SEQ ID NO: 9).

FIG. 8: the relative difference in the tumour gastric dry matter digestibility after 12H of fermentation due to the influence of muramidase and monensin supplementation is shown compared to the negative control (NC is negative control, PC is a positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:29, E is SEQ ID NO:30, F is SEQ ID NO:31, G is SEQ ID NO:32, H is SEQ ID NO:33, I is SEQ ID NO:34, J is SEQ ID NO:35, K is SEQ ID NO:36, L is SEQ ID NO:36, M is SEQ ID NO: 37).

FIG. 9: the relative difference in the rumen propionate concentration after 12H fermentation due to the influence of muramidase and monensin supplementation is shown compared to the negative control (NC is negative control, PC is positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:29, E is SEQ ID NO:30, F is SEQ ID NO:31, G is SEQ ID NO:32, H is SEQ ID NO:33, I is SEQ ID NO:34, J is SEQ ID NO:35, K is SEQ ID NO:36, L is SEQ ID NO:36, M is SEQ ID NO: 37).

FIG. 10 shows the relative difference in the concentration of tumouricbutyrate after 12H fermentation compared to the negative control due to the influence of muramidase and monensin supplementation (NC is negative control, PC is positive control containing monensin, A is SEQ ID NO:28, B is SEQ ID NO:21, C is SEQ ID NO:12, D is SEQ ID NO:29, E is SEQ ID NO:30, F is SEQ ID NO:31, G is SEQ ID NO:32, H is SEQ ID NO:33, I is SEQ ID NO:34, J is SEQ ID NO:35, K is SEQ ID NO:36, L is SEQ ID NO:36, M is SEQ ID NO: 37).

FIG. 11: showing the relative difference in total rumen VFA production after 12H fermentation compared to the negative control due to the influence of muramidase supplementation (NC is negative control, PC is positive control containing monensin, A is SEQ ID NO:38, B is SEQ ID NO:39, C is SEQ ID NO:40, D is SEQ ID NO:41, E is SEQ ID NO:42, F is SEQ ID NO:43, G is SEQ ID NO:44, H is SEQ ID NO:45, I is SEQ ID NO:46, J is SEQ ID NO:47, K is SEQ ID NO:48, L is SEQ ID NO:49, M is SEQ ID NO:50, N is SEQ ID NO:51, O is SEQ ID NO:52, S is SEQ ID NO:55, T is SEQ ID NO:56, U is SEQ ID NO:57, V is SEQ ID NO:58, W is SEQ ID NO:59, y is SEQ ID NO:53 and Z is SEQ ID NO: 54).

FIG. 12: showing the relative difference in carbon in the rumen VFA production after 12H fermentation compared to the negative control due to the influence of muramidase supplementation (NC is negative control, PC is a positive control containing monensin, A is SEQ ID NO:38, B is SEQ ID NO:39, C is SEQ ID NO:40, D is SEQ ID NO:41, E is SEQ ID NO:42, F is SEQ ID NO:43, G is SEQ ID NO:44, H is SEQ ID NO:45, I is SEQ ID NO:46, J is SEQ ID NO:47, K is SEQ ID NO:48, L is SEQ ID NO:49, M is SEQ ID NO:50, N is SEQ ID NO:51, O is SEQ ID NO:52, S is SEQ ID NO:55, T is SEQ ID NO:56, U is SEQ ID NO:57, V is SEQ ID NO:58, W is SEQ ID NO:59, y is SEQ ID NO:53 and Z is SEQ ID NO: 54).

FIG. 13: showing the relative difference in the amount of acetate produced by the rumen after 12H of fermentation compared to the negative control due to the influence of muramidase supplementation (NC is negative control, PC is a positive control containing monensin, A is SEQ ID NO:38, B is SEQ ID NO:39, C is SEQ ID NO:40, D is SEQ ID NO:41, E is SEQ ID NO:42, F is SEQ ID NO:43, G is SEQ ID NO:44, H is SEQ ID NO:45, I is SEQ ID NO:46, J is SEQ ID NO:47, K is SEQ ID NO:48, L is SEQ ID NO:49, M is SEQ ID NO:50, N is SEQ ID NO:51, O is SEQ ID NO:52, S is SEQ ID NO:55, T is SEQ ID NO:56, U is SEQ ID NO:57, V is SEQ ID NO:58, W is SEQ ID NO:59, y is SEQ ID NO:53 and Z is SEQ ID NO: 54).

FIG. 14: showing the relative difference in the production of rumen propionate after 12H of fermentation compared to the negative control due to the influence of muramidase supplementation (NC is negative control, PC is a positive control containing monensin, A is SEQ ID NO:38, B is SEQ ID NO:39, C is SEQ ID NO:40, D is SEQ ID NO:41, E is SEQ ID NO:42, F is SEQ ID NO:43, G is SEQ ID NO:44, H is SEQ ID NO:45, I is SEQ ID NO:46, J is SEQ ID NO:47, K is SEQ ID NO:48, L is SEQ ID NO:49, M is SEQ ID NO:50, N is SEQ ID NO:51, O is SEQ ID NO:52, S is SEQ ID NO:55, T is SEQ ID NO:56, U is SEQ ID NO:57, V is SEQ ID NO:58, W is SEQ ID NO:59, y is SEQ ID NO:53 and Z is SEQ ID NO: 54).

Brief description of the sequences

SEQ ID NO 1 is the cDNA sequence of a muramidase polypeptide isolated from Trichoderma pseudokoningii (Trichoderma koningiopsis).

SEQ ID NO 2 is the amino acid sequence as deduced from SEQ ID NO 1.

SEQ ID NO 3 is the amino acid sequence of a mature muramidase polypeptide from Trichoderma pseudokoningii.

SEQ ID NO:4 is the cDNA sequence of a muramidase polypeptide isolated from Thielavia terrestris.

SEQ ID NO 5 is the amino acid sequence as deduced from SEQ ID NO 4.

SEQ ID NO 6 is the amino acid sequence of a mature muramidase polypeptide from Thielavia terrestris.

SEQ ID NO:7 is a cDNA sequence of a muramidase polypeptide isolated from Tilletia indica (Tilletia indica).

SEQ ID NO 8 is the amino acid sequence as deduced from SEQ ID NO 7.

SEQ ID NO 9 is the amino acid sequence of a mature muramidase polypeptide from Tilletia indica.

SEQ ID NO 10 is a cDNA sequence of a muramidase polypeptide isolated from Acremonium alcalophilum.

SEQ ID NO 11 is the amino acid sequence as deduced from SEQ ID NO 10.

SEQ ID NO 12 is the amino acid sequence of a mature muramidase polypeptide from Acremonium alkalophilum.

SEQ ID NO 13 is a cDNA sequence of a muramidase polypeptide isolated from M.galbana (Cladorhinum bulbillasum).

SEQ ID NO 14 is the amino acid sequence as deduced from SEQ ID NO 13.

SEQ ID NO 15 is the amino acid sequence of the mature muramidase polypeptide from T.galamensis.

SEQ ID NO 16 is the cDNA sequence of muramidase polypeptide isolated from Methylocystis equinovi (oncogene equina).

SEQ ID NO 17 is the amino acid sequence as deduced from SEQ ID NO 16.

18 is the amino acid sequence of a mature muramidase polypeptide from A. marreum.

SEQ ID NO:19 is a cDNA sequence of a muramidase polypeptide isolated from Trichophaea fusca (Trichophaea saccharoata).

SEQ ID NO 20 is the amino acid sequence as deduced from SEQ ID NO 19.

SEQ ID NO 21 is the amino acid sequence of a mature muramidase polypeptide from Lachnospora lutescens.

SEQ ID NO:22 is the cDNA sequence of the muramidase polypeptide isolated from Pleurotus ostreatus.

SEQ ID NO 23 is the amino acid sequence as deduced from SEQ ID NO 22.

SEQ ID NO 24 is the amino acid sequence of the mature muramidase polypeptide from Pleurotus ostreatus.

SEQ ID NO:25 is the cDNA sequence of a muramidase polypeptide isolated from Cladosporium sp-9768.

SEQ ID NO 26 is the amino acid sequence as deduced from SEQ ID NO 25.

SEQ ID NO 27 is the amino acid sequence of the mature muramidase polypeptide from Cladosporium species-9768.

SEQ ID NO 28 is the amino acid sequence of the mature muramidase polypeptide from Chaetomium thermophilum var.

SEQ ID NO 29 is the amino acid sequence of a mature muramidase polypeptide from Acremonium alkalophilum.

SEQ ID NO 30 is the amino acid sequence of the mature muramidase polypeptide from Coprinopsis cinerea (Coprinopsis cinerea okayama).

SEQ ID NO 31 is the amino acid sequence of the mature muramidase polypeptide from Rasamsonia brevisipitata.

SEQ ID NO 32 is the amino acid sequence of a mature muramidase polypeptide from Acremonium alkalophilum.

SEQ ID NO 33 is the amino acid sequence of the mature muramidase polypeptide from a spotted well base shell (Poronia punctata).

SEQ ID NO 34 is the amino acid sequence of the mature muramidase polypeptide from Aspergillus elbow (Aspergillus deflectous).

SEQ ID NO 35 is the amino acid sequence of the mature muramidase polypeptide from the punctum locus shell.

SEQ ID NO 36 is the amino acid sequence of a mature muramidase polypeptide from a Paecilomyces species (Paecilomyces sp).

SEQ ID NO 37 is the amino acid sequence of a mature muramidase polypeptide from a species of Endosphaera (Hamigera sp).

SEQ ID NO 38 is the amino acid sequence of a mature muramidase polypeptide from Penicillium citrinum.

SEQ ID NO:39 is the amino acid sequence of a mature muramidase polypeptide from Pyrenophora cubensis (Pyronema domesticum).

SEQ ID NO 40 is the amino acid sequence of a mature muramidase polypeptide from a Thielavia sp.

SEQ ID NO 41 is the amino acid sequence of a mature muramidase polypeptide from Chaetomium sp.

SEQ ID NO 42 is the amino acid sequence of the mature muramidase polypeptide from Metarhizium anisopliae (Metarhizium iadini).

SEQ ID NO 43 is the amino acid sequence of the mature muramidase polypeptide from Aspergillus elbow.

SEQ ID NO 44 is the amino acid sequence of the mature muramidase polypeptide from Sporormia fimetaria.

SEQ ID NO:45 is the amino acid sequence of the mature muramidase polypeptide from Verticillium culmorum (Lecanicillium psalliotae).

SEQ ID NO 46 is the amino acid sequence of a mature muramidase polypeptide from Helminthosporium crassirhizoides (Lecanicillium psalliotae).

SEQ ID NO:47 is the amino acid sequence of the mature muramidase polypeptide from a species of the family Clavicipitaceae (Clavicipitaceae sp) -70249.

SEQ ID NO 48 is the amino acid sequence of a mature muramidase polypeptide from Thielavia terrestris.

SEQ ID NO:49 is the amino acid sequence of a mature muramidase polypeptide from Westerdykella (Westerdykella).

SEQ ID NO 50 is the amino acid sequence of a mature muramidase polypeptide from M.marmoreus.

SEQ ID NO:51 is the amino acid sequence of the mature muramidase polypeptide from Ovatospora brasiliensis.

SEQ ID NO 52 is the amino acid sequence of a mature muramidase polypeptide from purple-violet spore (Purpureocillium lilacinum).

SEQ ID NO 53 is the amino acid sequence of the mature muramidase polypeptide from Ovatospora brasiliensis.

SEQ ID NO:54 is the amino acid sequence of the mature muramidase polypeptide from Penicillium wellingtonnense (Penicillium wellingnense).

SEQ ID NO:55 is the amino acid sequence of the mature muramidase polypeptide from an Aspergillus species.

SEQ ID NO 56 is the amino acid sequence of a mature muramidase polypeptide from Chaetomium sp.

SEQ ID NO:57 is the amino acid sequence of a mature muramidase polypeptide from a Podospora species (Zopfiella sp).

SEQ ID NO:58 is the amino acid sequence of a mature muramidase polypeptide from Acremonium exiguum.

SEQ ID NO 59 is the amino acid sequence of a mature muramidase polypeptide from Chaetomium species.

Definition of

Acetate salt: acetate is used herein interchangeably with the term "acetic acid" and is one of the Volatile Fatty Acids (VFAs) produced in the rumen. It is a precursor of mammalian milk fat synthesis and is also used in muscle energy metabolism and body fat synthesis. The amount of acetate in the rumen is a measure of the rumen fermentation of ingested feed, and thus an increase in rumen acetate is an indication of an increase in energy supply in ruminants.

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. The antimicrobial activity may be, for example, bactericidal, intended to kill bacteria, or bacteriostatic, intended to prevent the growth of bacteria. The antimicrobial activity can include catalyzing hydrolysis of 1,4- β -linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan and between N-acetyl-D-glucosamine residues in the chitosan. Antimicrobial activity may also include muramidase binding to the surface of the microorganism and inhibiting its growth. Antimicrobial action may also include activation of bacterial autolysins by inhibition or reduction of bacterial toxins and by the action of bacteriophages using muramidases of the invention as immunostimulants.

Beef production: the term "beef production" is defined herein as the production of beef from cattle used to raise for meat production. Beef production can be measured, for example, by feed intake, daily feed intake, weight gain, average daily gain, carcass dressing present, carcass composition, and carcass score.

And (3) butyrate: butyrate is used herein interchangeably with the term "butyric acid" and is one of the Volatile Fatty Acids (VFAs) produced in the rumen. It is a precursor of beta-OH-butyrate for mammalian milk fat synthesis and also for muscle energy metabolism and body fat synthesis. The amount of butyrate in the rumen is a measure of the rumen fermentation of ingested feed, and thus an increase in butyrate is an indication of an increase in energy supply in ruminants.

Concentrating the mixture: the term "concentrate" means a feed with a high and rapid dry matter digestibility (DMd). Typically, the concentrate is a feed having a relatively high protein and/or energy concentration and a low concentration of nutrient wash fiber (NDF), such as molasses, oligosaccharides, sorghum, seeds and grains (e.g., whole from corn, oats, rye, barley, wheat or prepared from crushing, milling, etc.), oilseed filter cake (e.g., from cottonseed, safflower, sunflower, soybean (e.g., soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (e.g., wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

Dry matter digestibility (DMd): digestibility refers to the degree to which feed is degraded and absorbed into the animal's body as it passes through the digestive tract. The term "dry matter digestibility" means the disappearance of feed dry matter from the Gastrointestinal (GI) tract caused by a given animal at a specified level of feed intake. DMd was measured as the percentage difference in Dry Matter (DM) ratio between ingested feed and excretion from ingested feed. Thus, the rumen DMd is the percentage difference in the proportion of dry matter between ingested feed and digestate passing through the terminal compartment of the rumen and describes the potential of rumen microorganisms to retreat and digest feed DM.

Energy-corrected milk (ECM): energy schoolPositive milk "is a method of adjusting milk production for the amount of the main component in milk that affects the energy concentration (lactose, fat and protein) in milk, and determines to adjust the amount of milk produced to 3.5% fat and 3.2% protein. ECMS is calculated herein as described in the following documents: sjaunja, l.o., Baevre, l., Junkkarinen, l., Pedersen, j,

J. in p.gailon, y.chabert (editorial) "Performance Recording of animals: State of the Art [ behavioral records of animals: most front horizontal]Proceedings of the27th Biennial Session of the International Committee for Animal Recording [ International Animal records Committee, 27th collection of two-year conference Proceedings of the International Committee, 1990') ]Wageningen Academic Publishers]Root of Wachengning, the Netherlands; 1991 "A Normal propyl for an energycorrected milk (ECM) formula in 156-157 & Nordic proposal of Energy Corrected Milk (ECM) formula]”。

Feed Conversion Ratio (FCR): FCR is a measure of the efficiency of an animal (herein a ruminant) in converting feed quality to a desired output (e.g., body weight). FCR is calculated as feed intake divided by animal weight gain over a specified period of time. By "lower feed conversion ratio" or "improved feed conversion ratio" is meant that less feed is required to increase the weight of the animal and/or the milk production of the animal. A 2% improvement in FCR means a 2% reduction in FCR.

Feed efficiency: the term "feed efficiency" is the ratio of live weight gain to Dry Matter Intake (DMI), or energy corrected milk yield per kg dry matter intake (kg ECM/kg DMI). The higher the value, the better.

Forage preparation: the term "forage" as defined herein also includes coarse food grains. Forage is NDF-rich plant material, such as hay and silage from forage plants (grasses) and other forage plants (seaweeds and legumes) or any combination thereof. Examples of forage plants are Alfalfa (alfalfalfa, lucerne), lotus roots, brassica plants (e.g. kale, rapeseed (canola, turnip cabbage (sweden turnip), radish), clover (e.g. hetero-clover, red clover, subterranean clover, white clover), grass (e.g. bermuda grass, brome, pseudo-oat grass, fescue, carex grass (heath grass), meadow grass, dactylus glomerata (orchardgrass), ryegrass, grass ladder (Timothy-grass)), using whole wheat (maize), millet, barley, oats, rye, sorghum, soybean and wheat, and vegetables (e.g. beets). forage further includes crop residues from grain production (e.g. maize stalks; straws from wheat, barley, oats, rye and other grains), residues from vegetables like beets (beets), oil seed production like from soybean, Stems and leaves of rapeseed and other legumes.

Fungal muramidase: the term "fungal muramidase" refers to a polypeptide having muramidase activity, which is obtained or obtainable from a fungal source. Examples of fungal sources are fungi; that is, muramidase is obtained or obtainable from the kingdom fungi, wherein the kingdom is a taxonomic hierarchy. In particular, the fungal muramidase is obtained or obtainable from Ascomycota (Ascomycota), such as Pezizomycotina, wherein the terms phylum and subgenus are taxonomic grades.

If the classification grade of the polypeptide is not known, it can be readily determined by one of ordinary skill in the art by performing a BLASTP search of the polypeptide (using, for example, the National Center for Biotechnology Information, NCIB) website http:// www.ncbi.nlm.nih.gov /) and comparing it to the closest homolog. Unknown polypeptides that are fragments of known polypeptides are considered to belong to the same taxonomic species. Unknown native polypeptides or artificial variants comprising substitutions, deletions and/or insertions in up to 10 positions are considered to be from the same taxonomic species as known polypeptides.

An ionophore: the term "ionophore" is used herein for antibiotics (e.g. macrolide antibiotics) and/or feed additives for enhancing the growth of animals (e.g. ruminants) which can catalyse ion transport across hydrophobic membranes (such as lipid bilayers found in living cells) and exhibit a counter ion (such as for example Na) ⁺、H⁺、Ca²⁺、Mg²⁺And/or K⁺) High affinity of (2). Examples of ionophores include, but are not limited to, monensin, which is used, for example, in the beef and dairy industries to prevent coccidiosis, increase propionic acid production, and prevent bloating.

Separating: the term "isolated" means a substance in a form or environment not found in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance; (2) any substance that is at least partially removed from one or more or all of the naturally occurring components with which it is associated in nature, including but not limited to any enzyme, variant, nucleic acid, protein, peptide, or cofactor; (3) any substance that is modified manually by man relative to that found in nature; or (4) any substance that is modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a promoter that is stronger than the promoter naturally associated with the gene encoding the substance). The isolated material may be present in a sample of fermentation broth.

Mature polypeptide: the term "mature polypeptide" means a polypeptide that is in its final form following translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, and the like.

Milk yield: the term "milk yield" is used to describe the total milk yield of a cow. Milk production is measured as the total amount of milk produced, which may be expressed as daily milk production or as milk production per lactation, and lactation is defined as the period of time from the day of calving to the day of exhaustion (defined as the day on which the cow stops producing milk). The dry-out day is typically about 300 days after calving. Milk production is measured in Kg milk or Kg Energy Corrected Milk (ECM) to compensate for changes in milk solids.

Muramidase enzyme: the term "muramidase" is used for polypeptides having glycosidase activity and catalyzes the hydrolysis of the 1, 4-beta-bond between the N-acetylmuramic acid and the N-acetyl-D-glucosamine residues in the peptidoglycan. This hydrolysis in turn can compromise the integrity of the bacterial cell wall, leading to bacterial lysis. Other terms of muramidase include "lysozyme" and "N-acetylmuramidase hydrolase".

Muramidase activity: the term "muramidase activity" means the enzymatic hydrolysis of the 1,4- β -linkage between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in peptidoglycans or between N-acetyl-D-glucosamine residues in chitodextrins, leading to lysis due to osmotic pressure. Muramidases belong to the class of EC 3.2.1.17 enzymes. Muramidase activity is typically measured by turbidimetry. The method is based on the turbidity change of a suspension of Micrococcus luteus (ATCC 4698) induced by muramidase lysis. Under appropriate experimental conditions, these changes are proportional to the amount of muramidase in the medium (c.f. the Combined complex of Food and agricultural organizations in association with the INS 1105(www.fao.org) of the Food and agricultural organization in association with the Compendium for Food additives).

Organic matter digestibility (OMd): the digestibility of the organic matter was defined as DMd, but the amount of organic matter was calculated as: OM — DM-ash, the ash content is determined after complete combustion of the feed DM.

Propionate: propionate is used herein interchangeably with the term "propionic acid" and is one of the Volatile Fatty Acids (VFAs) produced in the rumen. Propionate is the main precursor for glucose synthesis in ruminants, glucose being used for lactose and energy metabolism. The amount of propionate in the rumen is a measure of the rumen fermentation of ingested feed, and thus an increase in rumen propionate is an indication of an increase in ruminant glucose supply.

Ruminant animals: the term "ruminant" means the following mammals: it digests plant-based feed primarily by bacterial action, first fermenting/degrading the plant-based feed within the first compartment of the animal's forestomach complex, then retaining the small particles and ruminating the long, semi-degraded mass (now referred to as "ruminant (cud)") and chewing again. The process of chewing the cud again to further break down plant matter and stimulate digestion is called rumination. Examples of ruminants are cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, reindeer, yaks, camels and llamas.

Feed for ruminants: the term "ruminant feed" or "animal feed for ruminants" refers to any compound, formulation, or mixture suitable for or intended for ingestion by a ruminant. Ruminant feed typically comprises forage (including fresh grass, roughage, and silage), and may further comprise concentrates as well as vitamins, minerals, enzymes, Direct Fed Microbial (DFM), amino acids, and/or other feed ingredients (e.g., premixes). The ruminant feed may be fed as a Total Mixed Ration (TMR) where all feed components are mixed and fed as a mixture prior to feeding, or as a Partial Mixed Ration (PMR) where most of the feed components are mixed and fed together but some of the concentrate is fed separately, or it may be fed as a separately fed feed with all components fed separately and not mixed.

Sequence identity: the degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

For The purposes of The present invention, sequence identity between two amino acid sequences is determined using The Needman-Wunsch algorithm (Needleman-Wunsch) (Needleman and Wunsch,1970, J.Mol.biol. [ J.Mol.Biol ]48:443-453), as implemented in The Niderl program of The EMBOSS Software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al 2000, trends Genet. [ genetic trends ]16:276-277) (preferably version 5.0.0 or more). The parameters used are gap opening penalty of 10, gap extension penalty of 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the non-reduced option) is used as the percent identity and is calculated as follows:

(same residue x 100)/(alignment length-total number of gaps in alignment). Ensiling: silage is a type of forage produced by the natural fermentation of wet plant material (e.g., fresh grass) and whole wheat (e.g., corn and barley). The fermentation process is carried out to preserve the wet material so that it can be used throughout the year.

A substantially pure polypeptide: the term "substantially pure polypeptide" means a preparation containing 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% by weight of other polypeptide material with which it is naturally or recombinantly associated. Preferably, the polypeptide is at least 92% pure, such as at least 94% pure, at least 95% pure, at least 96% pure, at least 97% pure, at least 98% pure, at least 99% pure, at least 99.5% pure, and 100% pure, by weight of the total polypeptide material present in the formulation. The polypeptides of the invention are preferably in a substantially pure form. This can be achieved, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

Variants: the term "variant" means a polypeptide having muramidase activity comprising an alteration (i.e., substitution, insertion, and/or deletion) of one or more (e.g., several) amino acid residues at one or more (e.g., several) positions. Substitution means the substitution of an amino acid occupying a position with a different amino acid; deletion means the removal of an amino acid occupying a position; and insertion means the addition of 1, 2, or 3 amino acids adjacent to and immediately following the amino acid occupying that position.

In one aspect, a muramidase variant according to the invention may comprise from 1 to 5; from 1 to 10; from 1 to 15; from 1 to 20; from 1 to 25; from 1 to 30; from 1 to 35; from 1 to 40; from 1 to 45; or from 1-50, i.e., 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, 45, 46, 47, 48, 49 or 50 alterations and having at least 20%, e.g., 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 a parent muramidase (e.g., SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, Muramidase activity of SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 46, SEQ ID NO 47, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52, SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, and SEQ ID NO 59).

Volatile Fatty Acids (VFA)/Short Chain Fatty Acids (SCFA): volatile Fatty Acids (VFAs), also known as Short Chain Fatty Acids (SCFAs), are fatty acids having less than six carbon atoms and include, for example, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid. Volatile Fatty Acids (VFAs) are produced by fermentation of carbohydrates in the rumen and provide the primary source of energy for ruminants. Thus, an increase in VFA can be used as an indication of an increase in ruminant energy and nutrient supply.

Detailed Description

Method for improving ruminant performance

Nutritional utilization of ruminant feed is important for optimal production and animal health in modern production systems. It has been surprisingly found that supplementation of ruminant feed with muramidase according to the invention results in an increased rumen dry matter digestibility, compared to when ruminant feed without muramidase is supplemented (as a control). By increasing the digestibility of rumen dry matter, ruminants are provided more nutrition for production.

Thus, the efficiency of nutrient utilization has been improved and the conversion of organic matter (e.g., dairy cows convert microbial proteins to milk or beef cattle convert them to meat) has been increased compared to the conversion obtained from ruminant feed without muramidase.

Furthermore, it was surprisingly found that the rumen dry matter digestibility was improved compared to that obtained when ruminants were provided with commonly used ionophores on the market.

In one aspect of the invention, the efficiency of nutrient utilization of ruminant feed is improved. By increasing the nutrient utilization of ruminant feed, the same amount of milk and/or meat can be produced from fewer ruminants, which reduces the use of natural resources and the amount of greenhouse gas (GHG) emissions produced per unit of milk and/or per unit of meat. This also results in a reduction in nitrogen and phosphate excretion per ruminant animal and thus a reduction in the total amount of phosphate and nitrogen excretion per unit yield.

Determination of the dry matter digestibility may be performed, for example, using an in vitro fermentation model adapted from Menke KH, Steingass H.1988(Estimation of the value of the high energy feed obtained from chemical analysis and in vitro gas production using rumen fluid) Anim Res Dev [ animal research and development ] (1988)28:7-55), as described in example 1.

In one aspect, the production of Volatile Fatty Acids (VFAs) in the rumen is increased as compared to the production of VFAs in the rumen of a ruminant that is not fed muramidase.

In one aspect, propionate production in the rumen is increased as compared to propionate production in the rumen of a ruminant that is not fed muramidase.

In one aspect, the production of acetate in the rumen is increased as compared to the production of acetate in the rumen of a ruminant that is not fed muramidase.

In one embodiment, the muramidase is administered at a level of 1 to 200mg enzyme protein/kg ruminant feed DM, such as 5 to 150mg, 5 to 125mg, 5 to 100mg, 5 to 75mg, 5 to 50mg, 5 to 40mg, 10 to 50, or 5 to 25mg enzyme protein/kg ruminant feed DM, or any combination of these intervals.

In one embodiment, the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels and llamas. In another embodiment, the ruminant is selected from the group consisting of cattle, dairy cows and beef cattle.

Muramidase is provided to ruminants during any period of time from birth until slaughter. In a preferred embodiment, muramidase is provided to the ruminant animal daily. In another embodiment, muramidase is provided to the ruminant every day during the lifetime of the ruminant.

In one embodiment, muramidase is provided to a ruminant selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas. In one embodiment, muramidase is provided to a growing ruminant. In one embodiment, muramidase is provided to the cow. In another embodiment, muramidase is provided to the cow during lactation. In one embodiment, muramidase is provided to beef cattle during the growth phase of beef cattle production. In one embodiment, muramidase is provided to beef cattle at the completion stage of beef cattle production.

Muramidase may be provided to the ruminant animal in any suitable manner. In one embodiment, the muramidase is fed to the ruminant animal as a feed, feed supplement, or feed additive. In another embodiment, muramidase is provided to the ruminant as drinking water. In yet another embodiment, the muramidase is provided to the ruminant animal as a bolus administration. In yet another embodiment, the muramidase is provided to the ruminant as a post-feed spray application to the ruminant feed. In one embodiment, muramidase is provided to the ruminant as a beverage in liquid form. In another embodiment, the muramidase is provided to the ruminant as a drench in liquid form. In another embodiment, the muramidase is provided to the ruminant as milk or milk substitute.

In one embodiment, the muramidase is of microbial origin. In another embodiment, the muramidase is of fungal origin. In one embodiment, the muramidase is obtained or obtainable from an Ascomycota (Ascomycota), such as the subdivision Sphaeria (Pezizomycotina).

In one embodiment, the muramidase comprises one or more domains from a Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and novel MUR polypeptides having muramidase activity.

In one embodiment, the muramidase comprises one or more domains from Glycoside Hydrolase (GH) family GH 24.

In one embodiment, the muramidase comprises one or more domains from Glycoside Hydrolase (GH) family GH 25.

In one embodiment, the muramidase comprises one or more domains from a novel MUR polypeptide having muramidase activity.

In a preferred embodiment, the present invention relates to a method of improving dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield in a ruminant animal, the method comprising administering to the ruminant animal a ruminant feed, a ruminant feed supplement or a ruminant feed additive comprising one or more muramidase enzymes, wherein:

(a) The muramidase is a muramidase comprising one or more domains from the Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and novel MUR polypeptides having muramidase activity, and is administered at a level of 1 to 200mg enzyme protein/kg ruminant feed DM;

(b) the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas; and

(c) an improvement in dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield of at least 1% compared to a control; and

(d) optionally providing muramidase to the ruminant daily for at least 30 days during the lifetime of the ruminant.

In one embodiment of the method, Energy Corrected Milk (ECM) production is improved by at least 1.25%, e.g., at least 1.5%, at least 1.75%, or at least 2.0% compared to a control. In another embodiment, the ECM production improvement is between 1% and 5%, such as between 1% and 4%, between 1% and 3%, between 1.25% and 2.5%, or between 1.5% and 2%, or any combination of these intervals, as compared to a control.

In one embodiment of the method, rumen dry matter digestibility (DMd) is improved by at least 1%, such as at least 1.5%, at least 1.75%, or at least 2.0% compared to a control. In another embodiment, the dry matter digestibility is improved by between 1% and 15%, such as between 1% and 10%, between 1% and 7%, between 1% and 5%, or between 2% and 5%, or any combination of these intervals, compared to a control.

In one embodiment of the method, the Volatile Fatty Acid (VFA) is selected from the group consisting of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and any combination thereof. In another embodiment, the Volatile Fatty Acid (VFA) is selected from the group consisting of acetate, propionate, butyrate, and any combination thereof. In yet another embodiment, the Volatile Fatty Acid (VFA) is an acetate and/or propionate.

In one embodiment of the method, the Volatile Fatty Acids (VFA) are improved by at least 1%, such as at least 1.5%, at least 1.75%, or at least 2.0% as compared to a control. In another embodiment, the volatile fatty acid improvement is between 1% and 15%, such as between 1% and 10%, between 1% and 7%, between 1% and 5%, or between 2% and 5%, or any combination of these intervals, as compared to a control.

In one embodiment of the method, the Volatile Fatty Acid (VFA) is an acetate salt. In another embodiment, the acetate is improved by at least 1%, such as at least 1.5%, at least 1.75%, or at least 2.0% as compared to a control. In another embodiment, the acetate improvement is between 1% and 15%, such as between 1% and 10%, between 1% and 7%, between 1% and 5%, or between 2% and 5%, or any combination of these intervals, as compared to a control.

In one embodiment of the method, the Volatile Fatty Acid (VFA) is propionate. In another embodiment, the propionate is improved by at least 1%, such as at least 1.5%, at least 1.75%, or at least 2.0% compared to a control. In another embodiment, the propionate improvement is between 1% and 15%, such as between 1% and 10%, between 1% and 7%, between 1% and 5%, or between 2% and 5%, or any combination of these intervals, as compared to a control.

In one embodiment of the method, the FCR is improved by at least 1%, e.g., at least 1.25%, at least 1.5%, at least 1.75%, or at least 2.0% as compared to the control. In another embodiment, the FCR improvement is between 1% and 10%, such as between 1% and 9%, such as between 1% and 8%, such as between 1% and 7%, such as between 1% and 6%, such as between 1% and 5%, such as between 1% and 4%, between 1% and 3%, between 1.25% and 2.5%, or between 1.5% and 2%, or any combination of these intervals, as compared to a control. A 1% improvement in FCR is defined as an 1/100 reduction in FCR for a ruminant supplemented with muramidase compared to the FCR for a ruminant which is not supplemented with muramidase.

In one embodiment, muramidase is provided to the ruminant during any period of time from birth to slaughter. In a preferred embodiment, muramidase is provided to the ruminant animal daily. In another embodiment, muramidase is provided to the ruminant every day during the lifetime of the ruminant.

In one embodiment, muramidase is provided to a growing ruminant. In one embodiment, muramidase is provided to the cow. In another embodiment, the muramidase is provided to the cow during lactation. In one embodiment, muramidase is provided to beef cattle during the growth phase of beef cattle production. In one embodiment, muramidase is provided to beef cattle at the completion stage of beef cattle production. In another embodiment, muramidase is provided to the calf in milk. In one embodiment, muramidase is provided to a ruminant selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas.

In one embodiment, the muramidase is of microbial origin. In another embodiment, the muramidase is of fungal origin. In one embodiment, the muramidase is obtained or obtainable from a phylum ascomycota, such as the subphylum discodermatum.

In one embodiment, the muramidase has at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to SEQ ID No. 3.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 3 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:3 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID No. 3.

In another embodiment, the muramidase is a variant of SEQ ID NO:3, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 3 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 3 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 3 does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 3 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 3 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 6 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:6 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 6.

In another embodiment, the muramidase is a variant of SEQ ID NO 6, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 6 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 6 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 6 does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO 6 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 6 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 9 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:9 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 9.

In another embodiment, the muramidase is a variant of SEQ ID NO:9, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 9 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 9 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, does not exceed 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 9 does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 9 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 9 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 12 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:12 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 12.

In another embodiment, the muramidase is a variant of SEQ ID NO:12, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 12 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 12 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 12 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 12 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 12 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 15 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:15 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 15.

In another embodiment, the muramidase is a variant of SEQ ID NO 15, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 15 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 15 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 15 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 15 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 15 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 18 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:18 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 18.

In another embodiment, the muramidase is a variant of SEQ ID NO:18, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 18 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 18 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, does not exceed 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 18 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 18 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 18 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 21 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:21 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 21.

In another embodiment, the muramidase is a variant of SEQ ID NO:21, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 21 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 21 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 21 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 21 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 21 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 24 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:24 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 24.

In another embodiment, the muramidase is a variant of SEQ ID NO:24, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO. 24 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 24 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 24 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 24 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 24 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 27 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:27 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 27.

In another embodiment, the muramidase is a variant of SEQ ID NO:27, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 27 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 27 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 27 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 27 does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 27 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 28 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:28 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 28.

In another embodiment, the muramidase is a variant of SEQ ID NO:28, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 28 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 28 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 28 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 28 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 28 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 29 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:29 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 29.

In another embodiment, the muramidase is a variant of SEQ ID NO:29, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO. 29 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 29 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 29 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 29 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 29 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 30 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:30 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 30.

In another embodiment, the muramidase is a variant of SEQ ID NO:30, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 30 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID NO 30 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 30 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 30 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 30 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 31 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:31 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 31.

In another embodiment, the muramidase is a variant of SEQ ID NO 31, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 31 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID NO 31 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 31 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 31 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 31 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 32 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:32 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 32.

In another embodiment, the muramidase is a variant of SEQ ID NO:32, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 32 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 32 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 32 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 32 does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 32 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 33 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:33 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 33.

In another embodiment, the muramidase is a variant of SEQ ID NO:33, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 33 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID NO 33 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 33 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 33 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 33 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 34 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:34 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 34.

In another embodiment, the muramidase is a variant of SEQ ID NO:34, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 34 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID NO 34 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 34 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 34 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 34 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 35 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:35 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 35.

In another embodiment, the muramidase is a variant of SEQ ID NO:35, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 35 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 35 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 35 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 35 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 35 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 36 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:36 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 36.

In another embodiment, the muramidase is a variant of SEQ ID NO:36, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 36 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 36 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 36 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:36 does not exceed 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 36 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO 37 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:37 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 37.

In another embodiment, the muramidase is a variant of SEQ ID NO 37, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 37 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 37 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 37 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:37 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 37 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 38 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:38 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 38.

In another embodiment, the muramidase is a variant of SEQ ID NO:38, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 38 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 38 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 38 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO 38 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 38 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 39 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:39 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 39.

In another embodiment, the muramidase is a variant of SEQ ID NO:39, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 39 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 39 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO:39 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:39 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 39 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 40 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:40 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 40.

In another embodiment, the muramidase is a variant of SEQ ID NO:40, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 40 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 40 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 40 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 40 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 40 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 41 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:41 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 41.

In another embodiment, the muramidase is a variant of SEQ ID NO:41, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 41 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 41 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 41 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 41 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 41 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 42 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:42 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 42.

In another embodiment, the muramidase is a variant of SEQ ID NO:42, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 42 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 42 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 42 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:42 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 42 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO 43 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:43 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 43.

In another embodiment, the muramidase is a variant of SEQ ID NO:43, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 43 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 43 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 43 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 43 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 43 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 44 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:44 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 44.

In another embodiment, the muramidase is a variant of SEQ ID NO:44, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 44 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 44 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 44 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 44 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 44 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:45 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:45 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 45.

In another embodiment, the muramidase is a variant of SEQ ID NO:45, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO:45 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 45 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 45 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:45 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 45 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 46 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:46 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 46.

In another embodiment, the muramidase is a variant of SEQ ID NO:46, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 46 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 46 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 46 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 46 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 46 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:47 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:47 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID No. 47.

In another embodiment, the muramidase is a variant of SEQ ID NO:47, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO:47 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 47 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 47 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 47 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 47 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 48 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:48 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 48.

In another embodiment, the muramidase is a variant of SEQ ID NO:48, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 48 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 48 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 48 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 48 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 48 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID No. 49 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:49 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 49.

In another embodiment, the muramidase is a variant of SEQ ID NO:49, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 49 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 49 that comprise one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 49 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 49 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 49 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 50 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:50 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 50.

In another embodiment, the muramidase is a variant of SEQ ID NO:50, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 50 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 50 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 50 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 50 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 50 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID No. 51 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:51 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO: 51.

In another embodiment, the muramidase is a variant of SEQ ID NO:51, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 51 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 51 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 51 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 51 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 51 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID No. 52 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:52 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID No. 52.

In another embodiment, the muramidase is a variant of SEQ ID NO 52, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 52 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 52 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 52 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO. 52 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 52 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO 53 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:53 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 53.

In another embodiment, the muramidase is a variant of SEQ ID NO:53, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 53 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 53 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 53 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO 53 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 53 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 54 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:54 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO: 54.

In another embodiment, the muramidase is a variant of SEQ ID NO:54, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 54 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 54 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 54 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:54 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 54 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 55 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:55 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 55.

In another embodiment, the muramidase is a variant of SEQ ID NO:55, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO:55 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID NO:55 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 55 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:55 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 55 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 56 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:56 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 56.

In another embodiment, the muramidase is a variant of SEQ ID NO:56, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 56 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 56 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 56 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:56 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO 56 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 57 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:57 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO. 57.

In another embodiment, the muramidase is a variant of SEQ ID NO:57, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 57 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 57 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO. 57 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:57 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO. 57 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 58 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:58 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO: 58.

In another embodiment, the muramidase is a variant of SEQ ID NO:58, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 58 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 58 that comprise one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, does not exceed 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO:58 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO:58 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ ID NO:58 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In one embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO. 59 or an allelic variant thereof; or is a fragment thereof having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids, or at least 200 amino acids. In another embodiment, the muramidase comprises or consists of the amino acid sequence of SEQ ID NO:59 or an allelic variant thereof and an N-terminal and/or C-terminal His-tag and/or HQ-tag. In another aspect, the polypeptide comprises or consists of amino acids 1 to 213 of SEQ ID NO 59.

In another embodiment, the muramidase is a variant of SEQ ID NO 59, wherein the variant has muramidase activity and comprises one or more substitutions, and/or one or more deletions, and/or one or more insertions, or any combination thereof, in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 positions. In another embodiment, the number of positions in SEQ ID NO 59 comprising one or more amino acid substitutions, and/or one or more amino acid deletions, and/or one or more amino acid insertions, or any combination thereof, is between 1 and 45, such as 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5 positions. In one embodiment, the number of positions in SEQ ID No. 59 comprising one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions, or any combination thereof, is NO more than 10, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of substitutions, deletions and/or insertions in SEQ ID NO 59 does not exceed 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another embodiment, the number of substitutions (preferably conservative substitutions) in SEQ ID NO 59 is NO more than 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, the number of conservative substitutions in SEQ

The amino acid change may be of a minor nature, i.e., a conservative amino acid substitution or insertion that does not significantly affect the folding and/or activity of the protein; a small deletion of typically 1 to 30 amino acids; small amino-terminal or carboxy-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by altering the net charge or another function (e.g., a polyhistidine segment, an epitope, or a binding domain).

Examples of conservative substitutions are within the following groups: 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 which do not generally alter specific activity are known in The art and are described, for example, by H.Neurath and R.L.Hill,1979, in The Proteins, Academic Press, N.Y.. Common substitutions 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.

Essential amino acids in polypeptides can be identified according to 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 resulting mutant molecules are tested for muramidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al, 1996, J.biol.chem. [ J.Biol ]271: 4699-4708. The active site of an enzyme or other biological interaction can also be determined by physical analysis of the structure, as determined by the following technique: nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutating putative contact site (contact site) amino acids. See, e.g., de Vos et al, 1992, Science [ Science ]255: 306-); smith et al, 1992, J.mol.biol. [ J.Mol.224: 899-); wlodaver et al, 1992, FEBS Lett. [ Provisions of the European Association of biochemistry ]309: 59-64. The identity of the essential amino acids can also be inferred from alignment with the relevant polypeptide.

The crystal structure of acremophilus alcalophilus CBS114.92 muramidase is As disclosed in WO 2013/076253. These abulilc coordinates can be used to generate a three-dimensional model depicting the structure or homologous structure (e.g., variants of the invention) of acremophilus alcalophilus CBS114.92 muramidase. Amino acid residues D95 and E97 were identified as catalytic residues using an x-ray structure.

(a) the muramidase is obtained or obtainable from Ascomycota and is administered at a level of 1 to 200mg enzyme protein per kg dry matter feed (DM) of ruminant;

(b) the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas;

(c) an improvement in dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield of at least 1% compared to a control; and

(d) Optionally providing muramidase to the ruminant daily for at least 30 days during the lifetime of the ruminant.

In one embodiment, the method is provided to a growing ruminant. In one embodiment, the method is provided to a cow. In another embodiment, the method is provided to a cow during lactation. In one embodiment, the method is provided to beef cattle during the growth phase of beef cattle production. In one embodiment, the method is provided to beef cattle at the completion stage of beef cattle production.

In one embodiment, Energy Corrected Milk (ECM) production is improved by at least 1.25%, e.g., at least 1.5%, at least 1.75%, or at least 2.0% as compared to a control. In another embodiment, the ECM production improvement is between 1% and 5%, such as between 1% and 4%, between 1% and 3%, between 1.25% and 2.5%, or between 1.5% and 2%, or any combination of these intervals, as compared to a control.

In one embodiment, the rumen dry matter digestibility (DMd) is improved by at least 1%, such as at least 1.5%, at least 1.75%, or at least 2.0% compared to a control. In another embodiment, the dry matter digestibility is improved by between 1% and 15%, such as between 1% and 10%, between 1% and 7%, between 1% and 5%, or between 2% and 5%, or any combination of these intervals, compared to a control.

In one embodiment of the method, the muramidase is administered at a level of 1 to 200mg enzyme protein/kg ruminant feed DM, such as 5 to 150mg, 5 to 125mg, 5 to 100mg, 5 to 75mg, 5 to 50mg, 5 to 40mg, 10 to 50, or 5 to 25mg enzyme protein/kg ruminant feed DM, or any combination of these intervals.

In one embodiment, muramidase is provided to a ruminant using one of the protocols disclosed herein.

In another preferred embodiment, the present invention relates to a method of improving dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield in a ruminant animal, the method comprising administering to the ruminant animal a ruminant animal feed, a ruminant animal feed supplement or a ruminant animal feed additive comprising one or more muramidase enzymes, wherein:

(a) The muramidase is a GH24 muramidase obtained or obtainable from ascomycota, administered at a level of 1 to 200mg enzyme protein per kg ruminant feed DM;

(b) the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas;

(c) an improvement in dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield of at least 1% compared to a control; and

(d) optionally providing muramidase to the ruminant daily for at least 30 days during the lifetime of the ruminant.

In one embodiment of the method, muramidase is provided to the ruminant using one of the protocols disclosed herein.

(a) the muramidase is a GH25 muramidase obtained or obtainable from ascomycota, administered at a level of 1 to 200mg enzyme protein per kg ruminant feed DM;

(b) the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas;

(c) an improvement in dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield of at least 1% compared to a control; and

(d) optionally providing muramidase to the ruminant daily for at least 30 days during the lifetime of the ruminant.

In one embodiment of the method, muramidase is provided to the ruminant using one of the protocols disclosed herein.

(a) the muramidase is a novel MUR polypeptide having muramidase activity, obtained or obtainable from Ascomycota, administered at a level of 1 to 200mg enzyme protein/kg ruminant feed DM;

(b) the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas;

(c) an improvement in dry matter digestibility (DMd) and/or Volatile Fatty Acid (VFA) yield and/or meat yield and/or milk yield of at least 1% compared to a control; and

(d) optionally providing muramidase to the ruminant daily for at least 30 days during the lifetime of the ruminant.

In one embodiment of the method, muramidase is provided to the ruminant using one of the protocols disclosed herein.

Formulation

The enzymes of the invention may be formulated as liquids or solids. For liquid formulations, the formulation may comprise polyols (like, for example, glycerol, ethylene glycol or propylene glycol), salts (like, for example, sodium chloride, sodium benzoate, potassium sorbate) or sugars or sugar derivatives (like, for example, dextrin, glucose, sucrose and sorbitol). Thus, in one embodiment, the composition is a liquid composition comprising a polypeptide of the invention and one or more formulations selected from the list consisting of: glycerol, ethylene glycol, 1, 2-propylene glycol, 1, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, dextrin, glucose, sucrose and sorbitol. The liquid formulation may be sprayed on already pelleted feed or may be added to the drinking water supplied to the ruminant.

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

In one embodiment, the solid composition is in particulate form. The particles may have a matrix structure (matrix structure) in which the components are homogeneously mixed. However, the particles typically comprise a core particle and one or more coatings, which are typically salt and/or waxy coatings. Examples of waxes are polyethylene glycol; polypropylene; carnauba wax; candelilla wax; beeswax; hydrogenated vegetable or ruminant oil such as hydrogenated tallow, hydrogenated palm oil, hydrogenated cottonseed oil, and/or hydrogenated soybean oil; a fatty acid alcohol; mono-and/or diglycerides, such as glycerol stearate, wherein stearate is a mixture of stearic and palmitic acid; microcrystalline wax; paraffin wax; and fatty acids, such as hydrogenated linear long chain fatty acids and derivatives thereof. Preferred waxes are palm oil or hydrogenated palm oil. The core particle may be a homogeneous blend of the muramidase of the invention, optionally in combination with one or more additional enzymes, and optionally together with one or more salts, or an inert particle (having the muramidase of the invention, optionally in combination with one or more additional enzymes applied thereto).

In one embodiment, the material of the core particle is selected from the group consisting of: inorganic salts (such as calcium acetate, calcium benzoate, calcium carbonate, calcium chloride, calcium citrate, calcium sorbate, calcium sulfate, potassium acetate, potassium benzoate, potassium carbonate, potassium chloride, potassium citrate, potassium sorbate, potassium sulfate, sodium acetate, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium sulfate, zinc acetate, zinc benzoate, zinc carbonate, zinc chloride, zinc citrate, zinc sorbate, zinc sulfate), starches or sugars or sugar derivatives (such as, for example, sucrose, dextrin, glucose, lactose, sorbitol), small organic molecules, starches, flours, cellulose and minerals, and clay minerals (also known as aqueous layered aluminum silicates). In a preferred embodiment, the core comprises a clay mineral, such as kaolinite or kaolin.

The salt coating is typically at least 1 μm thick and may be a particular salt or mixture of salts, such as Na₂SO₄、K₂SO₄、MgSO₄And/or sodium citrate. Further examples are those described in e.g. WO 2008/017659, WO 2006/034710, WO 1997/05245, WO 1998/54980, WO 1998/55599, WO 2000/70034, or polymer coatings as described in e.g. WO 2001/00042.

In another embodiment, the composition is a solid composition comprising a muramidase of the invention and one or more formulations selected from the list consisting of: sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, and cellulose. In a preferred embodiment, the formulation is selected from one or more of the following compounds: sodium sulfate, dextrin, cellulose, sodium thiosulfate, and calcium carbonate. In a preferred embodiment, the solid composition is in particulate form. In one embodiment, the solid composition is in the form of a granule and comprises a core particle, an enzyme layer comprising a muramidase of the invention, and a salt coating.

In another embodiment, the formulation is selected from one or more of the following compounds: glycerol, ethylene glycol, 1, 2-or 1, 3-propanediol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, glucose, sucrose, sorbitol, lactose, starch, kaolin, and cellulose. In a preferred embodiment, the formulation is selected from one or more of the following compounds: 1, 2-propanediol, 1, 3-propanediol, sodium sulfate, dextrin, cellulose, sodium thiosulfate, kaolin, and calcium carbonate.

Ruminant feed, ruminant feed supplement and ruminant feed additive

The ruminant feed composition or component according to the present invention has a crude protein content of between 50 and 800g/kg and further comprises one or more polypeptides having muramidase activity as described herein.

In addition or in the alternative (to the crude protein content described above), the ruminant feed composition of the present invention has a metabolizable energy of 5-30 MJ/kg.

In particular embodiments, the levels of metabolizable energy, crude protein, calcium, and/or phosphorus fall within any of ranges 2, 3, 4, or 5(r.2-5) in table B of WO 2001/058275.

In a particular embodiment, the ruminant feed comprises non-protein nitrogen obtained from, for example, urea.

The nitrogen content was determined by the Kjeldahl method (a.o.a.c.,1984, Official methods of Analysis [ Official analytical methods ] 14 th edition, Association of Official analytical chemists [ Official analytical chemists ], washington, d.c.) and the crude protein was calculated as nitrogen (N) multiplied by a factor of 6.25 (i.e. crude protein (g/kg) ═ N (g/kg) x 6.25).

Metabolizable energy can be calculated as follows: NRC publication Nutrients requirements in Swine [ nutritional requirements for ruminants ], seventh reissue 2001, Subcommittee on Swine nutrition, committee on animal nutrition, board of aggreculture, national research countci [ national institute of Nutrition for ruminants, division of Nutrition for ruminants, department of agriculture of the national research institute ] national academy of sciences Press, Washington, D.C., pages 2-6.

In a particular embodiment, the ruminant feed composition of the present invention comprises at least one vegetable protein as defined above.

The ruminant feed composition of the present invention may also comprise distillers dried Grains with Solubles (DDGS), typically in an amount of 0-30%.

In still other particular embodiments, the ruminant feed compositions of the present invention contain 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oat; and/or 0-40% soy flour; and/or 0-20% whey.

The ruminant feed may comprise vegetable proteins. In particular embodiments, the protein content of these vegetable proteins is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% (w/w). The vegetable protein may be derived from vegetable protein sources such as legumes and grains, for example, materials from plants of the families sphenoidaceae (fabaceae), cruciferae, chenopodiaceae, and poaceae, such as soybean meal, lupin meal, rapeseed meal, and combinations thereof.

In a particular embodiment, the vegetable protein source is material from one or more plants of the family leguminosae (e.g., soybean, lupin, pea or bean). In another particular embodiment, the vegetable protein source is material obtained from one or more plants of the family chenopodiaceae, such as beet, sugar beet, spinach or quinoa. Other examples of vegetable protein sources are rape and cabbage. In another particular embodiment, soy is a preferred vegetable protein source. Other examples of vegetable protein sources are cereals, such as barley, wheat, rye, oats, maize (corn), rice and sorghum.

In a particular embodiment, forage plants such as corn (maize), beans and grass have been chopped and stored anaerobically and fermentatively. This is called ensiling (silage or ensilage) and may constitute 90% of the bovine diet.

In a particular embodiment, the non-protein nitrogen (NPN) source may form part of the diet. An example is urea which makes up 25% of the total crude protein of a bovine diet.

Ruminant concentrates comprising a number of feeds may be formulated, for example, as mash feeds (non-pelleted) or pelleted feeds. Typically, the ground feed is mixed and sufficient amounts of essential vitamins and minerals are added according to the instructions for the species in question. The enzyme is added as a solid or liquid enzyme formulation. For example, for powdered feeds, solid or liquid enzyme formulations may be added before or during the ingredient mixing step. For pelleted feeds, the (solid or liquid) muramidase/enzyme preparation may also be added before or during the feed ingredient steps. Typically, a liquid enzyme formulation comprises muramidase of the invention, optionally accompanied by a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. Muramidase may also be incorporated into feed supplements, feed additives or premixes.

Alternatively, muramidase may be prepared by freezing a mixture of a liquid enzyme solution and a bulking agent (e.g., ground soybean meal), and then lyophilizing the mixture.

In one embodiment, the composition comprises one or more additional enzymes. In one embodiment, the composition comprises one or more microorganisms. In one embodiment, the composition includes one or more vitamins. In one embodiment, the composition includes one or more minerals. In one embodiment, the composition includes one or more amino acids. In one embodiment, the composition includes one or more other feed ingredients.

In another embodiment, the composition comprises one or more polypeptides of the invention, one or more formulating agents, and one or more additional enzymes. In one embodiment, the composition comprises one or more polypeptides of the invention, one or more formulating agents, and one or more microorganisms. In one embodiment, the composition comprises one or more polypeptides of the invention, one or more formulating agents, and one or more vitamins. In one embodiment, the composition comprises one or more polypeptides of the invention and one or more minerals. In one embodiment, the composition comprises a polypeptide of the invention, one or more formulating agents, and one or more amino acids. In one embodiment, the composition comprises one or more polypeptides of the invention, one or more formulating agents, and one or more other feed ingredients.

In another embodiment, the composition comprises one or more polypeptides of the invention, one or more blending agents, and one or more components selected from the list consisting of: one or more additional enzymes; one or more microorganisms; one or more vitamins; one or more minerals; one or more amino acids; and one or more other feed ingredients.

The final muramidase concentration in the diet is in the following range: 0.01 to 200mg enzyme protein per kg ruminant feed DM, such as 0.1 to 150mg, 0.5 to 100mg, 1 to 75mg, 2 to 50mg, 3 to 25mg, 2 to 80mg, 5 to 60mg, 8 to 40mg or 10 to 30mg enzyme protein per kg ruminant feed DM, or any combination of these intervals.

It is currently desirable to administer muramidase in one or more of the following amounts (dosage ranges): 0.01-200, 0.01-100, 0.5-100, 1-50, 5-100, 5-50, 10-100, 0.05-50, 5-25 or 0.10-10, all these ranges being in units of mg muramidase per kg feed DM.

To determine the mg of muramidase protein per kg of feed DM, muramidase was purified from the feed composition and the specific activity of the purified muramidase was determined using a correlation assay (see muramidase activity). The muramidase activity of a feed composition like this was determined using the same assay and the dose in mg of muramidase protein per kg of feed was calculated on the basis of these two assays.

In a particular embodiment, the ruminant feed additive of the present invention is intended to be included (or specified to be necessarily included) in a ruminant diet or feed at a level of from 0.01% to 10.0%, more particularly from 0.05% to 5.0% or from 0.2% to 1.0% (% means g additive per 100g feed). This is also true in particular for premixes.

The same rule was applied to determine the amount of muramidase protein mg in feed supplements and feed additives. Of course, if a sample is used for the muramidase for the preparation of a feed additive or feed, the specific activity can be determined from this sample (no purification of the muramidase from the feed composition, feed supplement or feed additive is required).

Additional enzymes

In another embodiment, the compositions described herein optionally comprise one or more enzymes. ENZYMEs can be classified according to handbook Enzyme Nomenclature [ ENZYMEs Nomenclature handbook ] (from NC-IUBMB,1992) and see also the Enzyme website on the internet: http:// www.expasy.ch/enzyme/. ENZYME is a repository of information about ENZYME nomenclature. It is based mainly on the recommendations of the International Commission on the Association of biochemistry and molecular biology nomenclature (IUB-MB), academic Press, Inc. [ academic Press Inc. ],1992 and describes each type of ENZYME characterized, for which EC (ENZYME Commission) numbers are given (Bairoch A. the ENZYME database, 2000, Nucleic acids SRes [ Nucleic acids research ]28: 304-. This IUB-MB enzyme nomenclature is based on their substrate specificity, sometimes based on their molecular mechanism; this classification does not reflect the structural features of these enzymes.

Another classification of certain glycoside hydrolases (e.g., endoglucanases, xylanases, galactanases, mannanases, glucanases, muramidases, and galactosidases) is described in Henrissat et al, "The carbohydrate-active enzymes database (CAZy) in2013[2013 carbohydrate-active enzymes database (CAZy) ], Nucl. acids Res. [ nucleic acids research ] (1/1 2014) 42(D1): D490-D495; see also www.cazy.org.

Thus, the composition of the invention may also comprise at least one other enzyme selected from the group consisting of: 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); amylases, such as, for example, alpha-amylase (ec 3.2.1.1); arabinofuranosidase (EC 3.2.1.55); beta-xylosidase (EC 3.2.1.37); acetyl xylan esterase (EC 3.1.1.72); feruloyl esterase (EC 3.1.1.73); cellulase (e.c. 3.2.1.4); cellobiohydrolases (ec 3.2.1.91); beta-glucosidase (EC 3.2.1.21); pullulanase (EC 3.2.1.41), alpha-mannosidase (EC3.2.1.24), mannanase (EC 3.2.1.25) and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any combination thereof.

In a particular embodiment, the composition of the invention comprises a phytase (EC 3.1.3.8 or 3.1.3.26). Examples of commercially available phytases include Bio-Feed^TMPhytase (Novozymes Inc.)

P、NP and

HiPhos (Dismamen Nutrition products), Natuphos^TM(BASF corporation)), (BASF),

And

blue (AB Enzymes Co. (AB Enzymes)),

(Huvepharma, Haowen pharmaceutical Co.)),XP (Verenium/DuPont) and

PHY (DuPont). Other preferred phytases include those described in, for example, WO 98/28408, WO 00/43503 and WO 03/066847.

In a particular embodiment, the composition of the invention comprises a xylanase (EC 3.2.1.8). Examples of commercially available xylanases include

WX andg2 (Disemann Products Co., Ltd.)),

XT and Barley (AB Vista, Inc.),(Verenium, Van Ennimu Co.) of,

X (Huvepharma) andXB (xylanase/β -glucanase, DuPont).

In a particular embodiment, the composition of the invention comprises a protease (EC 3.4). Examples of commercially available proteases include

ProAct (Dismaman Nutrition Products, DSM Nutrition Products).

In one particular embodiment, the compositions of the present invention comprise α -amylase (EC 3.2.1.1.) examples of commercially available α -amylases includeRumistar (imperial nutrition products).

Microorganisms

In one embodiment, the ruminant feed composition further comprises one or more additional microorganisms. In a particular embodiment, the ruminant feed composition further comprises bacteria from one or more of the following genera: lactobacillus, lactococcus, Streptococcus, Bacillus, Pediococcus, enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Mesococcus or any combination thereof.

In a preferred embodiment, the ruminant feed composition further comprises bacteria from one or more of the following strains: bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, bacillus cereus, bacillus pumilus, bacillus polymyxa, bacillus megaterium, bacillus coagulans, bacillus circulans, enterococcus and pediococcus species, Lactobacillus species, Bifidobacterium species, Lactobacillus acidophilus, pediococcus acidilactici, lactococcus lactis, Bifidobacterium bifidum, propionibacterium tereperi, Lactobacillus coli, Lactobacillus rhamnosus, clostridium butyricum, Bifidobacterium animalis subspecies (Bifidobacterium animalis ssp. animalis), Lactobacillus reuteri, Lactobacillus salivarius, escherichia coli, propionibacterium species.

In a more preferred embodiment, the composition, ruminant feed supplement, ruminant feed additive or ruminant feed further comprises a bacterium selected from one or more of the following strains: enterococcus faecium strain 8G-1(NRRL B-50173), enterococcus faecium strain 8G-73(NRRL B-50172), Bacillus pumilus strain 8G-134(NRRL B-50174), Megasphaera elsdenii strain NCIMB 41125, Propionibacterium strain P169(PTA-5271), Propionibacterium strain P170(PTA-5272) strain, Propionibacterium strain P179(NRRL B-50133), Propionibacterium strain P195(NRRL B-50132), Propionibacterium strain P261(NRRL B-50131), Propionibacterium jensenii strain P63(DSM22192), Propionibacterium strain P5(ATCC55467), Propionibacterium strain P54(NRRL B-50494), Propionibacterium strain P25(NRRL B-50497), Propionibacterium strain P49(NRRL B-50496), Propionibacterium strain P104(NRRL B-50495), Bacillus licheniformis strain 3-12a (NRRL B-50504), bacillus subtilis strain 4-7d (NRRL B-50505), Bacillus licheniformis strain 4-2a (NRRL B-50506), Bacillus subtilis strain 3-5h (NRRL B-50507), Bacillus 747(NRRL B-67257), or a strain having all the identifying characteristics of Bacillus 747(NRRL B-67257), Bacillus strain 1104(NRRL B-67258), Bacillus strain 1781(NRRLB-67259), Bacillus strain 1541(NRRL B-67260), Bacillus strain 2018(NRRL B-67261), and Bacillus strain 1999(NRRL B-67318).

In a more preferred embodiment, the composition, ruminant feed supplement, ruminant feed additive, or ruminant feed further comprises bacteria from one or more of the following bacillus subtilis strains: 3A-P4(PTA-6506), 15A-P4(PTA-6507), 22C-P1(PTA-6508), 2084(NRRL B-500130), LSSA01(NRRL-B-50104), BS27(NRRL B-50105), BS 18(NRRL B-50633), BS 278(NRRL B-50634), DSM29870, DSM 29871, NRRL B-50136, NRRL B-50605, NRRL B-50606, NRRL B-50622 and PTA-7547.

In a more preferred embodiment, the composition, ruminant feed supplement, ruminant feed additive or ruminant feed further comprises bacteria from one or more of the following bacillus licheniformis strains: NRRL B50015, NRRL B-50621 or NRRL B-50623.

In a more preferred embodiment, the composition, ruminant feed supplement, ruminant feed additive, or ruminant feed further comprises bacteria from one or more of the following bacillus amyloliquefaciens strains: DSM 29869, DSM 29872, NRRL B50607, PTA-7543, PTA-7549, NRRL B-50349, NRRL B-50606, NRRL B-50013, NRRL B-50151, NRRL B-50141, NRRL B-50147 or NRRL B-50888.

The bacterial count of each bacterial strain in the ruminant feed composition is 1x10⁴And 1x10¹⁴CFU/kg dry matter, preferably 1X10⁶And 1x10¹²CFU/kg dry matter, and more preferably at 1x10⁷To 1x10¹¹CFU/kg dry matter. In a more preferred embodiment, the bacterial count of each bacterial strain in the ruminant feed composition is at 1x10⁸And 1x10¹⁰CFU/kg dry matter.

The bacterial count of each bacterial strain in the ruminant feed composition is 1x10⁵And 1x10¹⁵CFU/ruminant/day, preferably at 1x10⁷And 1x10¹³Between CFU/ruminant/day, and more preferably at 1x10⁸And 1x10¹²CFU/ruminant/day. In a more preferred embodiment, the bacterial count of each bacterial strain in the ruminant feed composition is at 1x10⁹And 1x10¹¹CFU/ruminant/day.

In another embodiment, the one or more bacterial strains are present in a stable spore form.

Premix compound

In one embodiment, the ruminant feed may include a premix including, for example, vitamins, minerals, enzymes, amino acids, preservatives, antibiotics, other feed ingredients, or any combination thereof, mixed into the ruminant feed.

Amino acids

The compositions of the present invention may further comprise one or more amino acids. Examples of amino acids used in ruminant feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan.

Vitamins and minerals

In another embodiment, the ruminant feed may comprise one or more vitamins, such as one or more fat soluble vitamins and/or one or more water soluble vitamins. In another embodiment, the ruminant feed may optionally comprise one or more minerals, such as one or more trace minerals and/or one or more macrominerals.

Usually, fat-soluble vitamins and water-soluble vitamins and trace minerals form part of a so-called premix intended to be added to the feed, whereas the macrominerals are usually added separately to the feed.

Non-limiting examples of fat-soluble vitamins include vitamin a, vitamin D3, vitamin E, and vitamin K, such as vitamin K3.

Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, such as Ca-D-pantothenate.

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

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

In an alternative, the ruminant feed supplement or ruminant feed additive of the present invention comprises at least one of the individual components specified in table a of "nutritional requirements for ruminants [ nutrients in nutrients ]" seventh revision 2001, the committee for ruminant nutrition groups, the committee for ruminant nutrition, the council for agricultural committees, the National research council, the american National Academy of sciences Press ], washington, d.c. WO 01/58275. By at least one is meant any one, one or more of one or two or three or four etc. up to all thirteen or up to all fifteen separate components. More specifically, this at least one individual component is comprised in the additive of the invention in an amount to provide an in-feed-concentration (in-feed-concentration) within the ranges specified in the fourth or fifth or sixth column of table a.

Other feed ingredients

The composition of the present invention may further comprise natural or synthetic colorants, intestinal flora stabilizers, pH stabilizers/pH modifiers, digestibility enhancers, growth improving additives, aroma compounds/flavors, polyunsaturated fatty acids (PUFAs); essential oils, active oxygen producing substances, antifungal peptides, antifungal polypeptides, antimicrobial peptides, fungal fermentation extracts and cultures, immune modulating additives, antioxidant additives, metabolic enhancers, rumen fermentation modifiers, electron acceptors and rumen catalysts, other functional additives (zo/technical additives) (e.g., adhesives, anti-caking and coagulants, ammonia gas control agents, phyto-antimicrobials, anti-methanogens, and/or ionophores).

Examples of colorants include, but are not limited to, carotenoids, such as beta-carotene, astaxanthin, and lutein.

Examples of intestinal flora stabilizers and/or pH stabilizers include, but are not limited to, live yeast or yeast cultures, such as saccharomyces cerevisiae.

Examples of digestibility enhancers include, but are not limited to, enzymes such as alpha-amylase.

Examples of aromas/flavors include, but are not limited to, creosol, anethole, deca, undecalactone, ionone, irone, gingerol, piperidine, propylidene phthalide (propylidene phalide), butylidene phthalide (butylidene phalide), capsaicin, or tannin.

Examples of polyunsaturated fatty acids include, but are not limited to, C18, C20, and C22 polyunsaturated fatty acids, such as arachidonic acid, docosahexaenoic acid, eicosapentaenoic acid, and gamma-linoleic acid.

Examples of essential oils include, but are not limited to, anise, juniper, capsicum, cinnamon, clove, dill, garlic, eugenol or cinnamaldehyde and their active ingredients.

Examples of active oxygen generating substances include, but are not limited to, chemicals such as perborate, persulfate, or percarbonate; and enzymes such as oxidases, oxygenases or synthetases. Eicosatetraenoic acid, docosahexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of antifungal polypeptides (AFPs) include, but are not limited to, peptides of aspergillus megaterium and aspergillus niger, as well as variants and fragments thereof that retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384.

Examples of stabilizing agents (e.g., buffering agents and/or acidifying agents) include, but are not limited to, live yeast, sodium bicarbonate, calcareous seaweed, and lecithin

Examples of antimicrobial peptides (AMPs) include, but are not limited to, CAP18, lincomycin (Leucocin) a, trichrome (tritrpicin), Protegrin-1, Thanatin (Thanatin), defensins, lactoferrin, lactoferricin, and Ovispirin (Ovispirin), such as Novispirin (Novispirin) (Robert Lehrer,2000), myceliophycin (Plectasin), statins (including compounds and polypeptides disclosed in WO 03/044049 and WO 03/048148), and variants or fragments thereof that retain antimicrobial activity.

Examples of immunomodulators include, but are not limited to, B-glucan, Saccharomyces cerevisiae.

Examples of antioxidants include, but are not limited to, vitamin A, E and other natural antioxidants; such as lecithin.

Examples of electron acceptor agents include, but are not limited to, nitrates and organic compounds thereof.

Examples of fungal fermentation extracts and cultures include, but are not limited to, Aspergillus oryzae sold as Amaferm Vitaferm (Bio-enzyme Enterprise).

Examples of anticaking agents and binders include, but are not limited to, synthetic calcium aluminates.

Examples of functional additives include, for example, ammonia control.

Examples of ionophores include monensin, such as, for example, from the lilac company (Elanco)

The composition of the present invention may further comprise at least one amino acid. Examples of amino acids used in ruminant feed include, but are not limited to, lysine, alanine, beta-alanine, threonine, methionine, and tryptophan.

Use of muramidase to improve ruminant performance

In another aspect, the invention relates to the use of a ruminant feed supplement, or a ruminant feed for improving the Feed Conversion Ratio (FCR) of a ruminant, wherein the ruminant feed, ruminant feed supplement, or ruminant feed supplement comprises one or more muramidase enzymes, wherein the muramidase enzymes are administered at a level of 1 to 200mg enzyme protein/kg ruminant feed DM.

In a preferred embodiment, the improvement is compared to a ruminant feed, ruminant feed supplement, or ruminant feed supplement (referred to herein as a negative control) in which the muramidase is not present.

In one embodiment, the FCR is improved by at least 1%, e.g., at least 1.25%, at least 1.5%, at least 1.75%, or at least 2.0% as compared to a control. In another embodiment, the FCR improvement is between 1% and 5%, such as between 1% and 4%, between 1% and 3%, between 1.25% and 2.5%, between 1.5% and 2%, or any combination of these intervals, as compared to a control.

In one embodiment, the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels, and llamas. In a preferred embodiment, the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos and calves. In a more preferred embodiment, the ruminant is selected from the group consisting of: cattle, dairy cows and beef cattle.

In one embodiment, muramidase is provided to a growing ruminant. In one embodiment, muramidase is provided to the cow. In one embodiment, muramidase is provided to beef cattle during the growth phase of beef cattle production. In one embodiment, muramidase is provided to beef cattle at the completion stage of beef cattle production. In another embodiment, muramidase is provided to the calf in milk.

In one embodiment, the muramidase is of microbial origin. In another embodiment, the muramidase is of fungal origin. In one embodiment, the muramidase is obtained or obtainable from a phylum ascomycota, such as the subphylum discodermatum.

Preferred embodiments

The following is a list of preferred embodiments encompassed by the present invention:

1. a ruminant feed composition, such as a ruminant feed, a ruminant feed supplement, or a ruminant feed supplement, comprising one or more muramidase enzymes, wherein the muramidase enzyme is in an amount sufficient to be administered at a level of 1 to 200mg enzyme protein per kg of ruminant feed.

2. The ruminant feed composition of example 1, wherein the muramidase is administered at a level of from 1 to 200mg enzyme protein per kg dry matter of ruminant feed.

3. The ruminant feed composition of embodiment 1 or 2, wherein the muramidase is administered at a level of: from 5 to 150mg, from 5 to 125mg, from 5 to 100mg, from 5 to 75mg, from 5 to 50mg, from 5 to 40mg, from 10 to 50, from 5 to 25mg enzyme protein per kg dry matter of ruminant feed, or any combination of these intervals.

4. The ruminant animal feed composition of any of embodiments 1-3, wherein the Energy Corrected Milk (ECM) production of the ruminant animal is improved by at least 1.0%, preferably at least 1.5%, more preferably at least 2.0% after administration compared to a control.

5. The ruminant animal feed composition of any one of embodiments 1-4, wherein the ruminant animal is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels and llamas.

6. The ruminant animal feed composition of any of embodiments 1-5, wherein the ruminant animal is selected from the group consisting of: cattle, dairy cows and beef cattle.

7. The ruminant animal feed composition of any of embodiments 1-6, wherein the muramidase is provided to the ruminant animal daily during the lifetime of the ruminant animal.

8. The ruminant feed composition of any of embodiments 1-7, wherein the muramidase is of microbial origin.

9. The ruminant animal feed composition of embodiment 8, wherein the muramidase is of fungal origin.

10. The ruminant feed composition of any of embodiments 1-9, wherein the muramidase is obtained or obtainable from ascomycota.

11. The ruminant feed composition of any of embodiments 1-10, wherein the muramidase is obtained or obtainable from a subphylum sclerotinia.

12. The ruminant feed composition of any of embodiments 1-11, wherein the muramidase comprises one or more domains from a Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and a novel MUR polypeptide having muramidase activity.

13. The ruminant feed composition of any of embodiments 1-12, wherein the muramidase is selected from the group consisting of:

(a) a polypeptide having at least 50%, e.g., at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 to an amino acid sequence selected from the group consisting of: 3, 6, 9, 12, 15, 18, 21, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, and 59 SEQ ID NO;

(b) A variant of an amino acid sequence selected from the group consisting of: 3, 6, 9, 12, 15, 18, 21, 24, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, and 59, wherein the variant has muramidase activity and comprises one or more amino acid substitutions and/or one or more amino acid deletions and/or one or more amino acid insertions or any combination thereof in positions 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, 45, 46, 47, 48, 49, or 50;

(c) A fragment of the polypeptide of (a) or (b) having muramidase activity, wherein the fragment comprises at least 170 amino acids, such as at least 175 amino acids, at least 177 amino acids, at least 180 amino acids, at least 185 amino acids, at least 190 amino acids, at least 195 amino acids or at least 200 amino acids.

14. The ruminant feed composition of any of embodiments 1-13, wherein the ruminant feed composition further comprises one or more components selected from the list consisting of:

one or more carriers;

one or more additional enzymes;

one or more microorganisms;

one or more vitamins;

one or more minerals;

one or more amino acids;

one or more organic acids; and

one or more other feed ingredients.

15. The ruminant feed composition of any of embodiments 1-14, wherein the muramidase is in a granular form.

16. The ruminant animal feed composition of embodiment 15, wherein the pellet is coated.

17. The ruminant animal feed composition of embodiment 16, wherein the coating comprises a salt and/or a wax and/or a flour.

18. The ruminant feed composition of any of embodiments 1-17, wherein the muramidase is in a liquid formulation.

19. The ruminant feed composition of embodiment 18, wherein the liquid formulation is sprayed onto the feed after the feed has been pelletized.

20. The ruminant feed composition of any of embodiments 4-19, wherein the control is a ruminant feed composition that does not comprise a muramidase.

21. The ruminant feed composition of any of embodiments 4-20, wherein the control is a ruminant feed composition that does not comprise GH24 muramidase, GH25 muramidase, or a novel MUR polypeptide having muramidase activity.

22. The ruminant feed composition of any of embodiments 4-21, wherein the control is a ruminant feed composition comprising Hen Egg White Lysozyme (HEWL).

23. The ruminant feed composition of any of embodiments 4-21, wherein the control is monensin.

24. The ruminant feed composition of any of embodiments 1-23, wherein the ruminant feed composition comprising a muramidase is administered to a ruminant selected from the group consisting of: growing ruminants, dairy cows, beef cattle at the growth stage of beef production, beef cattle at the completion stage of beef production, and calves.

25. The ruminant feed composition of any of embodiments 1-24, wherein the ruminant feed composition comprising a muramidase is administered to a growing ruminant.

26. The ruminant animal feed composition of any of embodiments 1-24, wherein the ruminant animal feed composition comprising a muramidase is administered to a cow.

27. The ruminant feed composition of any of embodiments 1-24, wherein the ruminant feed composition comprising the muramidase is administered to a beef cattle at an increased stage of beef cattle production.

28. The ruminant animal feed composition of any of embodiments 1-24, wherein the ruminant animal feed composition comprising the muramidase is administered to a beef cattle at a completed stage of beef cattle production.

29. The ruminant feed composition of any of embodiments 1-24, wherein the ruminant feed composition comprising the muramidase is administered to a calf.

30. A method of improving Energy Corrected Milk (ECM) production in a ruminant, the method comprising administering to a ruminant feed composition as in any of embodiments 1-29.

31. A method of increasing dry matter digestibility (DMd) of a ruminant feed, a ruminant feed supplement, or a ruminant feed additive, the method comprising the steps of: a) providing at least one muramidase; b) providing a ruminant feed, a ruminant feed supplement, or a ruminant feed supplement suitable for a ruminant; c) applying a muramidase to a ruminant feed, a ruminant feed supplement, or a ruminant feed additive to form a ruminant feed composition; and d) feeding the ruminant feed composition, thereby achieving an increase in digestibility of the dry matter.

32. The method of embodiment 31 wherein DMd are measured according to example 2.

33. The method of any one of embodiments 31-32, wherein DMd is increased as compared to DMd in the feed prepared as in example 31 but without the muramidase added in step c).

34. The method of any one of embodiments 31-33, wherein the production of Volatile Fatty Acids (VFAs) in the rumen is increased as compared to the production of VFAs in the rumen of a ruminant that is not fed muramidase.

35. The method of any one of embodiments 31-34, wherein propionate production in the rumen is increased as compared to propionate production in the rumen of a ruminant that is not fed muramidase.

36. The method of any one of embodiments 31-35, wherein acetate production in the rumen is increased as compared to acetate production in the rumen of a ruminant that is not fed muramidase.

37. The method of any one of embodiments 31-36, wherein muramidase is administered at a level of from 1 to 310mg enzyme protein/kg ruminant feed dry matter.

38. The method of any one of embodiments 31-37, wherein the muramidase is administered at a level of: from 5 to 150mg, from 5 to 125mg, from 5 to 100mg, from 5 to 75mg, from 5 to 50mg, from 5 to 40mg, from 10 to 50, from 5 to 25mg enzyme protein per kg dry matter of ruminant feed, or any combination of these intervals.

39. The method of any one of embodiments 31 to 38, wherein Energy Corrected Milk (ECM) production by a ruminant is improved by at least 1.0%, preferably at least 1.5%, more preferably at least 2.0% after administration compared to a control.

40. The method of any one of embodiments 31-39, wherein the ruminant is selected from the group consisting of: cattle, cows, beef cattle, buffalos, calves, goats, sheep, lambs, deer, yaks, camels and llamas.

41. The method of any one of embodiments 31-40, wherein the ruminant is selected from the group consisting of: cattle, dairy cows and beef cattle.

42. The method of any one of embodiments 31-41, wherein muramidase is provided to the ruminant every day during the lifetime of the ruminant.

43. The method of any one of embodiments 31-42, wherein the muramidase is of microbial origin.

44. The method of any one of embodiments 31-42, wherein the muramidase is of fungal origin.

45. The method of any one of embodiments 31-44, wherein the muramidase is obtained or obtainable from Ascomycota.

46. The method of any one of embodiments 31-45, wherein the muramidase is obtained or obtainable from the subphylum discodermatum.

47. The method of any one of embodiments 31-46, wherein the muramidase comprises one or more domains from a Glycoside Hydrolase (GH) family selected from the list consisting of GH24, GH25, and a novel MUR polypeptide having muramidase activity.

48. The method of any one of embodiments 31-47, wherein the muramidase is selected from the group consisting of:

49. The method of any one of embodiments 31-48, wherein the muramidase is in a granular form.

50. The method of embodiment 49, wherein the particles are coated.

51. The method of embodiment 50, wherein the coating comprises salt and/or wax and/or flour.

52. The method of any one of embodiments 31-51, wherein the muramidase is in the form of a liquid formulation.

53. The method of embodiment 52 wherein the liquid formulation is sprayed onto the feed after the feed has been pelleted.

54. The method of any one of embodiments 39 to 53, wherein the control is a ruminant feed composition that does not comprise muramidase.

55. The method of any one of embodiments 39 to 53, wherein the control is a ruminant feed composition that does not comprise GH24 muramidase, GH25 muramidase, or a novel MUR polypeptide having muramidase activity.

56. The method of any one of embodiments 39 to 55, wherein the control is a ruminant feed composition comprising Hen Egg White Lysozyme (HEWL).

57. The method of any one of embodiments 39 to 55, wherein the control is monensin.

58. The method of any one of embodiments 31-57, wherein the ruminant animal feed composition comprising a muramidase is administered to a ruminant animal selected from the group consisting of: growing ruminants, dairy cows, beef cattle at the growth stage of beef production, beef cattle at the completion stage of beef production, and calves.

59. The method of any one of embodiments 31-58, wherein the ruminant feed composition comprising a muramidase is administered to a growing ruminant.

60. The method of any one of embodiments 31-58, wherein the ruminant animal feed composition comprising muramidase is administered to a cow.

61. The method of any one of embodiments 31-58, wherein the ruminant animal feed composition comprising a muramidase is administered to a beef cattle at an increased stage of beef cattle production.

62. The method of any one of embodiments 31-58, wherein the ruminant animal feed composition comprising the muramidase is administered to a beef cattle at a completion stage of beef cattle production.

63. The method of any one of embodiments 31-58, wherein the ruminant animal feed composition comprising a muramidase is administered to a calf.

Examples of the invention

Muramidase was cloned, expressed, characterized and tested for muramidase activity as described in WO 2013/076253.

Example 1-Effect of muramidase on rumen fermentation gas yield and feed Dry matter digestibility

The material and the method are as follows:

in vitro fermentation models were adapted from Menke and Steingass (Menke KH, Steingass H.) (the expression of the genetic feed expressed from chemical analysis and in vitro gapduction using a rumen fluid [ estimation of the high energy feed value obtained from chemical analysis and in vitro gas production using rumen fluid ] Anim Res Dev. [ animal research and development ] (1988)28: 7-55). The experimental design included treatment of negative controls (NC, feed only and rumen buffer), positive controls (PC, feed and rumen buffer with addition of commercial rumen modifier (monensin)), muramidase (SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24 and 27, negative control with muramidase), and blank (rumen buffer only), table 1. This experiment was repeated 2 times (experiment 1 and experiment 2) to increase the efficacy of the overall study.

TABLE 1

The method is based on the incubation of ruminant feed in buffer and rumen fluid solution for a period of 48h while obtaining the cumulative gas production and digestibility of feed DM. The buffered mineral solution used was prepared according to Menke and Steingass (1988) and heated in a water bath at 39 ℃ and treated with CO₂Purging was continued for 60 minutes. Sodium sulfite was used as a reducing agent in a buffer solution (0.33g/l solution). Rumen fluid was collected from 2 zeiss (Jersey) heifers (housed at an experimental farm at the university of copenhagen, denmark). Rumen fluid was collected by rumen intubation, poured into two hot flasks preheated to 39.0 ℃ ± 0.5 ℃ and immediately transferred to the laboratory. Rumen fluid was filtered through a 3-layer coarse filter cloth to eliminate feed particles and mixed with a buffered mineral solution at a ratio of 1 to 2 (Menke and Steingass, 1988).

90mL rumen fluid and buffer solution were dispensed into a Dulan flask fitted with a lid equipped with a wireless pressure sensor and gas valve (Ankom RF gas production System, Ankom

Maston (Macedon), N.Y., USA). After filling with 0.5000 ± 0.0010g of feed sample (corn silage, soybean meal cake in a ratio of 2:1 on dry matter) and preheating at 39 ℃ overnight. To a Duram flask was added 1.00mL of treatment solution (water or muramidase solution, or positive) Control solution), applied and mixed prior to closing the flask. The enzyme solutions (A to I) were all produced by Novixin and were administered in terms of mg of enzyme protein per kg of feed. Table 1 monensin (monensin sodium salt hydrate, batch BCBR9717V, sigma ornithch, Buchs, switzerland) was used as PC at a concentration of 22mg/kg feed, table 1. In use of CO₂All procedures were performed with continuous purging. The headspace of each flask after filling was 41 mL. Fermentation gas was automatically released from the flask through a gas valve each time the gas pressure increased by 0.75 PSI. Using the pressure differential associated with opening the gas valve, the cumulative gas production (Δ P) is calculated using the ideal gas law as follows: GP ═ Δ P/Po)' Vo (1), where: Δ P is the cumulative gas pressure change (kPa) in the bottle headspace; vo is the bottle headspace volume (41mL) and Po is atmospheric pressure. The data for gas production are expressed as ml/g DM incubated at standard pressure and are not included in the present example, but are used to ensure sufficient fermentation activity. The dry matter after fermentation was measured and correlated with the initial dry matter in the flask to determine the dry matter digestibility as (pre dry matter-post dry matter/pre dry matter). When considering blank background fermentations, the data are expressed as a relative improvement of DMd compared to the control. The initial model, including treatments, trials and interactions, was reduced to the main effect of the treatment after model evaluation by statistical analysis of the data based on ANOVA program in JMP 12.1.0(SAS institute) and expressed as LS mean.

Results

The results for relative DMd are presented in fig. 1 and show that the addition of muramidase enhances 48h in vitro rumen digestion of feed dry matter compared to the positive control (monensin). The data also show that addition of some muramidase can increase DMd in ruminant feed compared to NC.

Conclusion

In summary, some muramidase enzymes from 3 different classes showed improved rumen dry matter digestibility compared to the commonly used rumen additive monensin in two replicates of the same study. The data also show that most muramidase enzymes (compared to NC) improved rumen feed dry matter digestibility.

Examples 2-5 dose response Effect of muramidase on rumen fermentation and feed Dry matter digestibility

To summarize:

in vitro rumen fermentation using rumen fluid and buffer showed that muramidase can increase digestibility of dry matter, increase the yield of rumen acetate, propionate, total VFA, and increase total carbon pool in rumen VFA.

The material and the method are as follows:

the experiment was performed by Alimetrics (Alimetrics limited, Koskelontie 19B, FIN-02920 epsbo (Espoo), finland) using a 120mL serum bottle as the fermentation vessel. The fermentation preparation procedure is presented in Kettlen et al (Kettlen H., J.Vuorenmaa, D.Gaffney and J.Apajalahti (2016): Yeast hydrostate product enhancement in vitro rumen fermentation in vitro [ Yeast hydrolysate enhances rumen fermentation ]J.App.anim.Nutr. [ J.App.animal Nutrition]Volume 4; e 1; page 1, total 7). Briefly, the method is as follows: first, all bottles were filled with 1g of feed DM consisting of corn silage, barley flour, soybean flour in amounts of 0.5, 0.4 and 0.1g DM, respectively. Subsequently, the bottles were CO-treated₂(passing through a hot copper catalyst to scavenge O₂) Rinsed and sealed with a thick butyl rubber plug. Anaerobic, reduced and temperature-adjusted (+38 ℃) artificial saliva buffer solution (modified from agro manual No. 379 (department of agriculture, washington, d.1975), volume 37.65ml), freshly filtered rumen fluid (5% inoculum; volume 2ml), and test compound (volume 0.35ml) in anaerobic CO₂Rumen fluid was pumped directly from the rumen into a preheated thermos bottle and immediately closed and transferred to Alimetrics laboratories.

After inoculation of rumen fluid, buffer and test solution, the vessel was sealed with a butyl rubber septum. All fermentation vessels were inoculated in random order to prevent possible retardation. The inoculation time for each fermentation vessel was recorded and taken into account at each sampling to ensure that the fermentation duration was the same for each sample. The fermentation was continued for 12h in a rotary shaker at +38 ℃. At times 3, 6, 9 and 12h, the fermentation gas produced was measured and regenerated by collecting the fermentation gas in the syringe. The feed residue was measured after 12h fermentation.

The experimental design was a dose response, completely random design according to table 2 using 28 treatments with 150 fermenters. The 25 muramidase solutions were diluted to the required concentration of enzyme buffer (BSA, Tween 20, acetate buffer, calcium, NaOH, adjusted to pH 6). The negative control was supplemented with the same buffer used to dilute the enzymes to reach the same liquid volume as the supplemented fermentation vessel. The ionophore monensin (monensin sodium salt, 90% -95% TLC (Sigma-Oreochi; product code: M5273-1G)) was chosen as a positive control because it is a ruminant feed additive that affects the production of Volatile Fatty Acids (VFA).

Table 2: treatment, dose and study design

Sample collection

H.E.Valken and J.Apajalahti (2015) Natural resin acid-induced composition as a modulator of intestinal microbial and performance enhancing in broiler chicken [ Regulation of intestinal flora as broiler chickenNatural resin acid-rich compositions of moderating and performance enhancing agents]J.App.anim.Nutr. [ J.App.animal Nutrition]Volume 3; e 2; page 1 (total page 9)).

After 12h of fermentation, the digestibility of the dry matter was quantified by measuring the dry matter in the initial feed substrate and all fermentation vessels. The feed residue of the fermentation vessel was obtained by filtration through a tared sintered glass filter, washed with water and dried at 105 ℃ for 12 hours. Finally, the residual dry matter of the feed was weighed on a glass filter.

Computing and statistics

Dry matter digestibility was calculated as the ratio between feed residue and fed dry matter after final fermentation. The sum of VFA was calculated as the sum of acetate, propionate, butyrate and valerate, and the total carbon in the VFA was calculated by assigning the number of carbon atoms for each of acetate, propionate, butyrate and valerate to 2, 3, 4 and 5. The relative improvement in DMd and the concentration of total carbon in acetate, propionate, butyrate, total VFA, and VFA compared to NC was calculated.

Data were analyzed for linear and quadratic effects using a linear regression algorithm based on the proc mixing program of SAS (SAS institute). Unless otherwise stated, the data presented are linear regression estimates and standard error, as well as least squares means and standard error.

As a result:

the data show that muramidase can affect rumen dry matter digestibility and VFA production in vitro, fig. 2-7. All muramidase treatments increased DMd when compared to the control. Treatment C had the greatest improvement (9.5%) when supplemented with 0.8mg/40mL in the fermentation.

All muramidase treatments numerically increased rumen acetate and propionate production compared to the control. When a was treated with 0.4mg/40mL supplementation, acetate increased 16.0% and propionate increased 24% at the same dose, which resulted in an 11% increase in total VFA production (fig. 3 and fig. 4), respectively. Treatments a and B also reduced rumen butyrate production compared to controls, fig. 5. However, despite the reduction in butyrate, the carbon in VFA was still positively affected, with an improvement of up to 7.3% over NC, fig. 7.

Linear regression analysis identified that treatments A, B and C were most effective in increasing rumen acetate and propionic acid production. Linear regression analysis also identified treatments A, B and C as being most effective in reducing ruminal butyrate production. The effect of the doses of treatment A, B and C on acetate and propionate was secondary. The dose of muramidase had a secondary effect on total ruminal VFA production and total carbon in ruminal VFA for treatment C.

And (4) conclusion:

conclusion the data indicate that muramidase can increase digestibility of dry matter and increase the production of rumen acetate, propionate, total VFA and increase the total carbon pool in rumen VFA.

Examples 3-13 muramidase Effect on in vitro rumen fermentation and feed Dry matter digestibility

To summarize:

fermentation studies were performed using 13 muramidase enzymes from the Glycoside Hydrolase (GH) families GH24, GH25 and a novel MUR polypeptide having muramidase activity, the number of enzymes tested being 5, 4 and 4, respectively. In vitro fermentation was performed using rumen fluid and artificial saliva in a 120mL fermentor. It is hypothesized that muramidase can increase the yield of rumen fermentation products.

Taken together, the data indicate that muramidase from Glycoside Hydrolase (GH) families GH24, GH25 and novel MUR polypeptides having muramidase activity can affect the digestibility of ruminal dry matter and ruminal fermentation by increasing the production of propionate, total volatile fatty acids and total carbon in volatile fatty acids.

The material and the method are as follows:

The experimental design was a dose response, completely random design according to table 3 using 28 treatments with 150 fermenters. The 13 muramidase solutions were diluted to the required concentration of enzyme buffer (BSA, Tween 20, acetate buffer, calcium, NaOH, adjusted to pH 6). The negative control was supplemented with the same buffer used to dilute the enzymes to reach the same liquid volume as the supplemented fermentation vessel. The ionophore monensin (monensin sodium salt, 90% -95% TLC (Sigma-Oreochi; product code: M5273-1G)) was chosen as a positive control because it is a ruminant feed additive that affects the production of Volatile Fatty Acids (VFA).

Table 3: treatment, dose and study design

Sample collection

Natural resin acid-enriched compositions as a modulator of intestinal microbiota and performance enhancer for broiler chickens [ Natural resin acid-enriched compositions as modulators and performance enhancers]J.App.anim.Nutr. [ J.App.animal Nutrition]Volume 3; e 2; page 1 (total page 9)).

Computing and statistics

Dry matter digestibility was calculated as the ratio between feed residue and fed dry matter after final fermentation (DMd). The sum of VFA was calculated as the sum of acetate, propionate, butyrate and valerate, and the total carbon in VFA was calculated by assigning the number of carbon atoms of each of acetate, propionate, butyrate and valerate to 2, 3, 4 and 5, respectively. The data were calculated as the relative improvement in DM digestibility and the concentration of total carbon in acetate, propionate, butyrate, total VFA, and VFA compared to NC.

The data is divided into two data sets. Data set one including data from NC treatment and all muramidase treatment (dose 0.20mg/40mL) and PC (dose 0.01mg/40mL) was analyzed using the mixing program of SAS (SAS institute) and treatment was the primary effect. Data set two, including data from all 5 doses from treatments A, B and C, was subjected to linear and quadratic effects analysis using a linear regression algorithm based on the proc mixing program of SAS (SAS institute). Unless otherwise stated, the data presented are linear regression estimates and standard error, as well as least squares means and standard error.

As a result:

data from the current study show that muramidase can affect rumen DMd and VFA production in vitro, table 4 and figures 8-10. The DMd difference between unsupplemented NC and muramidase treatment was positive in 10 of 13 muramidase enzymes when evaluated at the same dose (0.2mg/40 mL). DMd the maximum improvement was 8.6%, FIG. 8. The increased response to DMd is evenly distributed over the 3 Glycoside Hydrolase (GH) families (GH24, GH25 and the novel MUR polypeptides with muramidase activity defined herein, 5/5, 2/4 and 3/4 respectively). The data also show that muramidase supplementation has a significant effect on propionate production. When evaluated at the same dose (0.2mg/40mL), 9 of the 13 muramidases increased propionate production compared to NC. The propionate production increased up to 14.4%, fig. 9. The improved response to propionate production is divided by the Glycoside Hydrolase (GH) families GH24, GH25 and a novel MUR polypeptide with muramidase activity, in this order (5/5, 3/4 and 1/4, respectively). Muramidase treatment in 9 of 13 muramidase enzymes reduced butyrate production compared to control when evaluated at the same dose (0.2mg/40 mL). The decline in butyrate production was as high as 49.3%, fig. 10. The response to decreased butyrate production is divided by the Glycoside Hydrolase (GH) families GH24, GH25 and a novel MUR polypeptide with muramidase activity, (4/5, 2/4 and 3/4, respectively). The effect of supplementation with 0.2mg muramidase/40 mL increased the total yield of VFA and increased total carbon in VFA for 8 of 13 treatments when compared to the control, table 4. The total VFA, and the increase in total carbon in VFA, are divided by the Glycoside Hydrolase (GH) families GH24, GH25, and a novel MUR polypeptide with muramidase activity, in that order (4/5, 2/4, and 2/4, respectively).

Regression analysis was performed for three muramidases A, B and C. Regression analysis showed that propionate production increased with increasing muramidase dose and butyrate production decreased with increasing muramidase dose, as also observed in fig. 9 and 10, respectively.

Table 4: effect of 13 muramidases (dose 0.2mg/40mL) on Dry matter digestibility and VFA production (mmol/L) after 12h rumen fermentation in vitro

And (4) conclusion:

taken together, the data indicate that muramidase from Glycoside Hydrolase (GH) families GH24, GH25 and novel MUR polypeptides having muramidase activity can affect the digestibility of ruminal dry matter and ruminal fermentation by increasing the production of propionate, total VFA, and total carbon in VFA.

Examples 4-22 muramidase effects on rumen fermentation in vitro and feed Dry matter digestibility

To summarize:

fermentation studies were performed using muramidase from Glycoside Hydrolases (GH) families GH24, GH25 and a novel MUR polypeptide having muramidase activity, the number of enzymes tested being 9, 8 and 7, respectively. In vitro fermentation was performed using rumen fluid and artificial saliva in a 120mL fermentor. It is hypothesized that muramidase can increase the yield of rumen fermentation products.

In summary, rumen fermentation is improved by supplementation with muramidase from three Glycoside Hydrolases (GH) families GH24, GH25 and a novel MUR polypeptide having muramidase activity. This improvement was observed as an increase in the production of total volatile fatty acids, acetate and propionate.

The material and the method are as follows:

the experiment was performed by Alimetrics (Alimetrics limited, Koskelontie 19B, FIN-02920 epsbo (Espoo), finland) using a 120mL serum bottle as the fermentation vessel. The fermentation preparation procedure is presented in Kettlen et al (Kettlen H., J.Vuorenmaa, D.Gaffney and J.Apajalahti (2016): Yeast hydrostate product enhancement in vitro rumen fermentation in vitro [ Yeast hydrolysate enhances rumen fermentation]J.App.anim.Nutr. [ J.App.animal Nutrition]Volume 4; e 1; page 1, total 7). Briefly, the method is as follows: first, all bottles were filled with 1g of feed DM consisting of corn silage, barley flour, soybean flour in amounts of 0.5, 0.4 and 0.1g DM, respectively. Subsequently, the bottles were CO-treated₂(passing through a hot copper catalyst to scavenge O₂) Rinsed and sealed with a thick butyl rubber plug. Anaerobic, reduced and temperature-adjusted (+38 ℃) artificial saliva buffer solution (modified from agro manual No. 379 (department of agriculture, washington, d.1975), volume 37.65ml), freshly filtered rumen fluid (5% inoculum; volume 2ml), and test compound (volume 0.35ml) in anaerobic CO₂Rumen fluid was pumped directly from the rumen into a preheated thermos bottle and immediately closed and transferred to Alimetrics laboratories.

The experimental design was a dose response, completely random design using 27 treatments with 145 fermenters according to table 5. The 24 muramidase solutions were diluted to the required concentration of enzyme buffer (BSA, Tween 20, acetate buffer, calcium, NaOH, adjusted to pH 6). The negative control was supplemented with the same buffer used to dilute the enzymes to reach the same liquid volume as the supplemented fermentation vessel. The ionophore monensin (monensin sodium salt, 90% -95% TLC (Sigma-Oreochi; product code: M5273-1G)) was chosen as a positive control because it is a ruminant feed additive that affects the production of Volatile Fatty Acids (VFA).

Table 5: treatment, dose and study design

Sample collection

Liquid samples obtained at 12h were analyzed for VFA (acetate, propionate, butyrate and valerate) concentration (ketstunn h., j.vuorenmaa, T) by GC-FID using a glass column packed with 80/120carbopack B-DA/4% Carbowax solid phase and helium gas as carrier gas.H.Natural resin acid-enriched compositions as a modulator of intestinal microbiota and performance enhancer for broiler chickens [ Natural resin acid-enriched compositions as modulators and performance enhancers]J.App.anim.Nutr. [ J.App.animal Nutrition]Volume 3; e 2; page 1 (total page 9)).

Computing and statistics

Dry matter digestibility was calculated as the ratio between feed residue and fed dry matter after final fermentation (DMd). The sum of VFA was calculated as the sum of acetate, propionate, butyrate and valerate, and the total carbon in the VFA was calculated by assigning the number of carbon atoms of each of acetate, propionate and butyrate to 2, 3, 4 and 5, respectively. The data were calculated as relative improvement in DMd and the concentration of total carbon in acetate, propionate, total VFA, and VFA compared to NC.

The data set was processed in that the amount of enzyme used was unknown. Thus, data from this process cannot be compared to other processes in this study.

Data were analyzed using a mixed program of SAS (SAS institute), which included the primary effects of processing.

Unless otherwise noted, the data lines presented are the least squares mean and standard error.

As a result:

the data show that muramidase supplementation improved rumen fermentation in vitro. This can be shown from the increase in total carbon in 23 total rumen VFA and VFA of the 24 muramidase treatments, table 6. Positive responses were evenly divided by three enzyme Glycoside Hydrolases (GH) families (GH24, GH25 and a novel MUR polypeptide with muramidase activity). Fig. 13 and 14 show up to 12.2% ± 1.99% increase in total VFA, and up to 12.5% ± 2.02% increase in total carbon in VFA, respectively. The increase in total carbon in total VFA and VFA was derived from the increase in rumen acetate and propionate, table 6. Fig. 13 and 14 show up to 10.7% ± 1.99% increase in acetate fermentation and 14.2% ± 2.21% increase in propionic acid fermentation, respectively.

Table 6: effect of 24 muramidases on Dry matter digestibility (DMd) and Volatile Fatty Acid (VFA) yield (mmol/L) after 12h rumen fermentation in vitro

Item	DMd	Acetic acid salt	Propionate salts	Butyric acid salt	Total VFA	Carbon in VFA
							NC	0.58	48.4	39.5	7.1	96	246
PC	0.51	44.2	42.8	3.7	91	234
							A	0.56	52.9	45.1	6.7	105	271
B	0.60	49.1	40.4	7.2	97	251
							C	0.58	52.0	44.3	7.2	104	269
D	0.57	50.1	43.0	5.9	99	255
							E	0.57	49.9	42.3	6.2	99	254
F	0.58	51.6	42.5	7.2	102	263
							G	0.56	49.7	41.1	7.3	99	255
H	0.59	52.6	44.7	5.3	103	264
							I	0.56	49.3	42.9	5.4	98	252
J	0.56	47.6	39.1	6.9	94	243
							K	0.57	49.5	40.9	7.2	98	254
L	0.56	50.7	42.0	7.3	101	259
							M	0.60	50.7	42.0	7.3	101	259
N	0.59	49.3	40.8	7.2	98	253
							O	0.59	51.1	42.0	7.3	101	260
S	0.58	49.4	41.2	7.1	98	254
							T	0.57	53.6	45.1	7.8	107	277
U	0.60	49.1	40.5	7.1	97	251
							V	0.59	49.2	40.6	7.1	97	252
W	0.59	48.9	40.9	6.7	97	250
							Y	0.57	50.8	43.1	6.1	100	258
Z	0.56	48.7	39.6	6.9	96	247
							SEM	0.02	0.96	0.87	0.29	1.90	4.96

And (4) conclusion:

in summary, rumen fermentation is improved by supplementation with muramidase from three Glycoside Hydrolases (GH) families GH24, GH25 and a novel MUR polypeptide having muramidase activity. This improvement was seen to be an increase in the production of total volatile fatty acids, acetate and propionate.