The subject matter of the invention is the polypeptide of antibacterial or lytic effect to bacterial cells or lytic or cytotoxic effect to eukaryotic cells, its encoding polynucleotide or expression vector, the new Staphylococcus pseudintermedius strain and their applications and production method of the said polypeptides.

Current state of art

Bacteria are capable of producing multiple compounds potentially lethal to the other microorganisms. One of such group of biologically active molecules are bacteriocins - ribosomally synthetized peptides or proteins produced by the bacteria, capable of killing of strains closely related to the producer strain in low concentrations. Both bacterial strains producing the bacteriocins and the bacteriocins alone have been currently widely used for food and feed preservation as well as for the selected medical and veterinary applications and in life sciences.

The bacterial species - Staphylococcus pseudintermedius - is a common opportunistic pathogen of domestic animals - in particular of dogs and horses, however it is isolated also from the other vascular organisms. Recently, a first case of infection with this bacterial species in human has been noticed (Bannoehr and Guardabassi, 2012, Garbacz et al., 2013, van Hoovels et al., 2006). An infection case resulting in patient's death was also described (Savini et al., 2013).

Bacteriocins are an important group of molecules produced and secreted by multiple bacteria. These are ribosomally synthetized peptides or bacteria, capable of killing of strains closely related to the producer strain in low concentrations (nanomols /liter). This activity makes bacteriocins one of the main effectors, thanks to which the producer strain gains an advantage over the other microorganisms competing in the same ecological habitat or in the same physiological niche. Bacteriocins are also one of bacterial metabolic regulators dependent on producer cell population density as well as on presence of the other bacteria. Due to lack or relative lack of toxic effect on the vascular organisms, bacteriocins are safe in use and widely used as food and feed preservatives. Safety of bacteriocins use is primarily related to their commonness, in particular with commonness of the producing bacteria, present primarily in milk products, silages and meat products, which are the components of the human diet for thousands of years. One should remember that bacteriocin molecules are not antibiotics. Bacteriocins and antibiotics are two different groups of compounds. Antibiotics are the products of secondary metabolism of microorganisms, synthetized by enzymes other than ribosomes and of wide spectrum of effect, with the activity generally lower comparing to bacteriocins (the lethal effect of antibiotic requires usually the several magnitudes higher concentrations comparing to corresponding bacteriocins concentrations). As opposed to bacteriocins, application of antibiotics results in many side effects, harmful to humans, animals and environment, both in short- and long-term perspective. Application of antibiotics is limited primarily to medical and veterinary purposes and must be strictly controlled (Riley and Chavan, 2007, Drider and Rebuffat, 2011,).

Bacteriocins are exceptionally diversified molecules. They have different sizes, structures, mechanisms of action and organization of encoding genes. This diversity hinders and complicates their classification, often different depending on the author. Bacteriocins are produced both by Gram-positive and Gram-negative bacteria, but the ones produced byGram- negative bacteria are much less diversified group of proteins and peptides than those produced by Gram-positive bacteria. Moreover, the bacteriocins produced by Gram-negative bacteria have narrower spectrum of action and in most cases bind with a specific sensitive cell receptor, absent in most Gram-positive bacteria.

Gram-negative bacteria-produced bacteriocins embrace three groups of proteins or peptides: colicins (produced by majority of so-called enterobacteria, dominating in human intestines), microcins and phage tail-like bacteriocins.

Gram-positive bacteria-produced bacteriocins (comprising the strain being the subject- matter of this invention) are divided into four classes: class I contains post-translationally modified peptides, class II contains heat-stable unmodified peptides of molecular mass below 10 kDa, class III contains large, heat-labile proteins, and class IV covers poorly recognized proteins of large molecular mass , covalently bounded with hydrocarbons or lipids. These classes are further divided into subclasses and groups, depending on more detailed criteria (Riley and Chavan, 2007).

Bacteriocins produced by Staphylococcus genus are called staphylococcins and are secreted both by coagulase-negative and coagulase-positive strains. Staphylococcins belong to four group of bacteriocins: subclass la called lantibiotics (nine representatives known), subclass lib and lid (four known representatives) and class II (two representatives)( Bastos et al., 2009).

In context of this invention one should draw its attention to the two groups of staphylococcins. The first one is a relatively recently described group of rare bacteriocins included into Gram-positive bacteria-produced lib subclass of bacteriocins, including aureocin A53 isolated from Staphylococcus aureus A53 and epidermicin NIOl obtained from Staphylococcus epidermidis 224. Both these peptide carry 51 amino acid residues, are linear, post-translationally unmodified, rich in tryptophan and in cationic amino acids and having high isoelectric point. All are plasmid-encoded, with operons encoding also the proteins involved in secretion of bacteriocins as well as in immunity against toxic effects of secreted bacteriocins . The mechanism of action of such bacteriocins remains unknown, however it is suggested that they induce formation of pores in the bacterial cell walls, causing metabolites outflow and, in consequence, death of cells. However, until now there have been no known examples of the commercial application of the a/m peptide staphylococcins (Netz et al., 2002, Sandiford and Upton, 2012).

The second group of staphylococcins worth notifying in context of this invention are lysostaphins. They are included into subclass Ilia of Gram-positive bacteria-produced bacteriocins, containing muramynolytic enzymes able to dissolution / lysis of sensitive bacteria and in consequence - their death. From functional perspective, lysostaphins are similar to lysozymes - common animal hydrolytic enzymes, hydrolyzing the peptidoglycans of bacterial cell walls. Subclass Ilia of staphylococcins includes two similar enzymes - lysostaphin produced by Staphylococcus simulans biowar simulans and endopeptidase ALE- 1, produced by Staphylococcus capitis EPK1. The first one discovered and best described staphylococcin is lysostaphin, being, in mature form, a protein of 25 kDa molecular mass with two domains: N-terminal peptidase, capable of digesting glycine-glycine peptide bonds, and C-terminal peptidase capable of binding with peptidoglycans of bacterial cell walls. Lysostaphin is a relatively rare example of bacteriocin used commercially in life science. This enzyme in a recombinant form is sold marketed by many companies as an effective bacteria dissolving agent , useful in molecular biology (Kumar, 1997, Sugai et al., 1997).

The market potential of bacteriocins is related to the high safety of its use and to the fact that they are classified as so-called 'natural' preservatives. According to different sources, it is estimated that within several years the annual value of the global sales market of such natural preservatives will reach USD 2.5 - 2.7 billion. In food and feed processing industry, bacteriocins are used both in purified form, directly as food additives, or as the products inoculated with bacterial strains producing the bacteriocins. The example of bacteriocin directly added to the products include for the most nisin, a peptide bacteriocin from subclass 1, produced primarily by Lactococcus lactis strains and approved as so called GRASS compound (generally recognized as safe) for use as food preservative in more than 50 countries. It kills the sensitive microorganisms in MIC (minimal inhibitory concentration) doses of nanomols/liter and is a safe and for the most stable preservative, resistant to heat and low pH values, commonly used in food processing (cheese, milk products, tinned food, meat and alcohol beverages) and in animal feed. Until now, nisin and nisin-producing bacterial strains are the only examples of such common and wide-scale commercial application. The other bacteriocins are applied much less frequently, and their use concerns for the most bacteriocin-producing bacteria application. This refers in particular to the strains which are able to produce microcins and colicins, used as protective probiotics in poultry and bovine production. Their administration in feeds reduces the harmful pathogens level in animal digestive tracts, significantly increasing the resistance to bacterial infections. The example of bacteriocin used commercially in life sciences is the a/m lysostaphin, sold by multiple companies as an effective bacteria dissolving agent, useful in molecular biology. Lysostaphin is also used in staphylococcus typing and is currently under clinical trials as anti- streptococcal drug (Riley i Gillor, 2007).

Thus, providing the new bacteriocins and the methods of their production as well as the bacterial strains applicable for such methods is of particular importance.

Unexpectedly, the a/m objective was obtained in this invention.

The subject-matter of invention is a polypeptide containing an amino-acid sequence selected from among Sequence 1 or Sequence 3 or their derivative of antibacterial or lytic effect to bacterial cells or lytic or cytotoxic effect to eukaryotic cells.

Another subject-matter of the invention is a polynucleotide containing a sequence coding the a/m polypeptide, advantageously comprising the nucleotide sequence selected from among Sequence 2 or Sequence 4.

A subsequent subject-matter of the invention is a polypeptide according to the invention or polypeptide according to the invention for application in therapy.

Another subject-matter of the invention is the expression vector containing the a/m polynucleotide, for the most plasmid comprising the sequence presented on Fig. 4A.

A subsequent subject-matter of the invention is the Staphylococcus pseudintermedius 222 strain deposited in the PCM under the accession No. B/00084.

Another subject-matter of the invention is application of the polypeptide according to the invention or polynucleotide according to the invention or bacterial strain according to the invention for production of formulation of antibacterial or lytic effect to bacterial cells or lytic or cytotoxic effect to eukaryotic cells. Another subject-matter of the invention is the production method of polypeptide according to the a/m invention, characterized in that the bacterial host culture containing the a/m expression vector is cultivated , followed by protein isolation from the post-culture fluid.

Advantageously, protein isolation is performed using the method selected from the following ones : salting-out, dialysis, concentration on semi-permeable membranes or reverse phase chromatography.

Advantageously, the Staphylococcus pseudintermedius 222 strain deposited in the PCM under the accessiin No. B/00084 is used as the bacterial host.

The new bacterial strain according to the invention belongs to the Staphylococcus pseudintermedius species and is capable to produce peptide and protein bacteriocins of wide spectrum of biological effect: antibacterial or lytic to Gram-positive bacteria as well as cytotoxic or lytic to eukaryotic cells. In addition, the significant features of one of the bacteriocins produced by the said strain is high resistance of its molecule to decomposition by proteolytic enzymes, heat resistance and maintaining bioactivity upon partial molecule fragmentation by chemical agents.

The new Staphylococcus pseudintermedius strain according to the invention of the working name Sp222 was isolated from pathological dermal lesions of dog and deposited on 14 January 2014 in the patent deposit of the Polish Collection of Microorganisms, located at the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wroclaw under the accession No. PCM B/00084.

The results of the studies performed by the authors of this invention demonstrated that Sp222 produces and secretes to the environment, among others, two new bacteriocins. The first one is the linear, tryptophan and cationic amino acid-rich antibacterial peptide of the working name BacSp222. This peptide has also hemolytic and cytotoxic properties to eukaryotic cells as well as manifests unique resistance to decomposition by proteolytic enzymes. The second bacteriocin isolated from Sp222 is the new enzyme of the working name lysostaphin Sp222, with lytic potential to bacterial cells and, in consequence , having with the ability to kill bacterial cells, (Mak and Wladyka, non-published results).

Another aspect of the invention concerns the new, third representatives of the a/m staphylococcins, i.e. peptide bacteriocin of the working name BacSp22, produced by the reserved Sp222 bacterial strain. Its amino acid sequence is unique and displays no statistically significant similarities to none of the currently known bacteriocins, however its physical and chemical properties (molecular mass , high amount of tryptophan, lysine and arginine residues) and antibacterial activity enable its classification into the group of peptides similar to the a/m aureocin A53 and epidermicin NIOl. The BacSp222 bacteriocin is capable of killing the Gram-positive bacterial cells in micromolar and sub-micromolar concentrations and demonstrates biological properties beyond the properties of typical bacteriocins. It is capable of erythrocyte hemolysis and demonstrates the cytotoxic effect to eukaryotic cells. These facts suggest that this peptide is capable of modifying the immunological response of host organism. Regardless of these activities, the molecule of BacSp222 bacteriocin differentiates from among many other bacteriocins with high resistance to decomposition by proteolytic enzymes - both eukaryotic and prokaryotic. It is also stable in the increased temperature. Its significant antibacterial activity after limited chemical fragmentation is also proved. All these properties - proteolytic resistance, temperature stability and maintaining bioactivity after limited chemical decomposition - are of importance for application (Moreno et al. 2000).

In the other aspect, the invention concerns the new representative of lysostaphins of the working name lysostaphin Sp222, produced by the Sp222 bacterial strain according to the invention. The enzymatic sequence is new, however with statistically significant similarities to already known lysostaphins of the other bacteria. Also the activity of the new enzyme is similar.

The description of the invention is accompanied with the following tables and figures.

Table 1 presents the MIC doses of BacSp222 bacteriocin against different microorganisms. The determined doses are the average from three independent measurements.

Table 2 presents the residual bactericidal activity of BacSp222 peptide bacteriocin digested with different proteolytic enzymes. This activity is presented as bacterial growth inhibition zone diameter (in mm) in the radial diffusion test. The tested samples include bacteriocin incubated with peptidases, the control samples include bacteriocin incubated without peptidases.

Fig. 1 presents the results of the experiment demonstrating the antibacterial activity of the Sp222 strain. On the left edge of a plate containing the solid nutrient medium, the wide vertical streak of Sp222 colonies was plated , whereas next to the streak, on the right, four indicator bacteria were plated in spots. After incubation of the plate for 24 hours in 37°C one may observe that the grown colonies of all four studied bacteria are asymmetric - in a form of crescent. Their growth was inhibited on the left by antibacterial substances secreted to the medium by the Sp222 strain.

Fig. 2 presents the results of the experiment demonstrating the lytic activity of Sp222 strain against bacterial cells. In two wells of 96-well microplate, the suspension of Staphylococcus aureus RN4220 bacterial cells was introduced. The first well contained no additives (the control), whereas the second contained 10 μΐ of the Sp222 post-culture medium. Then the microplate was incubated in temperature of 37°C with simultaneous measurement of decrease in optical density caused by bacterial lysis. In the well containing the Sp222 post-culture medium , this decrease was significantly higher comparing to the control well and proves the presence of an agent causing bacterial cell lysis in the Sp222 post- culture medium.

Fig. 3 presents the results of subsequent stages of BacSp222 peptide bacteriocin purification. In all panels, bacteriocin peak or band was marked with an arrow. A) Reverse phase - high-pressure liquid chromatography (RP-HPLC) on the Nucleosil C18 column B) RP-HPLC on the Kromasil C4 column C) SDS-PAGE denaturing electrophoresis of fractions collected at each purification stage (post-culture medium, after salting-out, after C18 column and final formulation after the C4 column). The figure illustrates that the final solution of purified bacteriocin contains a single and homogenous peptide band of molecular mass weight of ca. 5 kDa.

Fig. 4 presents the p222 plasmid sequence. The A panel contains nucleotide sequence of the entire plasmid, whereas the fragment encoding the BacSp222 peptide bacteriocin was highlighted. The B panel contains fragment of nucleotide sequence encoding the BacSp222 peptide bacteriocin along with respective amino acid residues. Fig. 5 presents the results of the experiment demonstrating the hemolytic activity of BacSp222 peptide bacteriocin. The activity was determined as percent of hemolysis of human erythrocyte suspension and calculated as average from three independent measurements.

Fig. 6 presents the results of the experiment demonstrating the cytotoxic activity of the BacSp222 peptide bacteriocin. The bacteriocin cytotoxic activity was tested in concentrations between 0.195 and 100 micromols / liter against human skin fibroblasts and human adipose- derived stem cells. The determination of cytotoxic activity was performed using two different biological tests - LDH and MTT.

Fig. 7 presents the results of the experiment concerning changes of the BacSp222 peptide conformation in different temperatures. Changes of conformation were observed by measurement the ellipticity at 220 nm in the circular dichroism spectrum of the peptide incubated in temperatures between 20 and 90 DC.

Fig. 8 presents the results of the experiment demonstrating the antibacterial activity of the BacSp222 peptide bacteriocin without formylated methionine at N-terminus. Antibacterial activity of native bacteriocin (marked as '+fM' on the figure) and deprived of methionine bacteriocin (marked as '-fM' on the figure), in amounts of 190, 380 and 760 picomols/well, were tested using the radial diffusion assay in the solid nutrient medium containing the sensitive bacteria suspension. The obtained diameters of bacterial growth inhibition zones demonstrate that bacteriocin in the form without N-terminak formylated methionine requires, in average, twice higher concentrations to achieve the diameter of growth inhibition zone specific for the native bacteriocin.

Fig. 9 presents the results of subsequent stages of Sp222 lysostaphin purification. In all panels, the peak or the band of the purified enzyme was marked with an arrow. A) Reverse phase - high-pressure liquid chromatography (RP-HPLC) on the Nucleosil C18 column B) RP-HPLC on the Discovery C5 column C) RP-HPLC on the Discovery C18 column. D) SDS-PAGE denaturing electrophoresis of fractions collected at each purification stage (post-culture medium, after salting-out, after Nucleosil C18 and Discovery C5 columns and final preparation after the Discovery C5 column). The figure illustrates that the final solution of purified lysostaphin contains a single and homogenous protein band of molecular mass of ca. 30 kDa.

Fig. 10 presents the Sp222 lysostaphin encoding sequence. The panel A contains the nucleotide sequence, whereas panel B contains the same nucleotide sequence with respective amino acid residues of the Sp222 lysostaphin molecule.

Fig. 11 presents the results of the experiment demonstrating the lytic activity of purified Sp222 lysostaphin against bacterial cells and its comparison with the commercial lysostaphin activity. In three wells of 96-well microplate, the suspension of Staphylococcus aureus RN4220 bacterial cells was introduced. The first well contained no additives (the control well), the second one contained 1 μg of Sp222 lysostaphin and the third one contained 1 of commercial lysostaphin. The microplate was then incubated in temperature of 37°C with simultaneous measurement of optical density decrease caused by bacterial lysis. In the well containing Sp222 lysostaphin this decrease is significantly higher comparing to the control well and comparing thr well with commercial lysostaphin, which proves high lytic activity of the Sp222 lysostaphin.

For better understanding of the substance of the invention, it was illustrated with the following examples, published only for illustrating and explaining the invention rather than limiting thereof. The examples cannot be identified with the entire range of the invention, which was specified in the patent claims. Example 1: Demonstration of antibacterial activity of substances secreted to the environment by the Sp222 strain.

To demonstrate the inhibiting effect of the Sp222 strain on the growth of the other bacteria, the culture of Sp222 on solid nutrient medium near the other bacteria was performed and the impact of Sp222 strain on the other bacteria growth was observed. For this purpose, a plate containing a sterile solid nutrient medium intended for bacteria was prepared (Tryptic Soy Agar, Sigma). On the left edge of a plate containing the solid medium, the wide vertical streak of Sp222 colonies was cultured. The plate was then incubated for 24 hours in 37°C. After this period, near of the Sp222 streak, on the right, four indicator bacteria were cultured in spots: Staphylococcus aureus RN4220, Staphylococcus pseudintermedius 223 (from the collection of the Department of Microbiology of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University), Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology) and Staphylococcus epidermidis ATCC 35547 (American Type Cell Culture Collection, USA). The plate was then incubated for 24 hours in 37°C. After incubation, the image presented on Fig. 1 was obtained. One may observe that the grown colonies of all four studied bacteria are asymmetric - in a form of crescent. Their growth was inhibited on the left by antibacterial substances secreted to the medium by Sp222.

Example 2: Demonstration of lytic activity of the substances secreted to the environment by the Sp222 strain against the other bacterial cells.

To demonstrate the lytic activity ( the ability to dissolve the cell walls leading to disintegration and death of bacterial cell) of the substances produced by Sp222 strain against the other bacterial cells, the suspension of studied bacteria was incubated in presence of liquid post-culture medium of Sp222 and the decrease of suspension turbidity was observed, proportional to bacterial lysis. For this purpose, a portion of sterile liquid bacterial nutrient medium (Trypticase Soy Broth, Sigma) was inoculated with Sp222 strain bacteria, followed by culturing for 15 hours in temperature of 37°C in the orbital shaker at 180 rpm/minute. The obtained bacterial suspension was centrifuged in 4°C at 4 000 g, followed by collection of clear supernatant with post-culture medium. This medium contained the substances secreted to the environment by the Sp222 strain. 10 μΐ of such post-culture medium was added to 96- well polystyrene microplate containing 150 μΐ of Staphylococcus aureus RN4220 suspension (from the collection of the Department of Microbiology of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University) in 20 mM Tris-HCl buffer of pH 7.5 containing 150 mM NaCl. The adjoining well contained 160 μΐ of Staphylococcus aureus RN4220 suspension in this buffer. This suspension contained no other additives and acted as a control. The microplate was then placed in the microplate reader (Synergy HI Hybrid Multi-Mode Microplate Reader, BioTek, USA) and the measurements of turbidity at 600 nm, every 2 minutes for 1 hour in temperature of 37°C were performed. Decrease of turbidity is proportional to bacterial cell lysis degree . The results of measurements are presented on Fig. 2. One may observe that in the well containing post-culture Sp222 medium, the turbidity decreased significantly higher comparing to the control well, which proves the presence of an agent secreted by Sp222 strain to the post-culture medium that is able to lyse bacterial cells .

Example 3: Procedure of purification of the BacSp222 peptide bacteriocin.

The procedure of BacSp222 peptide bacteriocin purification, present in liquid Sp222 post- culture medium embraces salting-out of peptide with high salt concentrations, followed by two stages of reverse phase chromatography. A portion of sterile liquid bacterial nutrient medium (Trypticase Soy Broth, Sigma) was inoculated with Sp222 strain bacteria and cultured for 15 hours in 37°C in an orbital shaker at 180 rpm/minute. The obtained bacterial suspension was centrifuged for 20 minutes in temperature of 4°C at 5 000 g, followed by collection of clear supernatant containing the post-culture medium. This medium contained the substances secreted to the environment by Sp222 strain, including the substance of lytic activity as specified in example 2. The medium was cooled to the temperature of 4°C and, to precipitate the proteins and peptides, the solid ammonium sulphate was continuously added until reaching 60% (in weight) saturation of the concentration. After this the medium was mixed for 30 minutes, followed by leaving it in an ice bath without mixing for 2 hours. The obtained protein deposit was centrifuged for 30 minutes in 4°C at 21 000 g. The supernatant was removed, whereas the deposit was dissolved in water containing 0.1 % (in volume) of trifluoroacetic acid (TFA) and 60 % (in volume) of acetonitrile. This solution was centrifuged for 5 minutes in 4°C at 16 000 g, the collected supernatant was filtered by a 0.45 μιη filter and separated by reverse phase high pressure liquid chromatography (RP-HPLC). The separation was performed on chromatographic set Ultimate 3000 (Dionex/Thermo, USA) and Nucleosil C18 300 A 250 x 8 mm column (Macherey-Nagel, Germany). The gradient elution with the use of two buffers was applied, A: 0.1 % (in volume) of TFA in water, B: 80 % (in volume) of acetonitrile in water with the addition of 0.07% (in volume) of TFA. The flow rate of 1.5 ml/minute and the spectrophotometric detection at 220 nm were applied. The separation was started on the column equilibrated at 60% of buffer B, followed by a linear gradient elution from 60 to 100% of buffer B during 20 minutes. The bacteriocin fraction eluted on the chromatogram as a peak of retention time within 18-20 minutes was collected to a test tube, lyophilized, dissolved in water containing 0.1% (in volume) of TFA and 30% (in volume) of acetonitrile and subject to re-chromatography on the Kromasil C4 100A 250 x 4.6 mm column (Sigma, USA). The flow rate of 1 ml/ and the spectrophotometric detection at 220 nm were applied. The separation was started on the column equlilibrated at 68% of buffer B, followed by linear gradient elution from 68 to 75% of buffer B during 20 minutes. The bacteriocin fraction eluted on the chromatogram as a peak of retention time within 11- 14.5 minutes was collected to a test tube, lyophilized, dissolved in water to obtain the final purified Sp222 bacteriocin solution. Peptide concentration in the solution was determined by the amino acid composition analysis. The stages of chromatographic purification are presented on Fig. 3. The figure contains also electrophoretic image of fractions collected at the particular purification stages. The image demonstrates that the final solution of purified bacteriocin contains a single and homogeneous peptide band of molecular mass of ca. 5 kDa.

Example 4: Determination of amino acid sequence of BacSp222 peptide bacteriocin.

The amino acid sequence of peptide purified according to the procedure described in example 3 was determined on the automatic protein sequencer Procise 491 (Applied Biosystems, USA) performing the Edman degradation of polypeptide chains. Before the sequence determination the formyl group at the N-terminus was chemically removed by incubation of the peptide in 0.6 M hydrochloric acid for 25 hours. The obtained amino acid sequence of BacSp222 bacteriocin was : fM AGLLRFLLS KGRALYNW AKS H VGKVWE WLKS G AT YEQIKEWIEN ALGWR . This sequence is presented in a form of single-letter amino acid residue abbreviations, provided that the letter "f" at the beginning of the sequence means that the alpha-amine group of N- terminal methionine residue is formylated. Molecular mass of BacSp222 bacteriocin was determined using the MALDI-ToF mass spectrometer, model ultrafleXtreme (Bruker, Germany). Obtained mass was equal to 5921.917 Da and agrees with the theoretical peptide molecular mass 5921.888 Da, calculated basing on the a/m amino acid sequence. This agreement proves that the a/m peptide amino acid sequence was determined correctly .

Example 5: Determination of the sequence of the gene encoding the BacSp222 peptide bacteriocin.

The sequence of the gene encoding the BacSp222 peptide bacteriocin was obtained using the bioinformatic shotgun sequence analysis of Sp222 genome. Complete DNA (desoxynucleic acid) isolated from Sp222 bacteria using the standard techniques known to branch specialists, was subject to shotgun sequencing using the MiSeq (Illumina) instrument, using the dedicated reagents and procedure set. The obtained short sequence readings were combined in longer sections (so called contigs) with the use of MIRA (http://mira-assembler.sourceforge.net/) and CLC Main Workbench (CLC bio) programmes. In one of contigs containing the plasmid sequence (p222), the BacSp222 peptide bacteriocin encoding sequence was identified. This was made by translating the plasmid DNA sequence based on genetic code into the sequences containing so called open reading frames (ORFs), i.e. amino acid residue sequences present in peptides and proteins. One of the obtained sequences was identical to the BacSp222 sequence determined by sequencing with the use of Edman degradation (see Example 4). The bacteriocin encoding sequence is as follows:

ATGGCAGGATTACTACGTTTTCTTTTAAGTAAAGGTCGCGCCTTATACAATTGGGC AAAG 60

AGTCATGTTGGAAAAGTTTGGGAGTGGCTTAAATCAGGAGCTACATATGAACAA ATTAAA 120

GAATGGATTGAAAACGC ATTAGGTTGGAGATAA 153 and is located between the ATG codon (8590-8592) for methionine and TAA codon (nucleotides 8740-8742), being the stop codon in the sequence of the entire p222 plasmid, illustrated on Fig. 4. The correctness of BacSp222 encoding sequence was confirmed by amplification of BacSp222 encoding DNA fragment by means of PCR reaction with the use of oligonucleotide starters complementary to the encoding sequence flanking fragments, followed by sequencing of the reaction product by Sanger method sequencing.

Example 6: Determination of antibacterial activity of the BacSp222 peptide bacteriocin. Antibacterial activity of BacSp222 peptide bacteriocin purified according to the procedure provided in example 3 was documented by determination of MIC doses (defined as the lowest bacteriocin concentration inhibiting the growth of the tested microorganism) for Gram- positive and Gram-negative bacteria as well as fungi representatives. The procedures of MIC doses determination were performed strictly according to the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI, 2006) or, for fungi, The European Committee on Antimicrobial Susceptibility Testing (EUCAST-AFST, 2012). List of microorganisms used for testing, including the precise description of their strains, is provided in Table 1. For MIC determinations, the microdilution procedure was applied, which embrace incubation of 100 μΐ of suspensions of the tested microorganism containing 5xl0 ⁴ colony forming units (CFU) in the 96-well microplates along with serial bacteriocin dilutions. Suspensions of the tested microorganisms were prepared in sterile Mueller-Hinton Broth medium normalized for cations (Cation-adjusted MHB, Sigma) or, in the case of Streptococcus pyogenes and Streptococcus sanguinis, in sterile MHB medium enriched in sterile 5% (in volume) horse blood lysate (Graso). Whereas in the case of yeast-like fungi Candida albicans, the suspensions were prepared in sterile RPMI 1640 (Sigma) medium enriched in 0.3 g/L of L-glutamine, 34.53 g of morpholino-propanesulfonic acid (MOPS)/L, 18 g/L of glucose, pH 7.0. After incubation of microplate for 24 hours in temperature of 37°C, visual assessment of wells turbidity was performed , providing the MIC doses as the lowest bacteriocin concentration completely inhibiting the growth of the tested microorganism (no turbidity ). The final MIC dose was calculated as an arithmetic mean from three independent measurements. Obtained results are presented in Table 1. These results demonstrate that BacSp222 bacteriocin kills many Gram-positive bacteria at MIC doses of 0.1 to ca. several micromols/L. The bacteriocin is incapable to kill the tested Gram-negative bacteria and fungi in the tested concentrations (up to 100 micromols/L).

Example 7: Determination of hemolytic activity of BacSp222 peptide bacteriocin.

Hemolytic activity (the ability to lyse erythrocytes ) of BacSp222 peptide bacteriocin purified according to the procedure specified in example 3 was documented by incubation of 5% (in volume) of human erythrocytes suspension with serial bacteriocin dilutions. Incubation was performed in phosphate buffered saline (PBS) for 1 hour in temperature of 37°C. Upon incubation, the erythrocytes were centrifuged (5 minutes at 2 000 g), the supernatant was transferred to the 96-well microplate wells and the amount of released hemoglobin was measured by reading of absorbance at 540 nm . As the control of 100% of lysis the erythrocyte suspension lysed in presence of 1% (in weight) detergent, sodium dodecyl sulfate, was used. The results of measurements are presented on Fig. 4 as a percent of erythrocyte lysis for each tested bacteriocin concentration, averaged from three independent measurements. The results demonstrated that BacSp222 bacteriocin has moderate hemolytic activity - at the highest tested peptide concentration, 100 μΜ, it causes lysis of 40% of erythrocytes. On the other hand, at concentrations below 10 μΜ, the hemolytic activity of peptide is very low (below 2% of hemolysis).

Example 8: Determination of cytotoxic activity of BacSp222 peptide bacteriocin.

Cytotoxic activity of BacSp222 peptide bacteriocin obtained as specified in example 3, was studied on two types of human cells, in parallel and independently, using two different tests. The human skin fibroblasts (HSF) and human afipose-like stem cells (ASC) were cultured in temperature of 37°C in atmosphere of 5% C0 ₂ in standardized PET T25 bottles. The cells were cultured in the Dulbecco's Modified Eagle's Medium (DMEM) with addition of phenol red, 10% (in volume) calf serum and 0.15 U/ml penicillin. The medium was replaced to fresh one every three days. After the third passage, 7000 cells were transferred to wells of 96-well microplate and incubated for 24 hours for sedimentation on the wells bottom . After this time, the medium was replaced with portions of 50 μΐ medium containing the serial bacteriocin dilutions in the range from 0.195 to 100 micromols/L. The negative control were the cells in the medium without bacteriocin, whereas the positive control (100% of mortality) the cells treated with 0.9 % (in volume) Triton X-100 detergent. After 4 hours of incubation, the survival ratio of cells was determined, in separate and independent manner by means of lactate dehydrogenase activity test (LHD, CytoTox 96 test by Promega) and by means of MTT tetrazolium salt (Sigma). The LDH test was performed using 30 μΐ portions of the medium transferred from the wells containing cells to the new 96-well microplate and mixed with 30 μΐ of LDH substrate. The reaction with the substrate was stopped after 30 minutes by adding 30 μΐ of 0.33 M acetic acid. The measurements of the absorbance of the produced colored product were performed using the PowerWave X (Bio-Tek) microplate reader at 490 nm wavelength, substracting the background absorbance at 690 nm. The MTT test was performed on cells sedimented in the wells, changing the bacteriocin-containing medium into 55 μΐ portions of the medium containing 0.5 mg/ml MTT. After 3 hours of incubation, the reaction was stopped by adding 100 μΐ of isopropanol/HCl mixture and the absorbance of the produced colour was measured using the PowerWave X (Bio-Tek) microplate reader at wavelength of 540 nm subtracting the background at 690 nm. The obtained absorbance values were averaged from three independent measurements and calculated into percentage values of cell mortality, with 0% of mortality as the absorbance value for cells from negative control wells and 100% of mortality for absorbance values from positive control wells (cells killed by detergent). The collected results are presented on Fig. 6. The results demonstrate that the BacSp222 peptide bacteriocin has a significant cytotoxic activity in concentrations above several micromols/L towart both types of tested cells and documented by two different cytotoxic activity tests.

Example 9: Determination of susceptibility of BacSp222 peptide bacteriocin on decomposition by proteolytic enzymes.

The sensitivity of BacSp222 peptide bacteriocin purified according to the procedure specified in example 3 to decomposition by proteolytic enzymes was studied by incubation of peptide with different peptidases, followed by determination of the residual antibacterial activity of bacteriocin using the radial diffusion technique. The following peptidases were applied: cathepsin G and elastase from human neutrophils (Biocentrum), bovine pancreatic trypsin (Sigma) and porcine pepsin (Sigma) as well as the following bacterial peptidases: proteinase K (A&A Biotechnology), proteinase StpC (in-house obtained enzyme) and proteinase V8 (Sigma). For incubation with cathepsin G, 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0 buffer was used, for incubation with elastase, trypsin and proteinase K 0.1 M Tris-HCl, pH 8.0 buffer was applied and for incubation with pepsin 0.1 M ammonium acetate of pH 4.0 were applied. For incubation with proteinase StpC 0.1 M Tris-HCl, 5 mM buffer of sodium salt of ethylenediaminetetraacetic acid (EDTA), 5 mM cysteine hydrochloride, pH 7.5 was applied and for incubation with proteinase V8 0.1 M phosphate buffer of pH 7.8 was used. The bacteriocin was incubated for 1 and 3 hours in temperature of 37°C in these buffers with enzymes at peptide : enzyme weight ratios of 50: 1 and 10: 1. After incubation, the portions of reaction mixtures were dosed to the wells cut-out in the sterile solid nutrient medium (Trypticase Soy Broth, Sigma) containing 1% (in weight) of agar and bacterial suspension of Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology). The separate control wells were filled with bacteriocin incubated in the respective buffers without peptidases addition. The plate with wells was incubated in temperature of 37°C for 24 hours, followed by measurement of bacteria growth inhibition zone diameters around each well. The obtained results are presented in Table 2. Analysis of these results demonstrates that, regardless of incubation time and peptide : enzyme weight ratio, the applied peptidases caused no inhibition of antibacterial activity of bacteriocin.

Example 10: Determination of sensitivity of BacSp222 peptide bacteriocin to heat

Sensitivity of BacSp222 peptide bacteriocin purified according to the procedure specified in example 3 on increased temperatures was studied by incubation of peptide in temperature of 100 DC, followed by verification of the residual antibacterial activity using the radial diffusion technique, and, independently, by observation of changes to molecule conformation in increased temperatures using the circular dichroism spectra measurements. To determine the residual antibacterial activity, the BacSp222 peptide bacteriocin solution was incubated in PBS in ambient temperature (control) and in temperature of 100°C for 30 and 60 min. After incubation, the portions of incubated solutions were dosed to the wells cut-out in sterile solid nutrient medium (Trypticase Soy Broth, Sigma) containing 1% (in weight) of agar and bacterial suspension of Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology). The plate with wells was incubated in temperature of 37°C for 24 hours, followed by measurement of bacteria growth inhibition zone diameters around each well. The obtained bacteria growth inhibition zone diameters were the same for control samples and for samples incubated in 100°C. To determine changes of conformation of the BacSp222 peptide bacteriocin molecule in increased temperatures, the bacteriocin solution in 50 mM sodium phosphate pH 7.4 containing 100 mM NaF was prepared. This solution was placed in the quartz thermostatic cell assembled in the spectropolarimeter J-715 (Jasco). Then continuous ellipticity measurements at wavelength of 220 nm were performed in temperature range between 20 and 90°C. The applied temperature changes rate was 1 DC / minute. The obtained curve of ellipticity changes is presented on Fig. 7. The chart demonstrates no significant changes of ellipticity, which proves that in the studied temperature range the spatial bacteriocin conformation is not subject to changes.

Example 11: Determination of antibacterial activity of the fragment of the BacSp222 peptide bacteriocin molecule. To determine antibacterial activity of the fragment of the BacSp222 peptide bacteriocin molecule purified according to the procedure specified in example 3, the N-terminally formylated methionine residue was cut-off using a chemical agent, followed by measurement of residual antibacterial activity using the radial diffusion technique. Cutting of N-terminal formylated methionine residue off from the BacSp222 peptide bacteriocin was performed by dissolving the peptide in 70% (in weight) trifluoro acetic acid (TFA), with addition of single cyanogen bromide crystal (CNBr) and incubation of such mixture in dark for 24 hours in ambient temperature. After incubation, the mixture was evaporated to dry in the vacuum centrifuge and dissolved in water containing 0.1 % (in volume) of trifluoroacetic acid (TFA) and 65 % (in volume) of acetonitrile. This solution was separated by reverse phase high pressure liquid chromatography (RP-HPLC). The separation was performed on chromatographic set Ultimate 3000 (Dionex/Thermo, USA) and Kromasil C4 250 x 4,6 mm column (Sigma). The gradient elution with the use of two buffers was applied, A: 0.1 % (in volume) of TFA in water, B: 80 % (in volume) of acetonitrile in water with the addition of 0.07% (in volume) of TFA. The flow rate of 1.5 ml/minute and the spectrophotometric detection at 220 nm were applied. The separation was started on the column equlilibrated at 65% of buffer B, followed by linear gradient elution from 65 to 72% of buffer B during 20 minutes. The Bac222Sp bacteriocin fraction, deprived of N-terminal formylated methionine eluted on the chromatogram as a peak of retention time within 9-10.5 minutes was collected to a test tube, lyophilized and dissolved in water. The antibacterial activity of the bacteriocin fragment and the native bacteriocin was determined using the radial diffusion method in agar nutrient medium containing the sensitive bacteria. To this end, the portions of serially diluted bacteriocin fragment and the native bacteriocin were dosed to wells cut in the sterile solid nutrient medium (Trypticase Soy Broth, Sigma) containing 1% (in weight) of agar and bacterial suspension of Bacillus subtilis LOCK 0816 (from the collection of the Institute of Fermentation Technology and Microbiology of the Lodz University of Technology). The plate with wells was incubated in temperature of 37°C for 24 hours, followed by measurement of bacteria growth inhibition zone diameters around each well. The measurement results demonstrated that the BacSp222 bacteriocin deprived of N-terminal formylated methionine requires averagely twice higher concentrations to reach the growth inhibition zone diameter identical as for native bacteriocin. Therefore it should be stated that the limited chemical fragmentation inhibits, in certain degree, the antibacterial activity of peptide, however does not completely suppress it. Example 12: Sp222 lysostaphin purification procedure

The procedure of purification of the lytic enzyme present in the liquid Sp222 post-culture medium embrce dialysis of the medium, salting-out the protein with high salt concentrations, followed by three stages of reverse phase chromatography. A portion of sterile liquid bacterial medium (Trypticase Soy Broth, Sigma) was inoculated with Sp222 bacterial strain, followed by culturing for 15 hours in temperature of 37°C in the orbital shaker at 180 rpm/minute. The obtained bacterial suspension was centrifuged for 20 minutes in 4°C at 5 000 g, followed by collection of a clear supernatant containing the post-culture medium. The medium contained substances secreted to the environment by Sp222 strain. The medium was dialyzed in distilled water in temperature of 4°C with the use of dialysis bag of 14 kDa cut-off limit and concentrated on the semi-permeable membrane of 10 kDa cut-off limit . Obtained preparation was frozen and lyophilized. The obtained lyophylizate was dissolved in small volume of water and its proteins were salted out with ammonium sulphate up to 60% (in weight) of saturation. Upon adding the salt, the medium was mixed for 30 minutes, followed by leaving in an ice bath without mixing for 2 hours. The obtained protein deposit was centrifuged for 30 minutes in 4°C at 21 000 g. Supernatant was removed, whereas the deposit was dissolved in water containing 0.1 % (in volume) of trifluoroacetic acid (TFA). This solution was centrifuged for 5 minutes in 4°C at 16 000 g, the collected supernatant was filtered by a 0.45 μηι filter and separated by reverse phase high pressure liquid chromatography (RP-HPLC). The separation was performed on chromatographic set Ultimate 3000 (Dionex/Thermo, USA) and Nucleosil C18 300 A 250 x 8 mm column (Macherey-Nagel, Germany). The gradient elution with the use of two buffers was applied, A: 0.1 % (in volume) of TFA in water, B: 80 % (in volume) of acetonitrile in water with the addition of 0.07% (in volume) of TFA. The flow rate of 1.5 ml/minute and the spectrophotometric detection at 220 nm were applied. The separation was started on the column equlilibrated at 38% of buffer B, followed by linear gradient elution from 38 to 53% of buffer B during 20 minutes. The bacteriocin fraction eluted on the chromatogram as a peak of retention time within 23.5 - 24.5 minutes, which was collected to a test tube, lyophilized, dissolved in water containing 0.1% (in volume) of TFA and 37% (in volume) of acetonitrile and subject to re-chromatography on the Discovery Bio Wide Pore C5 300A 250 x 4.6 mm column (Sigma, USA). The flow rate of 1 ml/ and spectrophotometric detection at 220 nm were applied. The separation was started on the column equilibrated at 37% of buffer B, followed by linear gradient elution from 37 to 45% of buffer B during 20 minutes. The bacteriocin fraction eluted on the chromatogram as a peak of retention time within 17 - 18 minutes, which was collected to a test tube, lyophilized and dissolved in water containing 0,1 % (in volume) TFA and 37 % (in volume) acetonitrile and was again re-separated on the Discovery Bio Wide Pore C18 300 A 250 x 4.6 mm column (Sigma, USA). The flow rate of 1 ml/minute and the spectrophotometric detection at 220 nm were applied. The separation was started on the column equlilibrated at 37% of buffer B, followed by linear gradient elution from 37 to 45% of buffer B in 20 minutes. The bacteriocin fraction eluted on the chromatogram as a peak of retention time within 15 - 16.5 minutes, which was collected to a test tube, containing 50 mM of sodium acetate, pH 5.5 and 1 M NaCl (to minimize lyophilization losses), and lyophilized. Final solution of purified Sp222 lysostaphin contained, apart from protein, 10 mM of sodium acetate, pH 5.5 and 200 mM NaCl. Enzyme concentration in final formulation was determined using the bicinchoninic acid assay (BCA, Sigma). The described Sp222 lysotaphin purification stages are illustrated on Fig. 9. The figure contains also the electrophoretic image of fractions collected at the particular purification stages. The figure demonstrates that the final solution of the purified enzyme contains a single and homogenous protein band of ca. 30 kDa molecular mass .

Example 13: Determination of N-terminal amino acid sequence of Sp222 lysostaphin.

The amino acid sequence of Sp222 lysostaphin purified according to the procedure described in example 12 was determined on the automatic proteinsequencer Procise 491 (Applied Biosystems, USA) performing the Edman degradation of polypeptide chains. Identification of 20 residues from N-terminus: A ATS TS GS A A WLNQ YPLNNG was carried out. The sequence is presented in a form of single-letter abbreviations of amino acid residues.

Example 14: Determination of the sequence of the gene encoding the Sp222 lysostaphin.

The Sp222 lysostaphin encoding sequence was obtained by way of bioinformatic shotgun sequence analysis of Sp222 genome. Complete DNA (desoxynucleic acid) isolated from Sp222 bacteria using the standard techniques known to branch specialist, was subject to shotgun sequencing using the MiSeq (Illumina) instrument, using the dedicated reagent and procedure set. The obtained short sequence readings were combined in longer sections (so called contigs) with the use of MIRA (http://mira-assembler.sourceforge.net/) and CLC Main Workbench (CLC bio) programmes. In one of contigs Sp222 lysostaphin encoding sequence was identified. This was made by translating the plasmid DNA sequence based on genetic code into the sequences containing so called open reading frames (ORFs), i.e. amino acid residue sequences present in peptides and proteins. One of the obtained sequences was identical to Sp222 lysostaphin sequence determined by sequencing with the use of Edman degradation (see example 13). The determined Sp222 lysostaphin encoding sequence and the sequence of the entire Sp222 lysostaphin protein is presented on Fig. 10.

Example 15: Determination of lytic activity of Sp222 lysostaphin against bacterial cells.

To demonstrate the lytic activity (the ability to dissolve the bacterial cell walls) of lysostaphin obtained according to the procedure obtained in example 12, the suspension of the studied bacterial strain was incubated in presence of liquid post-culture medium of Sp222 and the decrease of suspension turbidity was observed, proportional to the degree of bacterial lysis. For comparison purposes, the lytic activity of the other commerciall lysostaphin preparation was determined. To this end, 150 μΐ of suspension of Staphylococcus aureus RN4220 (from the collection of the Microbiology Division of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University) in 20 mM Tris-HCl buffer of pH 7.5 containing 150 mM NaCl was added to the wells of 96-well polystyrene plates . To the first well 1 microgram of Sp222 lysostaphin obtained according to example 12 was added, whereas to the second well 1 microgram of commercial lysostaphin (A&A Biotechnology) was added. In the third, control well, the suspension of studied bacteria with no additive was placed. The plate was then placed in the microplate reader (Synergy HI Hybrid Multi-Mode Microplate Reader, BioTek, USA) and the turbidity decrease at 600 nm was measured, every 2 minutes for 1 hour in temperature of 37°C. The decrease of turbidity is proportional to the degree of bacterial cell lysis. The results of measurements are presented on Fig. 11. One may observe that in the well containing the addition of purified Sp222 lysostaphin, turbidity decrease is significantly higher comparing to the control well, which proves the presence of an agent secreted by Sp222 in the post-culture medium and that it causes bacterial cell lysis. List of sequences

Sequence 1 (bacteriocin)

M ^* AGLLRFLLSKGRALYNWAKSHVGKVWEWLKSGATYEQIKEWIENALGWR 50 ^* Formylated methionine residue at N-terminus

Sequence 2

ATGGCAGGATTACTACGTTTTCTTTTAAGTAAAGGTCGCGCCTTATACAATTGGGCAAAG 60

AGTCATGTTGGAAAAGTTTGGGAGTGGCTTAAATCAGGAGCTACATATGAACAAATT AAA 120

GAATGGATTGAAAACGCATTAGGTTGGAGATAA 153

Sequence 3 ( lysostaphin)

MKNSKKIGISLATLALGSMLWGQVNAKELQVKPAPKTYTLAATSTSGSAAWLNQYPLNY 60

GFGSYNLPNNGGMHYGVDFGMSVGTPVKAITGGTVLNTAWDPYGGGNTITIKETDGV HTQ 120

WYMHLSKFNVQKGQKIKVGQVIGYSGNTGQSSGPHLHFQRMNGNPSNANAQDPLPFL KSI 180

GYGKSSGGSSNTKPSTSGGFKVNQYGTLYKAESATFTANTTI ITRYTGPFRSMPKAGTLK 240

SGQSIKYDEVMKQDGHVWVGYTNSAKKRVYVPVRTWNKNNNSLGSLWGTIK 291

Sequence 4

ATGAAAAATAGTAAAAAAATAGGTATCAGTTTAGCAACATTAGCTTTAGGTAGTATGTTA 60

GTAGTAGGACAAGTCAATGCGAAAGAATTACAAGTGAAGCCCGCTCCTAAAACTTAT ACT 120

CTTGCTGCTACTAGCACTAGTGGTTCTGCTGCTTGGTTAAATCAATATCCATTAAAT TAT 180

GGTTTCGGGAGTTATAACCTACCTAATAATGGTGGTATGCACTATGGGGTGGATTTT GGC 240

ATGAGTGTCGGAACTCCCGTGAAAGCAATAACTGGTGGGACAGTTCTTAATACCGCT TGG 300

GACCCTTATGGTGGTGGTAATACTATCACAATTAAAGAAACAGATGGCGTACATACA CAA 360

TGGTATATGCACTTGAGTAAATTTAATGTACAAAAAGGTCAAAAAATAAAGGTAGGA CAA 420

GTAATTGGATATTCAGGTAACACTGGTCAATCAAGTGGTCCACACCTTCATTTCCAA AGA 480

ATGAATGGAAATCCTAGTAACGCAAATGCTCAAGATCCTCTACCATTCTTAAAATCT ATT 540

GGTTATGGAAAATCAAGTGGTGGTTCTTCAAATACAAAACCATCAACATCTGGTGGT TTT 600

AAAGTAAACCAATATGGTACTTTATATAAAGCAGAATCTGCAACATTTACAGCAAAT ACT 660

ACAATCATAACTCGATATACAGGCCCATTTAGATCAATGCCAAAAGCTGGTACGTTA AAA 720

TCTGGTCAATCAATTAAATATGATGAAGTCATGAAACAAGATGGTCATGTTTGGGTT GGT 780

TATACTAATTCAGCAAAGAAAAGAGTTTATGTTCCAGTTAGAACATGGAATAAGAAT AAT 840

AACTCCCTGGGAAGTTTATGGGGAACAATAAAATAG 876

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Table 1: The MIC doses of BacSp222 bacteriocin for different microorganisms. The determined doses are the average from three independent measurements.

Strain sources:

* ATCC

** Institute of Fermentation Technology and Microbiology of the Lodz University of Technology

*** Polish Collection of Microorganisms, Polish Academy of Sciences, Wroclaw

**** Microbiology Division of the Faculty of Biochemistry, Biophysics and Biotechnology of the Jagiellonian University

***** Belgian Coordinated Collections of Microorganisms Table 2: The residual activity of BacSp222 peptide bacteriocin digested with different proteolytic enzymes. This activity is presented as bacteria growth inhibition zone diameter (in mm) in the radial diffusion test. The tested samples are bacteriocin incubated with peptidase, the control samples are bacteriocin incubated without peptidase.

Enzyme : peptide weight ratio

50: 1 50: 1 10: 1

Tested Control Tested Control Tested Tested sample, 1 sample, 1 sample, 3 sample, 3 sample, 1 sample, 3 hour of hour of hours of hours of hour of hours of

Enzyme: incubation incubation incubation incubatio incubation incubation n

Elastase 12.2 mm 13.1 mm 11.2 mm 11.4 mm 13.9 mm 14.6 mm

Trypsin 11.9 mm 13.1 mm 11.3 mm 11.4 mm 14.2 mm 15.1 mm

Pepsin 12.5 mm 13.1 mm 10.8 mm 11.3 mm 14.6 mm 14.1 mm

Cathepsin G 12.4 mm 13.7 mm 11.0 mm 11.6 mm 14.8 mm 14.4 mm

Proteinase 12.5 mm 14.0 mm 11.2 mm 11.3 mm 15.0 mm 15.3 mm V8

Proteinase 11.9 mm 13.1 mm 10.8 mm 11.4 mm 14.4 mm 14.8 mm K

Proteinase 12.4 mm 13.1 mm 10.8 mm 11.4 mm 14.2 mm 14.8 mm StpC