KR102035283B1 - Thermal stable endolysin LysCPD2 with lytic activity against Clostridium perfringens - Google Patents

Abstract

The present invention relates to a thermal stable endolysin LysCPD2 with lytic activity against Clostridium perfringens. Newly isolated endolysin LysCPD2 in the present invention shows a specific lytic activity against strains of the genus Bacillus and strains of the genus Clostridium, one of the causes of food poisoning and, in particular, exhibits stable antibacterial activity even when heat is applied. Therefore, endolysin of the present invention can be used for treating diseases caused by the infection of Clostridium perfringens, and, as a biological control agent, can contribute to the reduction of diseases in livestock and poultry caused by Clostridium perfringens, and also can improve the consumption stability of livestock and poultry through the development of a novel concept of controlling antibiotic resistant bacteria.

Description

Thermal stable endolysin LysCPD2 with lytic activity against Clostridium perfringens}

The present invention relates to a heat resistant endor lysine LysCPD2 with Clostridium perfringens solubility.

Clostridium perfringens is an anaerobic spore heat-resistant bacillus of the genus Clostitridium , which is a representative toxin-type food poisoning bacterium, and is also called gas bacterium. Gram-positive bacilli form ubiquitous follicles, but are rarely formed in medium. Organized anaerobes, but anaerobic demand is not very strict. It is widely distributed in soil, feces, etc. Invade trauma, create various toxins and infiltrate tissue, causing gas necrosis.

Depending on the type of toxin produced by Clostridium perfringens, it is divided into types A to F. It is a type A that causes human wound infection and food poisoning. Clostridium perfringens multiplies by spore germination in cooks left at room temperature for several hours after cooking, and can cause food poisoning with severe abdominal pain and diarrhea when grown to 1 × 10 ⁶ CFU / g or more. Clostridium perfringens form spores that are resistant to heat and low pH, making them difficult to inhibit and control.

Enterotoxemia, on the other hand, is an acute and fatal disease characterized by necrotic and hemorrhagic lesions caused by toxins produced by the growth of Clostridium perfringens in the intestines of cattle, sheep, goats and pigs. This is happening all over the world, and depending on the breeding mode, it is now possible to reduce the occurrence by adding antibiotics. However, after the onset of the antibiotic treatment, there is a problem that is difficult to expect a great effect.

Most of the existing synthetic antibiotics act in an inhibitory manner to inhibit the growth of bacteria, which causes the production of resistant bacteria. Stronger antibiotics have been developed to control bacteria, but the bacteria have evolved into more resistant bacteria, and the development of new and different antibiotics is needed to solve this problem.

Republic of Korea Patent No. 10-1200333 (2012.11.06), it is described with respect to the heat-resistant endolasine LysBPS13, food and feed composition comprising the same. Korean Patent Registration No. 10-1177358 (Aug. 21, 2012) discloses endolicin LysB4 having peptidase activity, and a food and feed composition comprising the same.

The recent tightening of government-wide regulations on the use of antibiotics has increased interest in the means of replacing them. In addition, since Clostridium perfringens forms spores, it exhibits resistance to heat and low pH, making it difficult to suppress and control. Accordingly, the present invention seeks to discover and provide a novel endolacin having lytic activity and heat resistance specific to Clostridium perfringens.

The present invention provides an endolisin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2.

Yen aldolase of the present invention, who is preferably preferably has an inhibitory activity against the Clostridium genus (Clostridium Spp.) Strains and Bacillus (Bacillus Spp.) Strain.

On the other hand, the present invention provides a food composition comprising an endolisin LysCPD2 consisting of an amino acid sequence.

The present invention is in Clostridium containing LysCPD2 who yen aldolase consisting of the amino acid sequence of SEQ ID NO: 2 (Clostridium Spp.) Strains and Bacillus (Bacillus Spp.) Provides a pharmaceutical composition for the prevention or treatment of strain infection do.

On the other hand, the present invention provides a feed composition comprising an endolisin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2.

On the other hand, the present invention provides a cleaning composition comprising an endolisin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2.

In the present invention, the newly isolated endolysine LysCPD2 shows specific lytic activity against Clostridium strains and strains of the genus Bacillus, one of the causative agents of food poisoning, and exhibits stable antibacterial activity even when heat is applied.

Therefore, the endorycin of the present invention can be used for treating diseases caused by the infection of Clostridium perfringens, and can contribute to the reduction of the incidence of livestock and poultry caused by Clostridium perfringens as a biological control agent. The development of a new concept of antibiotic-resistant bacteria control measures can improve the safety of livestock and poultry consumption.

1 is a result of an open reading frame (ORF) analysis of the bacteriophage CPD2 genome.
Figure 2 shows a schematic diagram of the present invention endolysine LysCPD2.
Figure 3 is the result of confirming the purification degree of the present invention endolysine LysCPD2.
Figure 4 is a result of confirming the dissolving ability of Clostridium perfringens ATCC 13124 and Clostridium perfringens FORC 25 according to the concentration (0nM, 50nM, 250nM, 500nM) treatment of the present invention LysCPD2.
5 is a result confirming the optimum lytic activity conditions of the present invention LysCPD2 (A: optimal PH, optimal Nacl concentration, C: optimal temperature).
6 is a result confirming the amidase activity of the present invention LysCPD2.
7 is a result of confirming the solubility of the spores activated by applying heat with LysCPD2 based on the heat resistance of the present invention LysCPD2.
8 is a result of measuring the heat resistance of the present invention LysCPD2 (A: 10 minutes, B: 30 minutes).

The present invention provides an endolisin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2. At this time, the endolicin of the present invention preferably has inhibitory activity against the Clostridium Spp. Strain and the Bacillus Spp. Strain.

Endoricin is an enzyme produced by bacteriophages, a bactericidal antibiotic, and a substance that destroys the cell walls of bacteria. Endorisine possesses a mechanism of cleaving specific linkages of peptidoglycans in bacterial cell walls, and these characteristics are fundamentally able to solve the problem of resistance, attracting attention as a new class of antibiotics. In addition, such as bacteriophages, high host specificity does not affect human and animal cells and beneficial bacteria, acts on both antibiotic-sensitive bacteria and resistant bacteria, and can provide a broader range of antimicrobial activity compared to bacteriophages. .

Accordingly, the present invention is to discover and provide a heat-resistant endor lysine LysCPD2 having a solubility of Clostridium perfringens, a food poisoning causing bacterium. In the present invention, the lysine endotolysin LysCPD2 isolated in the present invention through the following experiments show a specific lytic action against strains of Clostridium and strains of the genus Bacillus, which is one of the causes of food poisoning, especially when heat is applied, It was confirmed to exhibit a stable antimicrobial activity.

In particular, the endolysine LysCPD2 of the present invention shows an antimicrobial activity at pH 6 ~ 10, especially when the heat of 95 ℃, 80% activity and stable antibacterial activity, and exhibits the optimum activity at 37 ℃. Such a particularly high heat resistance may have a strong advantage when used as a feed additive or food additive that is easily exposed to heat in the future.

On the other hand, the present invention provides a food composition comprising an endolisin LysCPD2 consisting of an amino acid sequence. The food composition of the present invention is preferably meat, cereals, caffeine drinks, general beverages, chocolate, breads, snacks, sweets, pizza, jelly, noodles, gums, ice creams, alcoholic beverages, alcohol, vitamin complexes and other health supplements It may be added to any one selected from, but is not limited thereto.

The endolisin LysCPD2 of the present invention is preferably included 0.1% to 20% by weight relative to the food additive. If the amount is less than 0.1% by weight, the effect is insignificant, and if it exceeds 50% by weight, the effect increase compared to the amount of use is not economical.

The present invention is in Clostridium containing LysCPD2 who yen aldolase consisting of the amino acid sequence of SEQ ID NO: 2 (Clostridium Spp.) Strains and Bacillus (Bacillus Spp.) Provides a pharmaceutical composition for the prevention or treatment of strain infection do. The content of endolicin LysCPD2 included in the pharmaceutical composition of the present invention is preferably adjusted according to the method of use and the condition of the doser. The content of the microorganisms in the immunopotentiator of the present invention may be 0.1% by weight to 50% by weight compared to the immune enhancer, but is not necessarily limited thereto. However, when the content is less than 0.1% by weight, the immune effect may be insignificant, and when the content is more than 50% by weight, the effect increase rate relative to the amount used may be uneconomical.

On the other hand, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient in addition to the active ingredient. Carriers, excipients or diluents that can be used include lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium sulphate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and minerals, which may be used one or more. In addition, when the prophylactic and therapeutic agent is a medicament, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives may be further included.

On the other hand, the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, in particular by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It is good to formulate. Examples of specific formulations include PLASTERS, GRANULES, LOTIONS, LINIMENTS, LIMONADES, AROMATIC WATERS, POWDERS, Syrups ( SYRUPS), OPHTHALMIC OINTMENTS, LIQUIDS FORMULATIONS, AEROSOLS, EXTRACTS, ELIXIRS, OINTMENTS, FLUIDEXTRACTS, Emulsion (EMULSION) SUSPENSIONS, DECOCTIONS, INFUSIONS, EYE DROPS, TABLETS, Suppositories, SUPOSITORIES, INJECTIONS, SPIRITS, CATAPLASMA , Capsules (CAPSULES), creams (CREAMS), troches (TROCOES), tinctures (TINCTURES), pasta (PASTES), pills (PILLS), soft or hard gelatin capsules can be any one selected.

On the other hand, the dosage of the pharmaceutical composition of the present invention may be determined in consideration of the method of administration, the age, sex and weight of the recipient. For example, the present immunostimulant can be administered at least once at 0.1 to 100 mg / kg (body weight) per day based on the active ingredient. However, the above dosage is only one example to illustrate, and may be changed by the condition of the recipient.

On the other hand, the present invention provides a cleaning composition comprising an endolisin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2. The detergent composition of the present invention may further include components conventionally used in the detergent composition, and may include, for example, conventional auxiliaries such as stabilizers, solubilizers and flavorings, and carriers.

When the formulation of the cleaning composition of the present invention is a paste, cream or gel, the carrier component is animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. This can be used.

When the formulation of the detergent composition of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene Fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethyleneglycol or sorbitan may be used.

When the formulation of the detergent composition of the present invention is a suspension, as a carrier component, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester , Microcrystalline cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the cleaning composition of the present invention is a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular, in the case of spray, additionally chlorofluorohydrocarbon, Propellant such as propane / butane or dimethyl ether.

When the formulation of the detergent composition of the present invention is a surfactant-containing cleansing, the carrier component may be an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, methyltaurate, sarcosinate, Fatty acid amide ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

When the detergent composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be wiped off, peeled off or washed with water after application to the skin. As a specific example, the soap is liquid soap, powdered soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, the surfactant-free cleansing formulation is a cleansing cream , Cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.

On the other hand, the present invention provides a feed composition comprising an endolisin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2. In the feed composition, the total weight of the composition may contain 0.001 to 1.0% by weight of the endolacin LysCPD2. This is because if the content is less than 0.001% by weight relative to the total weight of the composition, the antimicrobial effect cannot be sufficiently exerted, and if it exceeds 1.0% by weight, it causes problems in the stability and safety of the formulation.

The composition of the present invention is mixed with a conventional formulated feed and fed by the method of oral administration, there is almost no problem of resistance or side effects such as immunocompromise even when continued or overdose.

Hereinafter, the configuration of the present invention will be described in detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following Examples and Experimental Examples, but includes modifications of equivalent technical ideas.

[ Example  One: CPD2's  Genome analysis]

Bacteriophage CPD2 was isolated from the edible chicken samples purchased at the mart, and the genome of the isolated CPD2 was analyzed. The genome of CPD2 was extracted and purified by phenol-chloroform extraction. Purified DNA was commissioned by 'Macrogen' to analyze the nucleotide sequence. Sequence analysis was performed using 'Sequencer FLX Titanium', and the assembly of each read was performed using the 'De Novo Assembler v. 2. carried out using 6 '.

In addition, the position of the ORF of the genome was predicted using '3.02, GeneMark.hmm, FgenesB'. In addition, the function of protein produced in each ORF was predicted through 'BLASTP' of 'NCBI'. According to the predicted function of the ORF, the proper names were named for each ORF, and all ORFs whose function was not identified were named hypothetical proteins. The location and function of the ORF are summarized through 'Genomic Workbench 6'.

As a result, the bacteriophage CPD2 genome has a total length of 18,212 bp and a G + C content of 27.11%. In addition, the ORF analysis predicted a total of 22 ORFs (FIG. 1). 1 is a result of an open reading frame (ORF) analysis of the bacteriophage CPD2 genome.

In addition, the analysis of the domain of the protein using the InterProScan program, it was predicted to have an amidase 3 domain having lytic activity in the N-terminal (N-terminal). This domain is a typical domain of bacteriophage endolysine (endolysin), and thus the protein expressed in the gene was confirmed as endolysine (endolysin) and named 'LysCPD2' (FIG. 2, Table 1). Figure 2 shows a schematic diagram of the present invention endolysine LysCPD2.

Endolysine LysCPD2 nucleic acid sequence and amino acid sequence Nucleic acid sequence ATGAAAATAGGTATTAGAGACGGACATAGTCCAAATTGTAAGGGTGCTATTGGTTTACGTGATGAACAATCATGTATGAGAGTTTTATGTAAAGAGGTTATAGAAATATTAGAAAAACATGGGCATGAGGTAGTTTATTGTGGTAGTGATGCAAGTACACAAAATGGTGAACTTTCAGAAGGTGTAAGAAAAGCTAATAATTCAAATGTTGATATATTTATTTCATTGCACATGAATAGCTTTAATGGGCAAGCCCAAGGAACAGAGGCACTTGTTACAGTTGGAGCAAGAAATTCTATAAAAGAAATTGCCTCAAGGTTATGCAAAAACTTTGCTAGTTTAGGTTTAGTAAATAGGGGTGTAAAAGAAGTTAATTTATATGAAATGAAGAACGTAAAAGCACCCAACATAATATTTGAAACTATGTTTTGCGATAACCCCCATGACATTAACGAAGTTTGGTCGCCTACACCATACGAAAAAATGGCTTTACTAATTGCAAATGCTATTGACCCAACTATTAAAGAAAATGAACTTTATAGAGTTGTTGTTCAATATTTTAACAACAAAAAAGATGCTGAAAACTGTCAACAAGAAATTGCTAAAAGATGGTATTGTTTTGTGGAGGAATGTAATTAA Amino acid sequence MKIGIRDGHSPNCKGAIGLRDEQSCMRVLCKEVIEILEKHGHEVVYCGSDASTQNGELSEGVRKANNSNVDIFISLHMNSFNGQAQGTEALVTVGARNSIKEIASRLCKNFASLGLVNRGVKEVNLYEMKNVKAPNIIFETMFCDNPHDINEVWSPTPKKNALLNEVYKNALL

[ Example  2: Endolysine Of LysCPD2  Separation, Expression, and Purification]

PCR was used to amplify the LysCPD2 gene. PCR primers were prepared based on the predicted nucleotide sequence information of LysCPD2, and were synthesized by inserting the cleavage sites of restriction enzymes at both ends of each primer for easy restriction enzyme treatment (Table 2).

PCR primer sequence primer  designation primer  order LysCPD2_N_F_BamHI 5'-GCG GGATCC ATGAAAATAGGTATTAGAGACGGAC-3 ' LysCPD2_N_R_SalI 5'-GCG GTCGAC TTAATTACATTCCTCCACAAAACAATAC-3 '

As the vector which cloned, the expression vector pET-28a plasmid was used. The PCR products and plasmids were treated with the restriction enzymes BamHI and SalI to form a linear state with a sticky end and purified using a 'pure PCR product purification kit'.

The purified PCR product and the vector were ligation using 'Ligation kit' of 'Takara' to make a recombinant plasmid pET-28a :: LysCPD2. The pET-28a :: LysCPD2 was inserted into Escherichia coli DH5α competent cells by a 'heat shock transformation' method, and the vector was cultured in kanamycin-added medium. Only cells that have entered are allowed to grow. In addition, colony PCR was performed on single colonies grown in antibiotic-added medium to distinguish between vectors into which the LysCPD2 gene was normally inserted.

The vector into which the LysCPD2 gene was normally inserted was extracted again to confirm that the gene sequence information exactly matches the intended. The vector having the gene sequence information confirmed was reinserted through heat shock transformation into E. coli BL21 (DE3), which is a protein expression strain.

Protein expression was inoculated with E. coli BL21 (DE3) clone in 50 ml of LB medium and grown at 37 ° C. and 220 rpm until the OD ₆₀₀ value was 1.0, then 0.2 mM IPTG was added and 30 ° C. By shaking incubation for 3 hours at. After incubation, the cells were allowed to settle through centrifugation (7000 × g, 10 min, 4 ° C.), and then resuspended in lysis buffer 50 mM Tris-Cl (pH 8.0), 500 mM NaCl 10 ml. And lysed through sonication.

After lysis, undissolved protein was removed by centrifugation (21,330 × g, 60 minutes, 4 ° C.), and it was placed in a chromatography column of 'Biorad' together with 1 ml of Ni-NTA resin. After mixing for 1 hour at 4 ° C. (inverting), the protein was not bound to the resin by passing through the column. Subsequently, 10 mM and 20 mM imidazole solutions were sequentially passed through a column of 10 ml and 5 ml, respectively, to remove non-specifically bound proteins. In the final step, an elution buffer containing 250 mM imidazole solution was removed. LysCPD2 bound to the resin was eluted by passage. The concentration of the obtained protein was confirmed by the 'Bradford assay', the purity was confirmed through SDS-PAGE (Fig. 3). Then, immediately stored at 4 ° C. using 'PD miditrap G-25 kit' from 'healthcare' using storage buffer 0mM Tris-Cl (pH 8.0), 500 mM NaCl, 30% glycerol. Figure 3 is the result of confirming the purification degree of the present invention endolysine LysCPD2.

[ Experimental Example  One: Endolysine Of LysCPD2 Clostridium On perfringens  Antibacterial effect on Of LysCPD2  Characterization]

The antimicrobial effect of the purified protein LysCPD2 on Clostridium perfringens was confirmed and analyzed to determine the optimal NaCl concentration, pH, and temperature conditions with the maximum antimicrobial effect. First, in order to check the spectrum of the host showing an antimicrobial effect, each host was incubated overnight, then dispensed in 0.7% DW soft agar, and then solidified on a plate, followed by LysCPD2. Representative hosts were identified (Table 3).

LysCPD2 Solubility Bacterial host LysCPD2 Clostridium perfringens  strain C. perfringens H3 + C. perfringens H9 + C. perfringens FD1 + C. perfringens ATCC 3624 + C. perfringens ATCC 13124 + C. perfringens FORC 25 + C. perfringens FORC 3 + Isolate human stool isolate 2582 - human stool isolate 2585 + human stool isolate 2589 + human stool isolate 2722 + Other Gram positive bacteria Listeria monocytogenes EGDe - Staphylococcus aureus RN4220 - Staphylococcus aureus newman - Bacillus cereus ATCC 10987 - Bacillus cereus ATCC 13061 + Bacillus subtilis ATCC 23857 + Clostridium histolyticum ATCC 19401 - Clostridium indolis ATCC 25771 -

C. perfringens ATCC 13124 and C. perfringens ATCC 13124 and C. perfringens FORC 25 were inoculated in 10 ml of BHI medium until the OD ₆₀₀ value was 1.0 at 37 ° C. Static culture It was allowed to settle through centrifugation (7000 × g, 10 min, 4 ° C.) and resuspended in 10 ml of lysis buffer used for protein purification. In a 96-well plate, 1 ml of suspension and LysCPD2 0nM, 50nM, 250nM, 500nM, treated with 500 μl of mineral oil to inhibit oxygen permeation, followed by stationary incubation at 37 ° C., OD every 5 minutes for 40 minutes _{The 600} value was measured to show the relative OD ₆₀₀ value relative to the control (FIG. 4). Figure 4 is a result of confirming the dissolving ability of Clostridium perfringens ATCC 13124 and Clostridium perfringens FORC 25 according to the concentration (0nM, 50nM, 250nM, 500nM) treatment of the present invention LysCPD2.

To determine the optimal NaCl concentration, a solution consisting of 50mM Tris-Cl (pH 8.0) and final concentrations of NaCl 0, 100mM, 200mM, 300mM, 400mM, 500mM and 1000mM was used as the reaction buffer. In order to confirm the optimal pH conditions, 5M NaCl and 5M HCl were added to a universal pH buffer (10mM KH ₂ PO ₄ , 10mM NaCitrate, 10mM H _3B O ₃ ) and pH 3 to pH 10 were used.

Clostridium perfringens ATCC 13124 ( C. perfringens ATCC 13124) was inoculated in 10 ml of BHI medium and incubated at 37 ° C. until the OD ₆₀₀ value was 1.0. It was allowed to settle through centrifugation (7000 × g, 10 minutes, 4 ° C.) and resuspended in 10 ml of reaction buffer. 96-well plates were treated with 1 ml of suspension and 250 nM of LysCPD2, and then incubated at 25 ° C. for 40 minutes, showing relative lytic activity (FIGS.

Finally, a solution consisting of 50mM Tris-Cl (pH 8.0) and 100mM NaCl was used as a reaction buffer to determine the optimum temperature conditions. 1.5 ml tube was treated with 1 ml of the cell suspension and 50 nM LysCPD2 treated as above, followed by incubation at 4, 25, 37, 45, 55, 65 ° C for 10 minutes, incubation at room temperature for 40 minutes, and then showed relative lytic activity (FIG. 5C). 5 is a result confirming the optimum lytic activity conditions of the present invention LysCPD2 (A: optimal PH, optimal Nacl concentration, C: optimal temperature).

As a result, LysCPD2 showed more than 50% antimicrobial activity between pH 7 and pH 10 and showed the strongest lytic activity at pH 9. In addition, the antibacterial activity was shown between NaCl concentration 0nM to 500mM showed the strongest lytic activity at 100mM concentration, it was confirmed that the maximum activity at 37 ℃ conditions.

In addition, the amidase activity of LysCPD2 was examined in this experiment. Amidase activity was assessed by measuring the lactic group liberated from N-acetylmuramic acid.

When purified LysCPD2 was treated with peptidoglycan extracted from Clostridium perfringens FORC 25, the amide bond between N-acetylmuramic acid and L-alanine of peptidoglycan was broken. It was confirmed that free muramic acid (muramic acid) was increased (FIG. 6). 6 is a result confirming the amidase activity of the present invention LysCPD2.

In addition, based on the heat resistance of LysCPD2 was confirmed by the heat dissolving ability to activate spores with LysCPD2. 1 ml of 1 × 10 ⁶ CFU / mL spore suspension was treated with 250 nM of LysCPD2, followed by incubation for 20 minutes at 75 ° C., the germination condition of spores, and colonies formed after incubation at 37 ° C. for 12 hours in BHI agar medium. The number was measured. As a result of the experiment, it showed a difference of about 7 log CFU / ㎖ compared to the control group not treated with LysCPD2 to confirm the solubility for heat activated spores. 7 is a result of confirming the solubility of the spores activated by applying heat with LysCPD2 based on the heat resistance of the present invention LysCPD2.

[ Experimental Example  2: Endolysine Of LysCPD2  Heat resistance measurement]

In order to determine the heat resistance of endolysine LysCPD2, the enzyme activity was measured after storing at 4 ° C, 25 ° C, 37 ° C, 45 ° C, 55 ° C, 65 ° C, 75 ° C, 85 ° C, 95 ° C.

Clostridium perfringens ATCC 13124 ( C. perfringens ATCC 13124) as inoculated in the procedure of Experimental Example 1 was inoculated in 10 ml of BHI medium and incubated at 37 ℃ until the OD ₆₀₀ value is 1.0. It was allowed to settle through centrifugation (7000 × g, 10 min, 4 ° C.) and again resuspended in reaction buffer 50 mM Tris-Cl (pH 8.0), 100 ml NaCl. LysCPD2 stored at 1 mL of suspension and each temperature in a 96-well plate was treated with 250 nM and incubated at room temperature for 40 minutes, showing relative lytic activity (FIGS. 8. A to B).

8 is a result of measuring the heat resistance of the present invention LysCPD2 (A: 10 minutes, B: 30 minutes). At this time, when the storage for 10 minutes showed an activity of about 80% of the maximum activity at 95 ℃, 30 minutes storage activity was reduced from 65 ℃, but maintained 50% of the activity to 85 ℃.

<110> Seoul National University R & DB Foundation <120> Thermal stable endolysin LysCPD2 with lytic activity against          Clostridium perfringens <130> YP-18-274 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 639 <212> DNA <213> Clostridium perfringens <400> 1 atgaaaatag gtattagaga cggacatagt ccaaattgta agggtgctat tggtttacgt 60 gatgaacaat catgtatgag agttttatgt aaagaggtta tagaaatatt agaaaaacat 120 gggcatgagg tagtttattg tggtagtgat gcaagtacac aaaatggtga actttcagaa 180 ggtgtaagaa aagctaataa ttcaaatgtt gatatattta tttcattgca catgaatagc 240 tttaatgggc aagcccaagg aacagaggca cttgttacag ttggagcaag aaattctata 300 aaagaaattg cctcaaggtt atgcaaaaac tttgctagtt taggtttagt aaataggggt 360 gtaaaagaag ttaatttata tgaaatgaag aacgtaaaag cacccaacat aatatttgaa 420 actatgtttt gcgataaccc ccatgacatt aacgaagttt ggtcgcctac accatacgaa 480 aaaatggctt tactaattgc aaatgctatt gacccaacta ttaaagaaaa tgaactttat 540 agagttgttg ttcaatattt taacaacaaa aaagatgctg aaaactgtca acaagaaatt 600 gctaaaagat ggtattgttt tgtggaggaa tgtaattaa 639 <210> 2 <211> 212 <212> PRT <213> Clostridium perfringens <400> 2 Met Lys Ile Gly Ile Arg Asp Gly His Ser Pro Asn Cys Lys Gly Ala   1 5 10 15 Ile Gly Leu Arg Asp Glu Gln Ser Cys Met Arg Val Leu Cys Lys Glu              20 25 30 Val Ile Glu Ile Leu Glu Lys His Gly His Glu Val Val Tyr Cys Gly          35 40 45 Ser Asp Ala Ser Thr Gln Asn Gly Glu Leu Ser Glu Gly Val Arg Lys      50 55 60 Ala Asn Asn Ser Asn Val Asp Ile Phe Ile Ser Leu His Met Asn Ser  65 70 75 80 Phe Asn Gly Gln Ala Gln Gly Thr Glu Ala Leu Val Thr Val Gly Ala                  85 90 95 Arg Asn Ser Ile Lys Glu Ile Ala Ser Arg Leu Cys Lys Asn Phe Ala             100 105 110 Ser Leu Gly Leu Val Asn Arg Gly Val Lys Glu Val Asn Leu Tyr Glu         115 120 125 Met Lys Asn Val Lys Ala Pro Asn Ile Ile Phe Glu Thr Met Phe Cys     130 135 140 Asp Asn Pro His Asp Ile Asn Glu Val Trp Ser Pro Thr Pro Tyr Glu 145 150 155 160 Lys Met Ala Leu Leu Ile Ala Asn Ala Ile Asp Pro Thr Ile Lys Glu                 165 170 175 Asn Glu Leu Tyr Arg Val Val Val Gln Tyr Phe Asn Asn Lys Lys Asp             180 185 190 Ala Glu Asn Cys Gln Gln Glu Ile Ala Lys Arg Trp Tyr Cys Phe Val         195 200 205 Glu Glu Cys Asn     210

Claims (6)

Endolicin LysCPD2 consisting of the amino acid sequence of SEQ ID NO: 2 having inhibitory activity against Clostridium perfringens .
The method of claim 1,
The endolicin is,
Endolicin LysCPD2, which further has an inhibitory activity against Bacillus cereus ATCC 13061 or Bacillus subtilis ATCC 23857.
A food composition comprising the endolacin LysCPD2 of claim 1.
A pharmaceutical composition for preventing or treating wound infection, food poisoning or intestinalemia caused by Clostridium perfringens infection comprising the endolicin LysCPD2 of claim 1.
Feed composition comprising the endolisin LysCPD2 of claim 1.
Cleanser composition comprising the endolacin LysCPD2 of claim 1.

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