Recombinant protein with antibacterial properties comprising an active chap catalytic domain, compositions comprising them, uses and methods utilizing thereof

Abstract

A recombinant protein with antibacterial properties comprising an active CHAP catalytic domain with an amino acid sequence having at least 80% identity with the sequence shown in SEQ. ID NO. 2 derived from Listeria monocytogenes, compositions comprising them, the use of the recombinant protein as an antibacterial agent and a method for inhibiting or limiting the growth of Gram (+) and / or Gram (-) bacteria with varied peptidoglycan structure.

Description

[0001] Recombinant protein with antibacterial properties comprising an active CHAP catalytic domain, compositions comprising them, uses and methods utilizing thereof

[0002] 5 TECHNICAL FIELD

[0003] The invention relates to a recombinant protein with antibacterial properties derived from the LM4 protein from Listeria monocytogenes and comprising an active CHAP (cysteine, histidine-dependent amidohydrolases / peptidases) catalytic domain and its use as an antiseptic agent, antibacterial agent, antiinflammatory agent, disinfecting agent for humans and animals. The invention may find use in the field of

[0004] 10 medicine and health care - in particular, for disinfection of surfaces, limiting the growth of pathogenic bacteria, treating and preventing diseases caused by Gram (+) and Gram (-) bacteria, in particular the recombinant protein with antibacterial properties will be used as a bactericidal agent for decontamination of surfaces in hospitals and medical facilities (removal of bacteria of the genus Staphylococcus, Klebsiella,

[0005] Pseudomonas and others), surfaces in contact with food (Listeria monocytogenes), as well as in fish

[0006] 15 aquaculture to inhibit the growth of various species of bacteria, including Yersinia ruckeri, thus preventing the development of yersiniosis in fish.

[0007] STATE OF ART

[0008] The resistance of microorganisms, including bacteria, to antimicrobial agents (antimicrobial resistance,

[0009] AMR) is one of the most serious problems and challenges for the modern medicine. Infections with

[0010] 20 microorganisms that do not respond to the used and established antimicrobial agents contributed to 1.27 mln deaths worldwide in 2019, generating costs both for the public healthcare system and for the economy due to prolonged patient recovery times [1], All surgical procedures, cancer chemotherapy and even minor skin infections, in the absence of an effective antibacterial agent, can develop into a life- threatening condition.

[0011] 25 Antibiotics constitute a significant part of antimicrobial compounds, the inappropriate use or overuse of which in medicine, agriculture and aquaculture causes a continuous increase in the AMR phenomenon worldwide [2,3,4], Data collected by the European monitoring network EARS-Net show that more than half (53.1%) of all E. coll strains, one third (34.3%) of K. pneumoniae strains and one fifth (18.7%) of P. aeruginosa strains [5] reported in 2021 were resistant to at least one group of antimicrobial agents. At the

[0012] 30 same time, a significant decline in the rate of new antibiotic development and their market entry is observed. Due to the rapid effect of resistance gaining by bacteria, new antibiotics are not widely used, so that they can become a weapon of last resort in the future.

[0013] 1 Currently, efforts of the scientists worldwide are focused on finding alternative, non-antibiotic bactericidal agents. Numerous enzybiotics, bacteriolytic enzymes derived from bacteria (bacteriocins, autolysins) or bacteriophages (endolysins) are known in the state of the art. Peptidoglycan hydrolases that cleave peptide bonds in the cross-linking bridges in the structure of peptidoglycan, the main component of

[0014] 5 bacterial cell walls, are also commonly known. Digestion of peptidoglycan causes cell wall breakdown, cell lysis, and its immediate death. However, because peptidoglycan-hydrolyzing enzymes exhibit specificity for specific bonds in the cell wall structure and because of differences in peptidoglycan structure among bacteria, peptidoglycan hydrolases can be more or less specific.

[0015] In the state of the art, various domains of the cysteine, histidine-dependent amidohydrolase / peptidase

[0016] 10 (CHAP), which are found in many peptidoglycan hydrolases of various species of bacteria and bacteriophages and are usually accompanied by other catalytic domains, most often the amidase domain, and peptidoglycan-binding domains, e.g. the SH3b or LysM domain, are known [6], The catalytic and amidase activity of CHAP domains, that is the hydrolysis of the bond between N-acetylmuramic acid in the sugar chain of peptidoglycans and the first amino acid of the peptide bridge - L-alanine [7] or the

[0017] 15 endopeptidase activity, i.e. the hydrolysis of the peptide bond between D-alanine of the peptide bridge and the glycine residue in the cross-linking bridge have been previously described in the literature [8],

[0018] Many catalytic domains of multidomain peptidoglycan hydrolases require the presence of peptidoglycan- binding domains for full activity, but some CHAP domains, e.g., the one that is a part of the endolysin LysK

[0019] (from bacteriophage K), when isolated, still retain bacteriolytic properties towards bacteria of the genus

[0020] 20 Staphylococcus [9], Endolysin LysK shows activity under conditions of high conductivity (4.3 mS / cm, 50 mM sodium acetate, pH 5.6) and in the presence of the substrate, which are staphylococcal cells. So far it has not been investigated, whether the enzyme is active against other bacterial species and under other conditions. Similarly, in the case of the isolated CHAP domain, which is part of the endolysin PlyC (from bacteriophage Cl), lytic activity was demonstrated, but much lower and only towards bacteria of the

[0021] 25 genus Streptococcus, when the test was performed under high conductivity conditions (17 mS / cm, PBS buffer)

[0010] ,

[0022] The state of the art lacks peptidoglycan hydrolases that are active against both Gram (+) and Gram (-) bacteria under low conductivity conditions, as well as those that are effective and non-toxic to living organisms, in particular in the breeding of aquatic organisms, e.g. fish. Therefore, there is a need to

[0023] 30 provide not only new enzymes of peptidoglycan hydrolase character that show high specificity towards a particular species or strain of bacteria, but also enzymes that act universally.

[0024] In the light of the described state of the art, the aim of the present invention is to overcome the indicated disadvantages and provide a bactericidal enzyme with a broad spectrum of activity against Gram(+) and

[0025] Gram(-) bacteria, with endopeptidase activity, which would be effective against many species of bacteria

[0026] 35 in a broad range of temperatures, low conductivity and a broad range of pH, and stable at the same time.

[0027] 2 The present invention may be an alternative to the commonly used antibiotics and bactericidal antiseptic agents effective against both Gram (+) and Gram(-) bacteria.

[0028] DISCLOSURE OF INVENTION

[0029] 5 The object of the invention is a recombinant protein with antibacterial properties comprising an active

[0030] CHAP catalytic domain having an amino acid sequence with at least 80% identity with the sequence shown in SEQ ID NO. 2 derived from Listeria monocytogenes.

[0031] Preferably, the recombinant protein comprises an active CHAP catalytic domain having an amino acid sequence with at least 85%, more preferably 90%, more preferably 95%, more preferably 99% identity

[0032] 10 with the sequence shown in SEQ ID NO. 2; most preferably, it comprises an active CHAP catalytic domain having the amino acid sequence SEQ. ID NO. 2.

[0033] Preferably, the recombinant protein is the active CHAP catalytic domain from Listeria monocytogenes having the amino acid sequence shown in SEQ ID NO. 2.

[0034] Preferably, the recombinant protein, additionally contains a bacterial cell wall binding domain attached

[0035] 15 to one of the termini, preferably the bacterial cell wall binding domain is a domain of the type SH3b, LysM,

[0036] AMIN, preferably the bacterial cell wall binding domain is a peptidoglycan binding domain.

[0037] Preferably, the recombinant protein further has an amino acid sequence useful for purification and / or isolation of the protein attached at one of the termini, wherein, preferably, the amino acid sequence useful for purification and / or isolation is located at the C -terminus side of the CHAP domain, preferably

[0038] 20 the amino acid sequence useful for purification and / or isolation of the recombinant protein is the oligohistidine sequence shown in SEQ ID NO. 5.

[0039] In the preferred embodiment, the recombinant protein consists of the sequence SEQ ID NO. 2 at the N- terminus of the recombinant protein and the oligohistidine sequence shown in SEQ ID NO. 5 at the C- terminus of the recombinant protein.

[0040] 25 Preferably, the recombinant protein has endolysin and / or bacteriocin and / or autolysin activity.

[0041] The invention also relates to a genetic construct encoding the recombinant protein according to the invention.

[0042] The invention also relates to a host cell comprising the genetic construct according to the invention.

[0043] The invention also relates to a composition comprising the recombinant protein according to the

[0044] 30 invention or a mixture thereof and a carrier, wherein, preferably, the composition is in the form of a solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, cream.

[0045] 3 The invention also relates to a non-medical use of the recombinant protein according to the invention as an antibacterial agent.

[0046] Preferably, in the non-medical use of the recombinant protein, the antibacterial agent is used in an aqueous environment with a conductivity of 0 to 2.5 mS / cm.

[0047] 5 Preferably, in the non-medical use of the recombinant protein, the antibacterial agent is used against bacteria of the genus Listeria spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., Corynebacterium spp., Bacillus spp., Escherichia spp., Klebsiella spp.,

[0048] Pseudomonas spp., Yersinia spp., Acinetobacter spp.; more preferably against the species Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus,

[0049] 10 Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus canis,

[0050] Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coll, Yersinia ruckeri, Acinetobacter heamolyticus.

[0051] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an antiseptic and disinfecting agent.

[0052] 15 The invention also relates to the use of a recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products.

[0053] The invention also relates to the use of a recombinant protein according to the invention and / or a

[0054] 20 composition according to the invention as an antibacterial agent in the food industry as a food additive for humans and / or animals, preferably as a feed additive, preferably as a fish feed additive.

[0055] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for disinfecting hospital surfaces, medical and

[0056] 25 veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

[0057] The invention also relates to a non-medical use of the recombinant protein according to the invention and / or the composition according to the invention as an in vitro bactericidal agent for inhibiting, eliminating bacteria, preventing the growth of Gram (+) and Gram(-) bacteria, preferably bacteria of the

[0058] 30 genus Listeria spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp., Enterococcus spp.,

[0059] Streptococcus spp., Corynebacterium spp., Bacillus spp., Escherichia spp., Klebsiella spp., Pseudomonas spp., Yersinia spp., Acinetobacter spp.; preferably bacteria of the species Listeria monocytogenes,

[0060] Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis,

[0061] Enterococcus faecium, Enterococcus faecalis, Streptococcus canis, Staphylococcus simulans, Streptococcus

[0062] 4 agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coli,

[0063] Yersinia ruckeri, Acinetobacter heamolyticus.

[0064] In the preferred non-medical use, the bactericidal agent is used in an aqueous environment with a conductivity of 0 to 2.5 mS / cm.

[0065] 5 The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as a bactericidal agent for inhibiting, eliminating bacteria, preventing the growth of bacteria in water, preferably bacteria of the species Escherichia coli,

[0066] Pseudomonas aeruginosa, Enterococcus feacalis, Yersinia ruckeri.

[0067] The invention also relates to the use of the recombinant protein as a bactericidal agent used in

[0068] 10 aquacultures.

[0069] The invention also relates to the use of the recombinant protein as a bactericidal agent in fish hatcheries and nurseries, closed water systems, in containers for transporting fish and / or fry, in containers for storing fish and / or fry, in containers with eggs and / or larvae of aquatic animals, in containers with aquatic plants and for equipment having contact with fish.

[0070] 15 In a preferred use of the recombinant protein as a bactericidal agent it is used in an aqueous environment with a conductivity of 0 to 2.5 mS / cm.

[0071] The invention also relates to the use of the recombinant protein according to the invention and / or the composition according to the invention as an antibacterial agent in the cosmetics industry as an additive to cosmetics improving their microbiological quality, preferably as a biopreservative, preferably as an

[0072] 20 additive to liquids, creams, milks, lotions, mists, gels.

[0073] The invention also relates to a cosmetic or care composition for cosmetic, care and hygiene uses in humans or animals, which comprises a recombinant protein according to the invention, wherein the composition is intended for external use, and wherein the recombinant protein is used in the cosmetic or care composition to reduce the occurrence of or eliminate bacteria from the composition.

[0074] 25 The invention also relates to a veterinary and / or pharmaceutical composition which comprises the recombinant protein according to the invention and a veterinarily and / or pharmaceutically acceptable carrier for use in the prevention and treatment of a disease and / or condition caused by bacteria, preferably bacteria of the genus Listeria spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp.,

[0075] Enterococcus spp., Streptococcus spp., Corynebacterium spp., Bacillus spp., Escherichia spp., Klebsiella

[0076] 30 spp., Pseudomonas spp., Yersinia spp., Acinetobacter spp.; more preferably caused by bacteria of the species: Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus canis,

[0077] 5 Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coli, Yersinia ruckeri, Acinetobacter heamolyticus.

[0078] The invention also relates to a veterinary composition comprising the recombinant protein according to the invention and a veterinarily acceptable carrier for use in the prevention and / or treatment of fish

[0079] 5 diseases caused by Yersinia ruckeri, preferably for use in the prevention and / or treatment of yersiniosis in fish.

[0080] In the preferred embodiment, the veterinary composition for use according to the invention is used for the treatment of yersiniosis in salmonid fish, preferably selected from rainbow trout (Oncorhynchus myk / ss), brook trout (Salvelinus fontinalis), brown trout (So / mo trutta morpha fario), sea trout (So / mo

[0081] 10 trutta morpha trutta), Atlantic salmon (Salmo salar).

[0082] The invention also relates to a method for inhibiting or limiting the growth of Gram (+) and / or Gram (-) bacteria with a varied peptidoglycan structure, which comprises the step in which the bacteria are contacted with the recombinant antibacterial protein according to the invention and / or the composition according to the invention in an environment with conductivity ensuring bactericidal activity of the

[0083] 15 recombinant protein.

[0084] In the preferred method of inhibiting or limiting bacterial growth, the contacting is carried out in an aqueous solution having a conductivity of 0 to 2.5 mS / cm.

[0085] In the preferred method of inhibiting or limiting bacterial growth, the bacteria are selected from the genus

[0086] Listeria spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp., Enterococcus spp., Streptococcus

[0087] 20 spp., Corynebacterium spp., Bacillus spp., Escherichia spp., Klebsiella spp., Pseudomonas spp., Yersinia spp., Acinetobacter spp.; preferably from bacteria of the species Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis,

[0088] Enterococcus faecium, Enterococcus faecalis, Streptococcus canis, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coli,

[0089] 25 Yersinia ruckeri, Acinetobacter heamolyticus.

[0090] The invention describes recombinant proteins comprising an active CHAP catalytic domain with an amino acid sequence having at least 80% identity with the sequence shown in SEQ. ID NO. 2 and its use or a composition comprising it for use as a medicine, antiseptic, antibacterial agent, disinfectant.

[0091] The recombinant protein according to the invention has peptidase properties and is capable of inhibiting

[0092] 30 the growth and degrading Gram (+) and Gram (-) bacteria. Bactericidal properties of the recombinant protein according to the invention for use as an antibacterial agent in the prevention and treatment of bacterial infections in humans and animals caused by Gram (+) bacteria, e.g., Listeria monocytogenes,

[0093] Staphylococcus aureus, Enterococcus faecalis, and Gram (-), e.g. Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia ruckeri has been described. The invention describes possible uses of the recombinant

[0094] 6 protein according to the invention as an antiseptic agent, an agent for decontamination of surfaces in public healthcare offices, public utilities, and food industry production facilities. Moreover, due to its effectiveness in eliminating the Yersinia ruckeri bacteria, the recombinant protein according to the invention can be used to prevent infections in salmonid fish.

[0095] 5 In the genome of the Listeria monocytogenes bacterium, the sequence of which was deposited in

[0096] GeneBank (NCBI, USA) on April 5, 2019 under the number PRJNA215355 [12, 13, 14], the EAF9399118 gene, which encodes a protein named by the authors of this use as the LM4 protein. (SEQ ID NO. 1), 487 amino acids long, was discovered and identified using sequence comparison and homology search methods. The protein encoded by this sequence was not previously known, nor was its function or possible

[0097] 10 uses known or suggested. Based on homology to other known proteins, using sequence comparison in

[0098] Blastp program, and modeling the structure and comparing it with known protein structures using

[0099] AlphaFold 2.0, the Authors concluded that this protein contains three functional domains: 1) N-terminal catalytic domain LM4_CHAP with a sequence homologous to proteins from the CHAP family; 2) LM4_Ami catalytic domain with a sequence characteristic for amidases; 3) C-terminal peptidoglycan-binding domain

[0100] 15 LM4_SH3b (Fig. 1).

[0101] Unexpectedly, after cloning, production in E. coli and purification to homogeneity of the isolated

[0102] LM4_CHAP domain (SEQ. ID NO. 2, Fig. 1), it was found that it exhibits bactericidal properties not only against Listeria monocytogenes, the genome of which it originates from, but also against many other

[0103] Gram(+) and Gram(-) bacteria. (Tab. 1). Moreover, it was found that the broad-spectrum bactericidal

[0104] 20 properties, i.e. against many bacterial species, are particularly well visible under low conductivity conditions (from 0 to 25 mM NaCI, i.e. from 0 to 2.5 mS / cm).

[0105] The essence of the invention is based on the unexpected finding of an active catalytic domain within the

[0106] EAF9399118 gene (SEQ ID NO. 1) in the genome of Listeria monocytogenes, which shows bactericidal properties against both Gram (+) and Gram (-) bacteria with different peptidoglycan structures. The

[0107] 25 LM4_CHAP domain is homologous to other peptidoglycan hydrolases that digest peptidoglycan between the amino acids that build peptide cross-linking bridges. CHAP endopeptidases are found in multidomain peptidoglycan hydrolases as one of the catalytic domains, and are generally specific to bacterial species whose unique cell wall structure they recognize and digest. Unexpectedly, the isolated catalytic domain

[0108] LM4_CHAP shows a very broad spectrum of bactericidal activity, recognizes and eliminates bacteria

[0109] 30 regardless of the structure of their cell wall.

[0110] An exemplary recombinant protein according to the invention constitutes an active enzyme having an amino acid sequence of the protein corresponding to the LM4_CHAP domain (SEQ ID NO. 2), not containing the signal sequences at the N-terminus of the sequence. A sequence that is at least 80% identical with it is the minimum sequence required for the bactericidal activity of the recombinant protein

[0111] 35 according to the invention. In the preferred embodiment of the recombinant protein according to the 7 invention, the LM4_CHAP sequence may be attached, e.g., at the C -terminus of the protein, with a sequence / sequences facilitating purification, e.g., an oligohistidine sequence that does not affect the activity of the LM4_CHAP protein. The bactericidal activity of the LM4_CHAP protein having SEQ. ID NO. 2 is the highest in an environment with pH 6.0-8.0 (Fig. 3) and NaCI concentration from 0 mM to 25 mM

[0112] 5 (Fig. 4).

[0113] In the preferred embodiment of the recombinant protein according to the invention, it has a bacterial cell wall binding domain attached to one of the termini, e.g., of the type SH3b, LysM, AMIN.

[0114] Examples of cell wall binding domains include:

[0115] - SH3b (Src Homology 3 domain superfamily, bacterial) domains, e.g., a binding domain found in

[0116] 10 bacteriocins, which is part of multidomain proteins such as lysostaphin, a bacteriocin secreted by bacteria Staphylococcus simulans (GenBank sequence: AAB53783.1, amino acids 402-475), domain binds cross-linking bridges in the peptidoglycan of staphylococci (pentaglycine); ALE-1, an endopeptidase produced by Staphylococcus capitis (PDB sequence: 1R77_A); endolysin from the

[0117] Staphylococccus aureus phage StB20 (GeneBank sequence: YP_007236686.1);

[0118] 15 - CBD (cell wall binding domain) domains, so-called SH3b-like domains that recognize cell wall elements of Listeria monocytogenes bacteria, found in bacteriophage endolysins, e.g., in the Ply endolysin from the PSA phage genome (PDB database, no. 1XOV);

[0119] - AMIN (Amidase N-terminal domain) domains, a domain that directs periplasmic proteins or proteins from the outside of the cell to the bacterial cell wall, most often to the site of cell division, the so-called

[0120] 20 septum, for example it occurs in the proteins: AmiC in E. coli (PDB 4BIN), AMIN~Amidase_3

[0121] (YP_001336853) from Klebsiella pneumoniae, AMIN (NP_938979) from Corynebacterium diphteriae;

[0122] - LysM domains, domains binding peptidoglycans in bacterial cell walls, for example transglycosylase

[0123] D (MltD) in E. coli (PDB 1E0G), N-acetylglucosaminidase AltA in Enterococcus faecalis.

[0124] The recombinant protein according to the invention can be used as a bactericidal and bacteriostatic agent

[0125] 25 to inhibit, eliminate and prevent the growth of both Gram (+) and Gram (-) bacteria, as it shows a broad spectrum of activity. A bactericidal or bacteriostatic agent comprising the recombinant protein according to the invention can be used as a product for disinfection of surfaces and instruments in health care facilities (clinics, hospitals), nursing homes, kindergartens, where there are large crowds of people, easy spread of pathogens, and, at the same time, many people with reduced immunity and exposed to

[0126] 30 infection. The main target pathogens are Klebsiella pneumoniae, Pseudomonas aeruginosa,

[0127] Staphylococcus aureus, Enterococcus faecalis. The invention, the recombinant protein according to the invention can also be used in the food industry as a component of products for disinfection of surfaces having contact with food, i.e. devices, tables, conveyor belts, etc. High antibacterial activity against Listeria monocytogenes allows for the elimination of bacteria during food production and the avoidance of food

[0128] 8 poisoning. The recombinant protein according to the invention can be administered in liquid form or as a powder for dissolving in aqueous solutions.

[0129] Equally high activity of the recombinant protein according to the invention was demonstrated against the bacteria Yersinia ruckeri, which allows its use in fish farming, especially industrial salmonid fish farming,

[0130] 5 for preventing infections caused by Yersinia ruckeri causing yersiniosis, especially in industrial farming of salmonid fish, such as rainbow trout (Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), brown trout (So / mo trutta morpha fario), sea trout (So / mo trutta morpha trutta) and Atlantic salmon (So / mo salary The recombinant proteins according to the invention can be formulated into a form convenient for administration, e.g., in the form of a solution, tablet, powder, suspension, which can be administered to

[0131] 10 water, water reservoirs, especially fish aquacultures, preferably salmonid fish aquacultures.

[0132] The recombinant protein according to the invention will be particularly preferably used for the removal of Yersinia ruckeri bacteria from water in aquacultures, in closed circuits, in containers for transporting fry, in containers for storing fish / fry during vaccinations, routine inspections, removal of parasites (e.g. sea lice), disinfection of eggs in hatcheries and during other experiments on fish.

[0133] 15

[0134] BRIEF DESCRIPTION OF DRAWINGS

[0135] Exemplary embodiments of the invention are presented in the drawings, in which:

[0136] FIG. 1. Shows a block diagram of the original protein encoded by the gene identified in the GeneBank database under the number EAF9399118 LM4 (SEQ. ID NO. 1) from Listeria monocytogenes, and a

[0137] 20 recombinant protein consisting of the isolated LM4_CHAP domain (SEQ ID NO. 2) and the recombinant protein LM4_CKAPHis Tag. LM4_CHAP - catalytically active domain of CHAP, LM4_Ami - catalytic domain of the LM4 protein homologous to amidase, LM4_SH3b - peptidoglycan-binding domain.

[0138] FIG. 2. Shows the bacteriolytic activity of the recombinant LM4_CHAP protein against Gram (+) and Gram

[0139] (-) bacterial strains administered at two concentrations, 0.5 and 1.0 pM. Bacterial cell lysis was observed

[0140] 25 as an increase in the fluorescence of the tested sample compared to the negative control containing no enzyme (0 pM).

[0141] FIG. 3. Shows the bactericidal activity of the recombinant LM4_CHAP polypeptide against Listeria monocytogenes MF7703 in buffers of different pH (5.0 - 9.0). The activity is presented as a reduction in the initial number of bacterial cells (on a logarithmic scale, CFU / ml logio).

[0142] 30 FIG. 4. Shows the dependence of the bactericidal activity of the recombinant LM4_CHAP protein on the conductivity of the reaction environment. The conductivity of the buffer is given in mS / cm. The activity is presented as a reduction in the initial number of bacterial cells (on a logarithmic scale, CFU / ml logio).

[0143] 9 FIG. 5. Shows the method of enzyme inactivation by incubation in the presence of EDTA solution. The activity is presented as a reduction in the initial number of bacterial cells (on a logarithmic scale, CFU / ml logio).

[0144] FIG. 6. Shows the stability of the enzyme. (A) The integrity of samples of the recombinant protein, stored

[0145] 5 for 30 days at different temperatures: -20°C, 4°C, and 25°C, was verified by SDS-PAGE gel electrophoresis under denaturing conditions. Ctrl - control, recombinant protein stored at -80°C. (B) The stability of the recombinant protein was checked in tests of the activity, which was presented as a reduction in the initial number of bacterial cells (on a logarithmic scale, CFU / ml logio). (C) The resistance of the recombinant protein to high temperature (100°C) in time from 30 to 180 minutes was investigated in bactericidal

[0146] 10 activity tests and presented as a reduction in the initial number of bacterial cells (logio CFU / ml).

[0147] FIG. 7. Shows the results of toxicity tests of the recombinant LM4_CHAP protein obtained in eukaryotic cells, an insect model and a fish model. (A) Cytotoxicity of the recombinant protein tested on human

[0148] HaCAT cell lines (keratinocytes). 1 - negative control without the recombinant protein added to the culture, 2 - positive control comprising 10% ethanol, 3 - cell culture treated with LM4_CHAP. (B) Normal

[0149] 15 development of Galleria mellonella on the seventh day after injection of LM4_CHAP. The graph shows the percentage of the total insect population of individuals in particular stages of insect development (larva, cocoon, pupa) under control conditions and in the presence of 1000 nM of the tested recombinant protein. (C) Normal morphology of Danio rerio fish exposed to 500 nM and 1000 nM of the LM4_CHAP protein solution, and control - without exposure to LM4_CHAP a) photos showing the lateral view of a

[0150] 20 three-day-old zebrafish larva; b) body length, and c) survival of zebrafish larvae. Scale bar = 1 mm.

[0151] The publications cited in this description and the references cited therein are hereby incorporated by reference in their entirety. The following examples illustrate the invention and are not intended to limit it in any way.

[0152] 25 EMBODIMENTS OF THE INVENTION

[0153] The following examples are provided merely for the purpose of illustrating the invention and explaining particular aspects thereof, and are not intended to be limiting and should not be identified with its entire scope, which is defined by the appended claims.

[0154] When the term "comprising" or "including" is used in this description and claims, it does not exclude other

[0155] 30 elements. For the purposes of the present invention, the term "consisting of" is considered to be the preferred embodiment of the term "comprising" or "including". If a group is defined below that includes at least a number of embodiments, this is also to be understood as disclosing a group that preferably consists of only those embodiments.

[0156] 10 The terms "about" or "approximately" in the context of the present invention mean a range of precision that one skilled in the art would understand to be sufficient to still provide the technical effect of the feature. The term typically means a deviation from the indicated numerical value of ±10%, and preferably

[0157] ±5%.

[0158] 5 Technical terms are used in accordance with their typical meaning. If a specific meaning is used, the meaning will be described in detail with reference to the context in which a given term is used.

[0159] EXAMPLES

[0160] In the following examples, unless otherwise indicated, standard materials and methods in the art were

[0161] 10 used or the manufacturer's recommendations for specific materials and methods were followed.

[0162] Example 1. Obtaining of the LM4_CHAP protein

[0163] In order to later isolate the LM4_CHAP protein, the LM4_CHAP gene sequence (SEQ ID NO. 6) was first cloned into the pLATE31 vector, which carries the ampicillin resistance gene and an additional oligohistidine sequence, which was added at the C-terminus of the cloned gene sequence. As a result of

[0164] 15 cloning, a gene for protein production (SEQ ID NO. 2) was created, containing an oligohistidine sequence at the C-terminus (SEQ. ID No. 4). The protein was obtained in E. coli ER2566 cells and then purified by affinity chromatography using an oligohistidine sequence (WorkBeads 40 Ni-NTA or Talon resin,

[0165] BioWorks). The oligohistidine sequence was not cleaved. The obtained LM4_CHAPHls Tagprotein preparations had a purity above 95%, were suspended in storage buffer, i.e., 50 mM Tris-HCI pH 8.0, 500

[0166] 20 mM NaCI, 10% glycerol at concentrations ranging from 4.2 to 8.5 mg / ml, and frozen in aliquots at -80°C.

[0167] Preparations stored in this manner were used for activity and toxicity tests at a concentration of 500 nM

[0168] (0.0134 mg / ml) or higher. Fig. 1 schematically shows the domain organization in the initial LM4 gene (SEQ

[0169] ID NO. 1) and the recombinant LM4_CHAP polypeptide. The produced recombinant LM4_CHAP protein with retained oligohistidine sequence (see Fig. 1, the construct named LM4_CHAPHis Tagin the figure) was

[0170] 25 used for further experiments. The presence of the oligohistidine sequence in LM4_CHAPHis Tagdid not affect the change of the activity compared to the LM4_CHAP protein. Therefore, for easier understanding, its abbreviated name LM4_CHAP was used in the following sections.

[0171] Example 2. An enzyme active against various species of pathogenic bacteria

[0172] To verify whether the obtained recombinant LM4_CHAP protein exhibits bactericidal properties, activity

[0173] 30 tests were performed. The bactericidal activity of the LM4_CHAP enzyme (obtained in Example 1) was verified against various bacterial strains listed in Tab. 1. Bacteria were grown in TSB medium (Graso

[0174] BioTech) at 37°C, except Yersinia ruckeri, which was grown at 28°C, with shaking, and harvested in the logarithmic growth phase (OD6oo=0.6), washed and suspended in 50 mM glycine-NaOH buffer, pH 8.0 to

[0175] 11 reach a density of 1x10sCFU / ml. Each of the bacterial strains prepared in this way was incubated for 2 hours at room temperature in the presence of 500 nM of the LM4_CHAP enzyme. The reaction mixtures were plated on TSB-agar plates (LB-agar for E. coli) in 10-fold dilutions, by the so-called droplet method.

[0176] Tab. 1 shows the results of the experiment, defining the enzyme activity as a reduction in the initial

[0177] 5 number of bacteria expressed on a logarithmic scale (reduction by 1 log = 90% reduction).

[0178] Tab. 1. Bacteriolytic activity of the LM4_CHAP enzyme (SEQ. ID NO. 2) against various bacterial species. The activity was defined as a reduction in the initial number of bacterial cells expressed in logio CFU / ml by 4 logs (99.99%), 3 logs (99.90%), 2 logs (99.00%), 1 log (90.00%), 0 logs (0%, no reduction = no activity)

[0179] Bacterial species Bacterial group Bactericidal activity (reduction in Gram + / Gram - the initial number of bacteria)

[0180] CFU / ml logio % CFU / ml

[0181] Listeria monocytogenes MF7703# 4 99.99

[0182] Listeria monocytogenes DSM 19094 4 99.99

[0183] Listeria monocytogenes MF7839 4 99.99

[0184] Listeria monocytogenes MF3860 4 99.99

[0185] Listeria monocytogenes MF4001 4 99.99

[0186] Listeria monocytogenes MF4588 4 99.99

[0187] Listeria monocytogenes MF4804 4 99.99

[0188] Listeria monocytogenes MF7035 4 99.99

[0189] Staphylococcus aureus NCTC 8325-4 4 99.99

[0190] Staphylococcus epidermidis DSM28319 4 99.99

[0191] Staphylococcus simulans DSM20037 4 99.99

[0192] Staphylococcus simulans CCM 3583 4 99.99

[0193] Enterococcus f aecium DSMZ2146 4 99.99

[0194] Enterococcus faecalis DSM 20376 4 99.99

[0195] Streptococcus canis DSM 20715 4 99.99

[0196] Streptococcus agalactiae 123 B 4 99.99

[0197] Streptococcus dysgalactiae DSM 20662 4 99.99

[0198] Corynebacterium striatum DSM20668 4 99.99

[0199] Bacillus subtilis DSM 10 2 99.00

[0200] Escherichia coli DSM 1103 4 99.99

[0201] Klebsiella pneumoniae DSM 789 4 99.99

[0202] Pseudomonas aeruginosa DSM 939 4 99.99

[0203] Yersinia ruckeri CCM 4620 3 99.90

[0204] Acinetobacter haemolyticus DSM6962 4 99.99 # isolate from the food industry (11)

[0205] 10 An unexpectedly high bacteriolytic activity of the recombinant LM4_CHAP protein was demonstrated against many Gram (+) and Gram (-) bacterial species. The LM4_CHAP protein eliminated from 99% of the initial 1 million bacterial cells in the case of Bacillus subtilis to 99.99% for the other strains tested within 2 hours of incubation at room temperature. The research results indicate the possibility of using the recombinant protein LM4_CHAP as an antibacterial agent for decontamination of surfaces, tools and

[0206] 15 devices in medical facilities, but also in the food industry, veterinary medicine and aquaculture.

[0207] 12 Example 3.

[0208] The lytic activity of the enzyme in the form of the recombinant LM4_CHAP protein (obtained in Example

[0209] 1) was verified against the following bacterial strains Listeria monocytogenes MF7703, Staphylococcus aureus NCTC 8325-4, Staphylococcus epidermidis DSM 28319, Escherichia coli DSM 1103 and Yersinia

[0210] 5 ruckeri CCM 4620. Bacteria were grown in TSB medium (Graso BioTech) at 37°C, except Yersinia ruckeri, which was grown at 28°C, with shaking, and harvested in the logarithmic growth phase. (ODgoo=0.6), washed twice and suspended in 50 mM glycine-NaOH buffer, pH 8.0 to reach a density of lxlO8CFU / ml.

[0211] Each bacterial strain prepared in this way was incubated in the presence of 5 pM SYTOX Green (Invitrogen) and 0 pM, 0.5 pM or 1.0 pM of LM4_CHAP enzyme at room temperature for 1 hour. For fluorescence

[0212] 10 measurement, 100 pl of the reaction was transferred to a 96-well plate, where the fluorescence intensity was measured (Infinite M 1000 Pro fluorescence plate reader, Tecan Trading AG, excitation at the wavelength of 504 nm, emission at the wavelength of 523 nm).

[0213] The LM4_CHAP enzyme showed lytic activity against Gram(+) bacteria. The results presented in Fig. 2 show the increase in the level of fluorescence resulting from cell lysis of Gram(+) strains of bacteria:

[0214] 15 Listeria monocytogenes MF7703, Staphylococcus aureus NCTC 8325-4 and Staphylococcus epidermidis

[0215] DSM 28319. The LM4_CHAP enzyme does not show lytic activity against Gram(-) bacteria, while

[0216] LM4_CHAP is bactericidal against them. In Fig. 2, there was not observed an increase in the fluorescence level for Gram(-) bacteria, which means that the bactericidal effect of the enzyme in the form of the recombinant LM4_CHAP protein caused by the enzyme against Gram(-) bacteria results from other

[0217] 20 mechanisms of toxicity, and cell lysis is not a direct cause of their elimination.

[0218] Example 4. The effect of buffer conditions on the bacteriolytic activity of the LM4_CHAP protein against bacteria Listeria monocytogenes.

[0219] In order to test the effect of the pH of the reaction environment on the bactericidal activity of the recombinant LM4_CHAP protein (obtained in Example 1), its bactericidal activity was tested in buffers

[0220] 25 with different pH (5.0 - 9.0). Listeria monocytogenes DSM 19094 cells were grown in TSB medium at 37°C with shaking, harvested in the logarithmic growth phase (ODgoo=0.6), washed and suspended in buffers with increasing pH: 10 mM sodium acetate, pH 5.0; 10 mM sodium acetate, pH 6.0; 5 mM Tris-HCI, pH

[0221] 7.0; 50 mM glycine buffer, pH 8.0; 50 mM Tris-HCI, pH 9.0. The conductivity of the above-mentioned buffers was approx. 0.095 mS / cm - 1.7 mS / cm. The bacterial cell suspension (1x10sCFU / ml) was

[0222] 30 incubated in the presence of 0.5 pM of the LM4_CHAP enzyme in the appropriate buffer at room temperature for 2 hours. The reaction mixtures were plated on TSB-agar plates in 10-fold dilutions, using the so-called droplet method.

[0223] 13 The LM4_CHAP enzyme showed the highest bactericidal activity against bacteria Listeria monocytogenes in buffers with pH ranging from 6 to 8 (Fig. 3), thus in a wide, commonly found range of pH, also in the pH range corresponding to the physiological conditions on the skin, in wounds, etc.

[0224] Example 5. The influence of the conductivity of the reaction environment on the bactericidal activity of

[0225] 5 the LM4_CHAP protein against Listeria monocytogenes.

[0226] To determine the sensitivity of the recombinant LM4_CHAP protein obtained in Example 1 to the conductivity of the reaction environment, the bactericidal activity of the LM4_CHAP enzyme was tested in buffers with different conductivities (0-10 mS / cm). Listeria monocytogenes DSM 19094 cells were grown in TSB medium at 37°C with shaking, harvested in the logarithmic growth phase, washed and

[0227] 10 suspended in 50 mM glycine buffer, pH 8.0 with the addition of NaCI at concentrations of 0 mM, 25 mM,

[0228] 50 mM and 100 mM, which corresponds to conductivity values of 0, 2.5, 6.1 and 10.8 mS / cm, respectively.

[0229] Bacterial cell suspension (lxlOsCFU / ml) was incubated in the presence of 0.5 pM recombinant LM4_CHAP protein in an appropriate buffer at room temperature for 2 hours. The reaction mixtures were plated on

[0230] TSB-agar plates in 10-fold dilutions, using the so-called droplet method.

[0231] 15 It was found that the enzyme in the form of the recombinant LM4_CHAP protein showed bactericidal activity in buffers containing from 0 to 25 mM NaCI, which corresponds to conductivity in the range from

[0232] 0 to 2.5 mS / cm (Fig. 4). This indicates a particularly advantageous use of the LM4_CHAP protein in aqueous solutions, in which the concentration of ions, e.g. sodium, does not exceed 25 mM.

[0233] Example 6. Enzyme deactivation.

[0234] 20 Inactivation by incubation in the presence of EDTA in solution. The LM4_CHAP enzyme obtained in

[0235] Example 1, diluted in glycine buffer (50 mM glycine-NaOH, pH 8.0) to a concentration of 0.5 pM, was preincubated in glycine buffer with the addition of EDTA at a concentration from 0 mM to 100 mM for 15 min. After this time, the bactericidal activity of the enzyme against Listeria monocytogenes MF7703 was determined according to the procedure described in Example 2. It was shown that 5 mM EDTA

[0236] 25 concentration completely inactivates the LM4_CHAP enzyme (Fig. 5).

[0237] Deactivation by increased NaCI concentration in solution. As demonstrated in Example 5, increasing the conductivity of the reaction environment to 6.1 mS / cm, which corresponds to 50 mM NaCI concentration

[0238] (0.3% NaCI solution), completely inhibits the activity of the LM4_CHAP enzyme. It was shown that increasing the conductivity of the reaction mixture by adding NaCI or other ions is one of the simple

[0239] 30 methods of enzyme deactivation (Fig. 4), which may be useful in its future uses.

[0240] Example 7. Stability of the enzyme stored in a solution at different temperatures

[0241] To test the stability of the LM4_CHAP enzyme, the protein was stored in solution for one month at different temperatures. The recombinant LM4_CHAP enzyme obtained in Example 1 was suspended in a

[0242] 14 buffer of 50 mM Tris-HCI pH 8.0, 500 mM NaCI, 10% glycerol at a concentration of 0.0134 mg / ml and stored for 1 month at -80°C, -20°C, 4°C and 25°C. The recombinant protein preparations were then subjected to bactericidal activity and integrity tests by loading samples onto a polyacrylamide gel and separated by SDS-PAGE. Enzyme activity against Listeria monocytogenes MF7703 was determined as

[0243] 5 described in Example 2. SDS-PAGE analysis revealed minor protein degradation during storage at 4°C and room temperature (Fig. 6A). LM4_CHAP retained full activity during storage at -20°C and 4°C, but its activity decreased upon storage at 25°C (Fig. 6B).

[0244] In order to test the stability of the LM4_CHAP protein at high temperature, the preparation obtained in

[0245] Example 1 was diluted in storage buffer (50 mM Tris-HCI pH 8.0, 500 mM NaCI, 10% glycerol) to a

[0246] 10 concentration of 0.0134 mg / ml, incubated at 100°C for 10, 30, 60 min and 3 h, and then cooled and subjected to a bactericidal activity test using the Listeria monocytogenes MF7703 strain according to the protocol described in Example 2. The enzyme activity remained at the control level (untreated enzyme) even after 3 h of incubation at 100°C (Fig. 6C).

[0247] The recombinant LM4_CHAP protein is very stable in aqueous solutions, also is thermostable, and can be

[0248] 15 used for long-term uses at various temperatures, e.g., high-temperature decontamination.

[0249] Example 8. Biological safety of the LM4_CHAP protein (in vitro tests using eukaryotic cells and in vivo tests using insect and fish models).

[0250] The toxicity of the recombinant LM4_CHAP protein enzyme was tested in vitro on eukaryotic cells and in vivo in insect and fish models.

[0251] 20 The cytotoxicity of the LM4_CHAP protein obtained in Example 1 was tested in vitro. Using the MTT assay, the metabolic activity of human keratinocytes (HaCAT) cultured for 24 hours in DMEM medium with 10%

[0252] FBS (Fig. 7 A, 1 - control, untreated cells), and additionally supplemented with 10% ethanol (Fig. 7 A, 2 - positive control) or 3 -treated with 5 pM of the LM4_CHAP protein (Fig. 7 A, 3) was compared. The formed formazan crystals were dissolved with DMSO, and then the absorbance was measured

[0253] 25 spectrophotometrically at 570 nm. As expected, the cytotoxic effect of ethanol was correlated with a significant decrease in absorbance values (Fig. 7 A, column 2). In turn, cells treated with the LM4_CHAP protein remained metabolically active, at least to an extent equal to that of the untreated control

[0254] (compare Fig. 7 A, columns 1 and 3). Thus, the test results confirm the lack of harmful effects of the protein on the metabolism of eukaryotic cells.

[0255] 30 In order to exclude the potential acute or lethal toxicity of the recombinant LM4_CHAP protein, the protein was tested in two in vivo models: wax moth (Galleria mellonella) caterpillars and zebrafish (Danio rerio) embryos and larvae. In the insect model, wax moth caterpillars > 2 cm in length were used. 20 pl

[0256] (microliters) of a 1000 nM LM4_CHAP solution in 50 mM glycine buffer, pH 8.0, was injected into the body cavity of the G. mellonella caterpillar using a 0.5 ml G30 insulin syringe. Control caterpillars were injected

[0257] 15 with 20 pl of glycine buffer. The caterpillars were then incubated in an incubator at 28°C with >60% humidity. For a week following injection, any changes in appearance were assessed daily, and the survival rate of G. mellonella was monitored. No pigmentation, a visual indicator of declining health, was observed during the seven-day observation period. Caterpillars injected with LM4_CHAP continued their

[0258] 5 development and, as in the control, formed cocoons and then began pupation. The emergence of motile pupae from the cocoon during the experiment indicates that metamorphosis was proceeding correctly

[0259] (Fig. 7B). Differences in the number of G. mellonella at subsequent developmental stages (caterpillar, cocoon, pupa) result from slight differences in the age of the injected caterpillars. A toxicity test using moth caterpillars confirmed that the recombinant LM4_CHAP protein had no effect on the development

[0260] 10 of these insects.

[0261] In the fish tests, wild-type Danio rerio were used. Groups of 12 normally developing embryos, at the developmental stage up to 4 hours after fertilization, were placed in 3-cm diameter Petri dishes filled with

[0262] 2 ml of fluid. In the experiment, a 500 nM or 1000 nM solution of LM4_CHAP in 50 mM glycine buffer at pH 8.0 was used. In the control, embryos were incubated in the presence of glycine buffer alone or in 50

[0263] 15 mM glycine buffer at pH 8.0 supplemented with the buffer in which the LM4_CHAP protein is stored, added in a volume corresponding to 1000 nM LM4_CHAP. The embryo dishes were incubated for three days in an incubator at 28.5°C. The fluids were replaced daily with freshly prepared solutions, and the developing fish embryos were observed. Survival and morphology were assessed using the criteria described in OECD test no. 236. During the experiment, neither morphological changes nor increased

[0264] 20 mortality were observed in the control fish nor in fish treated with 500 nM and 1000 nM LM4_CHAP (Fig.

[0265] 7C). The obtained results indicate that the use of LM4_CHAP protein below a concentration of 1000 nM in short-term uses (the study herein was conducted for 3 days) does not pose a biological threat.

[0266] Example 9. Bactericidal activity of the recombinant LM4_CHAP protein against bacteria Yersinia ruckeri under conditions similar to those prevailing in aquaculture.

[0267] 25 Due to the fact that, as shown in Example 8, the LM4_CHAP protein is safe for use in Danio rerio fish and that the protein is bactericidal for the Gram (-) bacterium Yersinia ruckeri, which is a pathogen causing a dangerous disease, yersiniosis in fish, it was verified whether the LM4_CHAP protein would eliminate the

[0268] Y. ruckeri bacterium under conditions similar to aquaculture in the presence of 1-day-old or 4-day-old

[0269] Danio rerio fish.

[0270] 30 For this purpose, Y. ruckeri CCM 4620 bacterial cells were prepared according to the protocol in Example

[0271] 2 and suspended in 50 mM glycine-NaOH buffer, pH 8.0 to a final concentration of 1 x 107CFU / ml. The experiment was conducted on Danio rerio fish 24-48 or 96-120 hours after fertilization. Petri dishes contained 10 individuals in 2.5 ml of glycine buffer as above. Fish were treated with the recombinant

[0272] LM4_CHAP protein preparation obtained in Example 1 at concentrations of 0.5 pM, 1.0 pM and 1.5 pM

[0273] 35 suspended in the above-mentioned glycine buffer and incubated at 28°C for 2 hours. Then, bacteria were 16 plated on 5 ml TSB-Agar plates in the so-called droplet test, and the reduction in the initial bacterial count was estimated as the order of magnitude of logio CFU / ml. The negative control consisted of Y. ruckeri bacteria incubated in the presence of fish without added LM4_CHAP enzyme, while the positive control consisted of bacteria treated with identical concentrations of LM4_CHAP bactericidal protein without the

[0274] 5 presence of fish. The results shown in Tab. 2 indicate the high bactericidal efficacy of the tested recombinant protein LM4_CHAP, eliminated Y. ruckeri bacteria in the presence of Danio rerio fish almost as effectively as under optimal conditions, especially when added at higher concentrations. The protein concentrations used were not toxic to the fish.

[0275] Tab. 2. Bacteriolytic activity of the LM4_CHAP protein / enzyme (SEQ ID NO. 2) at three different

[0276] 10 concentrations against the Yersinia ruckeri CCM 4620 strain in the presence of 1-day-old Danio rerio fish. Activity is presented as a reduction in the initial number of bacterial cells expressed in no. of logs decrease in cell number (logio CFU / ml) by 4 logs (99.99%), 3 logs (99.90%), 2 logs (99.00%), 1 log (90.00%), 0 logs (0%, no reduction = no activity).

[0277] LM4_CHAP protein concentration Bactericidal activity (reduction in the initial number of bacteria)

[0278] CFU / ml logio % reduction in the number of cells

[0279] 0.5 pM - fish 2 99.00

[0280] 0.5 pM + fish 1 90.00

[0281] 1.0 pM - fish 4 99.99

[0282] 1.0 pM + fish 2 99.00

[0283] 1.5 pM - fish 4 99.99

[0284] 1.5 pM + fish 4 99.99

[0285] 15 The presented results indicate the high effectiveness of the recombinant LM4_CHAP protein in aquaculture uses and the possibility of using the invention in fish farming, especially industrial salmonid fish farming, for the treatment and prevention of fish diseases, especially yersiniosis, caused by the bacteria Yersinia ruckeri.

[0286] Example 10. Recombinant protein LM4_CHAP_L2_CBD

[0287] 20 The recombinant LM4_CHAP_L2_CBD protein (SEQ ID NO. 9) was produced by fusion of LM4_CHAP and

[0288] LM8_CBD domains and was shown to be active against various species of pathogenic bacteria at conductivities above 2.5 mS / cm.

[0289] To test whether the fusions of the recombinant LM4_CHAP protein (SEQ ID NO. 2) with the peptidoglycan- binding domain exhibit bactericidal properties under broad conductivity conditions, including conditions

[0290] 25 with conductivity > 2.5 mS / cm, a recombinant LM4_CHAP_L2_CBD protein was produced by fusing

[0291] LM4_CHAP (SEQ ID NO. 2) with a domain derived from the LM8 protein (CBD, SH3b-like, SEQ ID NO. 8). A

[0292] 17 recombinant protein was created containing the LM4_CHAP protein sequence (SEQ ID NO. 2) at the N- terminus and the CBD-binding, SH3b-l ike domain of the LM8 protein (SEQ ID NO. 8) at the C -terminus, connected by a L2 linker from the sequence of the LM4 gene (aa 371-392 of SEQ ID NO. 1).

[0293] In order to later isolate the recombinant LM4_CHAP_L2_CBD protein, the synthetic gene (BioCat) was

[0294] 5 cloned into the pET22b vector (Promega), which carries the ampicillin resistance gene and an additional oligohistidine sequence added at the C -terminus of the gene sequence. The cloning resulted in a gene for protein production (SEQ. ID NO. 9) containing an oligohistidine sequence (SEQ ID NO. 10) at the C- terminus. The protein was produced in E. coli ER2566 cells and then purified by affinity chromatography using the oligohistidine sequence (WorkBeads 40 Ni-NTA resin (BioWorks) or Talon (Cytiva). The

[0295] 10 oligohistidine sequence was not cleaved. The obtained LM4_CHAP_L2_CBDHls Tagprotein preparations had a purity above 95%, were suspended in storage buffer, i.e., 50 mM Tris-HCI, pH 8.0, 500 mM NaCI, 10% glycerol at a concentration of 7.1 mg / ml, and frozen in aliquots in liquid nitrogen and then stored at -80°C.

[0296] The preparations stored in this manner were used for activity assays at a concentration of 2 pM (0.076 mg / ml).

[0297] 15 Activity tests of the recombinant LM4_CHAP_L2_CBD protein were performed. The bactericidal activity of the LM4_CHAP_L2_CBD protein was verified against various bacterial strains listed in Tab. 3. Bacteria were cultured in TSB medium (Graso BioTech) at 37°C, with the exception of Yersinia ruckeri, which was cultured at 28°C with shaking and harvested in the logarithmic growth phase (ODgoo = 0.6), washed and suspended in 50 mM glycine-NaOH buffer, pH 8.0, 100 mM NaCI to achieve a density of 1 x io6CFU / ml.

[0298] 20 Each bacterial strain prepared in this manner was incubated for 24 hours at room temperature in the presence of 2 pM (0.076 mg / ml) of the enzyme being tested. The reaction mixtures were plated on TSB- agar plates in 10-fold serial dilutions, using the droplet method. Tab. 3 presents the experimental results, defining enzyme activity as a reduction in the initial number of bacteria expressed on a logarithmic scale

[0299] (reduction by 1 log = 90% reduction).

[0300] 25

[0301] 18 Tab. 3. Bacteriolytic activity of the LM4_CHAP_L2_CBD protein against various bacterial species. Activity was defined as a reduction in the initial number of bacterial cells expressed in logio CFU / ml by 4 logs (99.99%), 3 logs (99.90%), 2 logs (99.00%), 1 log (90.00%), or 0 logs s (0%, no reduction = no activity).

[0302] A group of Bactericidal activity (reduction in bacteria the initial number of bacteria)

[0303] Bacterial species Gram + / Gram loglO CFU / ml % CFU / ml

[0304] Listeria monocytogenes MF 7703 + 4 99.99

[0305] Staphylococcus aureus NCTC 8325-4 + 4 99.99

[0306] Staphylococcus epidermidis DSM 28319 + 4 99.99

[0307] Staphylococcus simulans CCM 3583 + 4 99.99

[0308] Enterococcus faecalis DSM 20376 + 4 99.99

[0309] Streptococcus agalactiae 123B + 4 99.99

[0310] Yersinia ruckeri CCM 4620 0 00.00

[0311] Escherichia coli DSM 1103 1 90.00

[0312] 5

[0313] The obtained results indicate the high efficiency of the recombinant chimeric protein LM4_CHAP_L2_CBD in the elimination of various Gram (+) bacteria, similarly to the single-domain LM4_CHAP protein. The chimera, unlike the single-domain LM4_CHAP protein, is active in conditions with conductivity above 2.5 mS / cm (i.e. at approximately 10 mS / cm), which ensures the possibility of its bactericidal use in

[0314] 10 physiological conditions.

[0315] NOTATION LISTING:

[0316] LM4 - proper name of a protein identified by the Authors in the Listeria monocytogenes genome, which amino acid sequence no. EAF9399118 is available in the GeneBank database (SEQ ID NO. 1)

[0317] CHAP - cysteine, histidine-dependent amidohydrolase / peptidase

[0318] 15 LM4_CHAP - CHAP catalytic domain derived from the full-length LM4 protein

[0319] LM4_Ami - amidase domain (Ami), being a fragment of the full-length LM4 protein

[0320] LM4_SH3b - SH3b (src Homology-3 bacterial domain) peptidoglycans binding domain, located at the C- terminus of the full-length LM4 protein

[0321] LM8 - the proper name of a protein identified by the Authors in the Listeria monocytogenes genome,

[0322] 20 which amino acid sequence is available in the GeneBank database EAG2511929.1 (SEQ. ID NO. 8)

[0323] TSB - tryptic soy broth

[0324] MTT - 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

[0325] EDTA- ethylenediaminetetraacetic acid

[0326] DMEM - cell culture medium (Dulbecco's Modified Eagle Medium)

[0327] 25 FBS - fetal bovine serum

[0328] CFU - bacterial colony-forming unit

[0329] 19 LITERATURE:

[0330] 1. Antimicrobial Resistance Collaborators. (2022). Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet; 399(10325): P629-655. DOI:

[0331] 2. World Bank. 2017. "Drug-Resistant Infections: A Threat to Our Economic Future." Washington, DC: World Bank. License: Creative Commons Attribution CC BY 3.0 IGO

[0332] 3. McKernan C, Benson T, Farrell S, Dean M. Antimicrobial use in agriculture: critical review of the factors influencing behaviour. JAC Antimicrob Resist. 2021 Nov 30;3(4):dlabl78. doi: 10.1093 / jacamr / dlabl78. PMID: 34859222; PMCID: PMC8634307.

[0333] 4. Schar, D., Klein, E.Y., Laxminarayan, R. et al. Global trends in antimicrobial use in aquaculture. Sci Rep 10, 21878 (2020). https: / / doi.org / 10.1038 / s41598-020-78849-3

[0334] 5. European Centre for Disease Prevention and Control. Surveillance report. Antimicrobial resistance in the EU / EEA (EARS-Net)) - Annual Epidemiological Report for 2021. Stockholm: ECDC; 2022 www.ecdc.europa.eu

[0335] 6. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 2012 Oct;7(10):1147-71. doi: 10.2217 / fmb.l2.97. PMID: 23030422; PMCID: PMC3563964.

[0336] 7. Nelson D, Schuch R, Chahales P, Zhu S, Fischetti VA. PlyC: a multimeric bacteriophage lysin. Proc Natl Acad Sci U S A. 2006 Jul ll;103(28):10765-70. doi: 10.1073 / pnas.0604521103. Epub 2006 Jul 3. PMID: 16818874; PMCID: PMC1487170.

[0337] 8. Becker SC, Dong S, Baker JR, Foster-Frey J, Pritchard DG, Donovan DM. LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells. FEMS Microbiol Lett. 2009 May;294(l):52-60. doi: 10.1111 / j.1574-6968.2009.01541.x. Epub 2008 Mar 10. PMID: 19493008.

[0338] 9. Horgan M, O'Flynn G, Garry J, Cooney J, Coffey A, Fitzgerald GF, Ross RP, McAuliffe O. Phage lysin LysK can be truncated to its CHAP domain and retain lytic activity against live antibiotic-resistant staphylococci. Appl Environ Microbiol. 2009 Feb;75(3):872-4. doi: 10.1128 / AEM.01831-08. Epub 2008 Dec 1. PMID: 19047377; PMCID: PMC2632115.

[0339] 10. Shang X, Nelson DC. Contributions of Net Charge on the PlyC Endolysin CHAP Domain. Antibiotics (Basel). 2019 May 28;8(2):70. doi: 10.3390 / antibiotics8020070. PMID: 31142020; PMCID: PMC6628322.

[0340] 11. Fagerlund A, Wagner E, Mpretrp T, Heir E, Moen B, Rychli K, Langsrud S. Pervasive Listeria monocytogenes Is Common in the Norwegian Food System and Is Associated with Increased Prevalence of Stress Survival and Resistance Determinants. Appl Environ Microbiol. 2022 Sep 22;88(18):e0086122. doi: 10.1128 / aem.00861-22. Epub 2022 Aug 25. PMID: 36005805; PMCID: PMC9499026.

[0341] 12. Pirone-Davies C et al., "Genes significantly associated with lineage II food isolates of Listeria monocytogenes.", BMC Genomics, 2018 Sep 25;19(l):708

[0342] 13. Li Z et al., "Draft Genome Sequences of Listeria monocytogenes Strains from Listeriosis Outbreaks Linked to Soft Cheese in Washington State.", Genome Announc, 2017 Sep 7;5(36)

[0343] 14. Burall LS et al., "Whole-Genome Sequencing Identifies an Atypical Listeria monocytogenes Strain Isolated from Pet Foods.", Genome Announc, 2014 Dec 24;2(6)

[0344] SEQUENCE LISTING:

[0345] SEQ ID NO. 1 The amino acid sequence encoded by the EAF9399118 LM4 gene originating from Listeria monocytogenes genome. The sequence comprises the CHAP catalytic domain (amino acids in bold) at positions (aa 1-208), the LM4_Ami catalytic domain (amino acids without additional markings) at positions

[0346] (aa 209-395), and the SH3b peptidoglycan-binding domain (underlined amino acids) at positions (aa 396-

[0347] 487).

[0348] 20 MKTQSQINKRLRDYKNGWDSPYRVKSWTSYDASFGAMEPGAIDVDRSYHAQCADLPTDYILWLTDNKYR

[0349] AWGNAKDFPNNKFPEGWKIVENKPSTIPKEGWIAVFTSGTYAQYGHIGIVYDGGNTNSFQILEQNWNGWA

[0350] NKKPSLRWDNYYGLTHFIVPPVAKEIEEPKKDAKSAPKQSVKENTNIKVNANHIKGWTMTKRGRKPKGVV

[0351] IHNDAGTMNSKQYYNNLVNVDYNRLARGIAHAYADRNGIWEAISEDRIAWHVSDGVQPGSGNFEFYGIEV

[0352] NQSMYVGDKDFLKNEQTALKFAAHKLKKWGLPANRNTVRLHNEFSYTACPHRSAKLHAGIDPTKQAWTKS

[0353] T QL KL KD Y F I KQ I KAYM.KGDKPKITTVKNKPGSAS rPA7VRAL>MZVGWKVNKYGTYYKTEHATFTPNTPIKT

[0354] HYVGPFRSCPVNGVLQPGQTVRYDTVCKQDGHIWISYTAYNGKDVWLAIRTWDKNTDSLGKLWGTIK

[0355] SEQ ID NO. 2 Amino acid sequence of the LM4_CHAP domain

[0356] MKTQSQINKRLRDYKNGWDSPYRVKSWTSYDASFGAMEPGAIDVDRSYHAQCADLPTDYILWLTDNKYR

[0357] AWGNAKDFPNNKFPEGWKIVENKPSTIPKEGWIAVFTSGTYAQYGHIGIVYDGGNTNSFQILEQNWNGWA

[0358] NKKPSLRWDNYYGLTHFIVPPVAKEIEEPKKDAKSAPKQSVKENTNIKVNANHIKGWTMTKRGRKPKG

[0359] SEQ ID NO.3 Amino acid sequence of the LM4_Ami domain

[0360] VVIHNDAGTMNSKQYYNNLVNVDYNRLARGIAHAYADRNGIWEAISEDRIAWHVSDGVQPGSGNFEFYGI

[0361] EVNQSMYVGDKDFLKNEQTALKFAAHKLKKWGLPANRNTVRLHNEFSYTACPHRSAKLHAGIDPTKQAWT

[0362] KSTQLKLKDYFIKQIRAYMKGDKPKITTVKNKPGSASTPANRRDMNG

[0363] SEQ ID NO. 4 Amino acid sequence of the LM4 SH3b domain

[0364] WKVNKYGTYYKTEHATFTPNTPIKTHYVGPFRSCPVNGVLQPGQTVRYDTVCKQDGHIWISYTAYNGKDV

[0365] WLAIRTWDKNTDSLGKLWGTIK

[0366] SEQ ID NO. 5 Oligohistidine amino acid sequence from the pLATE31 vector

[0367] GHHHHHHG

[0368] SEQ ID NO. 6 DNA nucleotide sequence encoding the LM4_CKAPHis Tagdomain, a fragment of the

[0369] EAF9399118 LM4 gene originating from Listeria monocytogenes genome (SEQ. ID No. 1), from which, as a result of translation the LM4_CHAP amino acid sequence (SEQ. ID No. 2) is formed, additionally comprising the oligohistidine seguence at the C -terminus (SEQ. ID No. 7)

[0370] ATGAAAACCCAGTCTCAGATCAACAAACGCCTGCGTGATTACAAAAACGGCGTTGTCGATAGCCCGTACC

[0371] GCGTTAAATCCTGGACCTCTTATGACGCGAGCTTCGGTGCCATGGAACCGGGTGCAATCGACGTTGACCG

[0372] TTCTTACCACGCGCAGTGCGCCGATCTGCCTACCGATTACATCCTGTGGCTGACCGACAACAAATACCGT

[0373] GCCTGGGGTAACGCTAAAGATTTCCCGAACAACAAATTCCCGGAAGGCTGGAAGATTGTCGAAAACAAAC

[0374] CAAGCACCATCCCGAAAGAAGGTTGGATTGCAGTCTTCACGTCCGGCACCTACGCTCAGTACGGCCACAT

[0375] CGGTATCGTTTACGATGGCGGCAACACCAACTCCTTCCAGATCCTGGAACAGAACTGGAATGGCTGGGCC

[0376] AACAAGAAACCGTCTCTGCGTTGGGACAACTACTACGGCCTGACCCACTTCATTGTTCCGCCGGTCGCCA

[0377] AAGAGATCGAAGAGCCGAAAAAAGATGCTAAATCCGCGCCTAAACAGAGCGTTAAAGAGAACACCAACAT

[0378] CAAGGTTAACGCGAACCACATTAAAGGCTGGACGATGACCAAACGTGGTCGTAAACCGAAAGGTGGCCAP CACCATCACCACCACGGC

[0379] SEQ ID NO. 7 Nucleotide sequence of the oligohistidine tag from the pLATE31 vector

[0380] GGCCATCACCATCACCACCACGGC

[0381] SEQ ID NO. 8 Amino acid sequence of the LM8 protein encoded by the EAG2511929.1 gene originating from Listeria monocytogenes genome. The sequence comprises the Ami catalytic domain (amino acids 21 in bold) at positions 30-199, the CBD, SH3b-like peptidoglycan-binding domain, (underlined amino acids) at positions 205-336, and a domain comprising the oligohistidine tag (amino acids in italics) at positions

[0382] 1-29

[0383] PfAGSHHffHffHGMASPfPGGGQMGPSGPvEECATVNGVSFRQNLVSSSKYGIKAPNTMKPKKITVHNTYNDAT AQNETDYCKNNNNEVSFHVAVDDKEAIQWPFDRNAWHCGDGGNGYGNRNTVGVEICYSKSGGSKYTKSE QNAIKYIAGLCVQQGIAATKDTIKKHQDWSGKYCPHRVLAEKRWPALQQAI IDEYKRI TS KNPNRHSGAV

[0384] VDSVPMLSKTDFKSSPVRMYKAGTAILVYEHNKYWYKAYINDKLCYIYKSFCVSNGKKDAKGRIPVKIKS

[0385] AKDLRIPVWDNTRLNSGKIKWYAPYTKLSWYDNRKGYLELWYTKDGWYYTANYFLK

[0386] SEQ ID NO. 9 Amino acid sequence of the LM4_CHAP_L2_CBD chimera. The sequence comprises the CHAP domain (amino acids in bold, positions 1-164), the 12 linker (amino acids in italics, positions 165-189), the

[0387] CBD, SH3b-like peptidoglycan-binding domain (underlined amino acids, positions 190-321), and the oligohistidine tag domain (font highlithted in gray, positions 322-329).

[0388] MKTQSQINKRLRDYKNGWDSPYRVKSWTSYDASFGAMEPGAIDVDRSYHAQCADLPTDYILWLTDNKYR

[0389] AWGNAKDFPNNKFPEGWKIVENKPSTIPKEGWIAVFTSGTYAQYGHIGIVYDGGNTNSFQILEQNWNGWA

[0390] NKKPSLRWDNYYGLTHFIVPPVAKKPKTTTVKZVKPGSASPPAAIRAPTWGRHSGAVVDSVPMLSKTDFKSS

[0391] PVRMYKAGTAILVYEHNKYWYKAYINDKLCYIYKSFCVSNGKKDAKGRIPVKIKSAKDLRIPVWDNTRLN

[0392] SGKIKWYAPYTKLSWYDNRKGYLELWYTKDGWYYTANYFLKLEHHHHHH

[0393] SEQ ID NO. 10 Oligohistidine amino acid sequence from the pET22b vector

[0394] LGHHHHHH

[0395] 22

Claims

CLAIMS1. A recombinant protein with antibacterial properties characterized in that it comprizes an active CHAP catalytic domain with the amino acid sequence shown in SEQ. ID NO. 2 derived from Listeria monocytogenes, and an amino acid sequence attached at one of the termini, useful for purification and / or isolation of the protein.

2. The recombinant protein according to claim 1, characterized in that it additionally contains a bacterial cell wall binding domain attached to one of the termini, preferably the bacterial cell wall binding domain is a domain of the type SH3b, LysM, AMIN, preferably the bacterial cell wall binding domain is a peptidoglycan binding domain.

3. The recombinant protein according to claims 1-2, characterized in that the amino acid sequence useful for purification and / or isolation of the protein attached at one of the termini is located at the C-terminus of the CHAP domain, preferably the amino acid sequence useful for purification and / or isolation of the recombinant protein is the oligohistidine sequence shown in SEQ ID NO. 5.

4. The recombinant protein according to claim 3, characterized in that it consists of the sequence SEQ IDNO. 2 at the N-terminus of the recombinant protein and the oligohistidine sequence shown in SEQ ID NO.5 at the C-terminus of the recombinant protein.

5. The recombinant protein according to claims 1-4, characterized in that it has endolysin and / or bacteriocin and / or autolysin activity.

6. A genetic construct characterized in that it encodes the recombinant protein as defined in claims 1-5.

7. A host cell characterized in that it comprises the genetic construct as defined in claim 6.

8. A composition characterized in that it comprises the recombinant protein as defined in claims 1-5 or a mixture thereof and a carrier, wherein preferably, the composition is in the form of a solution, tablet, powder, granulate, suspension, emulsion, aerosol, gel, cream.

9. A non-medical use of the recombinant protein as defined in claims 1-5 and / or the composition as defined in claim 8 as an antibacterial agent.

10. The non-medical use according to claim 9, characterized in that the antibacterial agent is used in an aqueous environment with a conductivity of 0 to 2.5 mS / cm.

11. The non-medical use according to claims 9-10, characterized in that the antibacterial agent is used against bacteria of the genus Listeria spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp.,Enterococcus spp., Streptococcus spp., Corynebacterium spp., Bacillus spp., Escherichia spp., Klebsiella spp., Pseudomonas spp., Yersinia spp., Acinetobacter spp.; more preferably against the species Listeria23monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus,Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus canis,Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coli, Yersinia ruckeri, Acinetobacter heamolyticus.

12. Use of the recombinant protein as defined in claims 1-5 and / or the composition as defined in claim 8 as an antiseptic and disinfecting agent.

13. Use of the recombinant protein as defined in claims 1-5 and / or the composition as defined in claim 8 as an antibacterial agent for decontamination of surfaces and rooms in food processing, preferably for decontamination of surfaces and / or tools that come into contact with food or semi-finished food products.

14. Use of the recombinant protein as defined in claims 1-5 and / or the composition as defined in claim 8 as a decontaminating agent for surfaces in health care in medicine, veterinary medicine, diagnostics, preferably for disinfecting hospital surfaces, medical and veterinary offices, laboratory surfaces, surfaces of tools, surfaces of devices used in medicine, veterinary medicine, diagnostics.

15. A non-medical use of the recombinant protein as defined in claims 1-5 and / or the composition as defined in claim 8 as an in vitro bactericidal agent for inhibiting, eliminating bacteria, preventing the growth of Gram (+) and Gram (-) bacteria, preferably bacteria of the genus Listeria spp., Klebsiella spp.,Pseudomonas spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., Corynebacterium spp.,Bacillus spp., Escherichia spp., Klebsiella spp., Pseudomonas spp., Yersinia spp., Acinetobacter spp.; preferably bacteria of the species Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecium, Enterococcus faecalis, Streptococcus canis, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coli, Yersinia ruckeri, Acinetobacter heamolyticus.

16. The non-medical use according to claim 15, characterized in that the bactericidal agent is used in an aqueous environment with a conductivity of 0 to 2.5 mS / cm.

17. Use of the recombinant protein as defined in claims 1-5 and / or the composition as defined in claim 8 as a bactericidal agent for inhibiting, eliminating bacteria, preventing the growth of bacteria in water, preferably bacteria of the species Escherichia coli, Pseudomonas aeruginosa, Enterococcus feacalis,Yersinia ruckeri.

18. The use according to claim 17, characterized in that the bactericidal agent is used in aquacultures.

19. The use according to claims 17-18, characterized in that the bactericidal agent is used in fish hatcheries and nurseries, closed water systems, in containers for transporting fish and / or fry, in24containers for storing fish and / or fry, in containers with eggs and / or larvae of aquatic animals, in containers with aquatic plants and for equipment having contact with fish.

20. The use according to claims 17-19, characterized in that the bactericidal agent is used in an aqueous environment with a conductivity of 0 to 2.5 mS / cm.

21. A veterinary and / or pharmaceutical composition comprising the recombinant protein as defined in claims 1-5 and a veterinarily acceptable carrier for use in the prevention and / or treatment of fish diseases caused by Yersinia ruckeri, preferably for use in the prevention and / or treatment of yersiniosis in fish.

22. The veterinary and / or pharmaceutical composition for use according to claim 21, characterized in that it is used for the treatment of yersiniosis in salmonid fish, preferably selected from rainbow trout(Oncorhynchus mykiss), brook trout (Salvelinus fontinalis), brown trout (So / mo trutta morpha fario), sea trout (So / mo trutta morpha trutta), Atlantic salmon (So / mo salar).

23. A method for inhibiting or limiting the growth of Gram (+) and / or Gram (-) bacteria with a varied peptidoglycan structure, characterized in that it comprises a step in which the bacteria are contacted with the recombinant antibacterial protein as defined in claims 1-5 and / or the composition as defined in claim8 in an environment with conductivity ensuring bactericidal activity of the recombinant protein.

24. The method according to claim 23, characterized in that the contacting is carried out in an aqueous solution with a conductivity of 0 to 2.5 mS / cm.

25. The method according to claims 23-24, characterized in that the bacteria are selected from the genusListeria spp., Klebsiella spp., Pseudomonas spp., Staphylococcus spp., Enterococcus spp., Streptococcus spp., Corynebacterium spp., Bacillus spp., Escherichia spp., Klebsiella spp., Pseudomonas spp., Yersinia spp., Acinetobacter spp.; preferably from bacteria of the species: Listeria monocytogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis,Enterococcus faecium, Enterococcus faecalis, Streptococcus canis, Staphylococcus simulans, Streptococcus agalactiae, Streptococcus dysgalactiae, Corynebacterium striatum, Bacillus subtilis, Escherichia coli,Yersinia ruckeri, Acinetobacter heamolyticus.25

Images