KR19990077430A - Novel peptide isolated from catfish and the use thereof - Google Patents

Abstract

The present invention relates to a novel antimicrobial peptide isolated from the damaged mucosa of catfish (Parasilurus asotus) and its derivatives, and to the use thereof. The pure antimicrobial peptide Paracin I was isolated and purified, and then the molecular weight and amino acid sequence were determined, the derivatives of the antimicrobial peptide Paracin I were prepared, and then the structure of the antimicrobial peptide Paracin I was analyzed. As a result of measuring the antimicrobial activity and hemolytic activity of the derivatives, the antimicrobial peptide Paracin I contained 19 amino acid residues having a molecular weight of about 2,000.4Da in whole or in part and showed strong antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi. It strongly inhibits the expression of microorganisms at the site and shows little hemolytic activity. Because it does not have outstanding effects that can be used as an antibacterial agent such as wound healing promoters, foreign agents, mouthwashes, and eye drops.

Description

Novel peptide isolated from catfish and the use

The present invention relates to novel antimicrobial peptides isolated from catfish (Parasilurus asotus). More specifically, the present invention relates to novel antimicrobial peptides isolated from catfish that exhibit potent antimicrobial activity against a wide range of microorganisms, derivatives thereof and uses thereof.

As a result of research on the defense mechanism of insects against the invasion of microorganisms, since the discovery of a new peptide antimicrobial cecropin in silkworm larvae, the importance of peptides as a biologically active substance was greatly recognized. Recent years have shown that almost all higher organisms accumulate or secrete antimicrobial peptides in the body, independent of the immune system, as a defense against pathogenic microbes. To date, more than 2,000 peptide antimicrobial agents have been found, and these peptides have different amino acid compositions for each species found, but are known to have similar antimicrobial mechanisms. Cecropin, magainin, bombinin, defensin, tachyplesin and buforin are known as representative antimicrobial peptides. It is composed of 17-24 amino acids and has antimicrobial activity against gram-negative bacteria, gram-positive bacteria, as well as prokaryotes and fungi, and some are effective against cancer cells and viruses. In particular, magainin, a peptide antimicrobial, is a peptide with 23 amino acid compositions isolated from the amphibian epidermis (Zasloff, 1987), and has been reported to act on human lung cancer cells as well as pathogens. In addition, most of the antimicrobial peptides specifically target cells and kill rapidly and mostly exhibit a broad spectrum of activity (Park, CB, Lee, JH, Park, IY, Kim, M. S and Kim, SC, 1997, Park, CB, Kim, MS and Kim, SC 1996) have been reported to inhibit cytoplasmic responses with the microbial activity of other antimicrobial peptides such as promoting wound healing and promoting chemotaxis in monocytes.

On the other hand, the mucous surface of living organisms is under constant attack from microorganisms, but infection by bacterial invasion rarely occurs, is limited to local areas, and heals quickly. Recently, antimicrobial peptides have been reported, such as host defense effector molecules that protect the mucous epithelium from invading bacteria (Bevins, 1994). Examples of such mucosal immunologic agents include andropin obtained from ductal epithelial cells of the reproductive organs of Drosophila, mucous skin surface of Xenopus laevis and merginine from the gastrointestinal tract, buforin I from Bufo bufo gargarizans, and small mucous ducts. Antimicrobial peptides from and proorosidine from Pleuronectes have been reported (Cole, AM, Weis, P. and Diamond, G., 1997). However, although many antimicrobial peptides have been reported in the mucous layers of amphibians, insects and mammals (Park, CB, Kim, MS and Kim, SC, 1996, Samakovlis, C., Kylsten, P., Kimbrell, DA, 1991, Diamond) , G., Zasloff., M., Eck., Brasseur, M., Maloy, WL and Bevins, 1991) Only a few minority have been found in the mucous layers of aquatic organisms (Park, CB, Lee, JH). , Park, IY, Kim, M. S and Kim, SC, 1997, Cole, AM, Weis, P.and Diamond, G. 1997, Chen, H., Brown, JH, Morell, JL and Huang, CM 1998) .

Antimicrobial peptides have strong antimicrobial activity against a wide range of microorganisms, and second, host cells act only on pathogens that invade from the outside without destroying them, acting as antibiotics that are harmless to the human body. The antimicrobial activity is very different from the conventional antibiotics, so it is less likely to induce resistance. Fourth, since there is no secondary modification such as glycosylation, it can be mass-produced through genetic modification. It has strong physicochemical stability against heat, acid, alkali, etc., and therefore has great industrial utility in the pharmaceutical and food fields.

Therefore, the inventors of the present invention isolate the antimicrobial peptides from the mucosa layer of the catfish, which is a kind of fish, and confirm that the substance has antimicrobial activity against a wider range of microorganisms than the conventional antimicrobial peptides. By identifying, the present invention has been completed.

Accordingly, an object of the present invention is to provide a novel antimicrobial peptide and its derivatives isolated from the damaged mucosa of catfish, a kind of fish. Another object of the present invention is to provide a novel antimicrobial peptide isolated from the wound mucosa layer of catfish and its derivatives as an excellent antimicrobial agent against various microorganisms.

In order to achieve the above object, the present invention randomly wounds the epidermis of a catfish, which is a kind of fish, and separates and purifies the pure antimicrobial peptide from the wound epithelial mucosa, and then determines molecular weight and amino acid sequence. It was achieved by measuring the antimicrobial and hemolytic activity of the antimicrobial peptides and derivatives thereof and analyzing the structure.

Hereinafter, the configuration and operation of the present invention.

1A and 1B show the results of reverse-phase high performance liquid chromatography (HPLC) analysis of a novel antimicrobial peptide paracin I isolated from catfish.

Figure 2 is a mass spectrometric spectrum showing the molecular weight measurement of the novel antibacterial peptide paracin I isolated from catfish.

Figure 3 shows the results of comparing the amino acid sequence identity of the novel antibacterial peptide paracin I and buporin I and human histone H2A.5 isolated from catfish.

Figure 4 shows the circular dichroism measurement of the novel antimicrobial peptide paracin I isolated from catfish.

5 shows the axial projection diagram of the novel antimicrobial peptide Paracin I isolated from catfish at 170 ° rotation / residual.

In the present invention, after rubbing the epithelial layer of catfish with sandpaper, the mucus of the wound epithelial mucosa is collected, concentrated with a Sep-Pak C18 cartridge, and applied to a C18 reversed phase high-performance liquid chromatography column to isolate and purify a novel antimicrobial peptide. step; Using the MALDI-MS (matrix associated laser ionization mass spectroscopy), the molecular weight of the novel antimicrobial peptide isolated and purified in this step was measured, and the amino acid of the antimicrobial peptide was analyzed using an automatic amino acid sequence (Fullerton, USA). Analyzing the sequence; Preparing derivatives in which the amino acid sequences of the novel antimicrobial peptides are reversed by deriving derivatives having two and four residues removed from the carboxyl termini of the novel antimicrobial peptides and amino acid sequences of the novel antimicrobial peptides; Measuring the antimicrobial activity of the novel antimicrobial peptides and their derivatives by radial diffusion analysis using B.subtilis ATCC 62037 and measuring the hemolytic activity according to the method of Park et al .; It is composed of the step of determining the secondary structure of the novel antimicrobial peptide using the spectrometer in the presence or absence of trifluoroethanol (TFE).

The microorganisms used in the present invention are Bacillus subtilis ATCC 62037, Staphylococcus aureus ATCC 15752, Streptococcus mutans ATCC 25175, Pseudomonas putida ATCC 17426, Escherichia coli ATCC 27325, Salmonella typhimurium ATCC 15277, Serratia sp. ATCC 21074, Cryptococcus neoformans ATCC 34881, Saccharomyces cerevisiae ATCC 44774 and Candida albicans ATCC 10231 and these microorganisms were obtained from the American Type Culture Collection (ATCC).

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples and Experimental Examples, but the scope of the present invention is not limited to the following Examples and Experimental Examples.

Example 1; Preparation of Novel Antibacterial Peptides and Their Derivatives from Catfish

Step 1: Purification of Peptide Isolation from Epithelial Wounds of Catfish

Catfish skin (16 cm ² ) was scratched with sandpaper and stunned with electric shock after 5 hours, followed by scraping the proteinaceous epithelial mucosa of the wounded and unwounded portions of the catfish. Mucus (20 g) obtained from catfish skin was homogenized using a Waring blender in 200 mL of extract solution (0.2M sodium acetate, 0.2% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride). Homogenates were centrifuged at 20,000 x g for 30 minutes in Himac SCR20BR (Hitachi, Tokyo, Japan) and supernatants collected. Reversed phase concentration of peptide in supernatant using Sep-Pak C18 cartridge (Millipore, Milford, MA, USA) activated with 80% acetonitrile containing 0.1% (v / v) TFA (buffer A) and 0.1% (v) / v) excess acetonitrile was removed by treatment with TFA (buffer B). The supernatant concentrated with the Sep-Pak C18 cartridge was washed with 20 mL of buffer B, and the peptide inserted into the cartridge groove was eluted with 6 mL of buffer A. The eluate was affinity and then resuspended in buffer B. The resuspended sample was applied to a C18 reversed phase high performance liquid chromatography (HPLC) column (3.9 × 300 mm, Delta Pak, Millipore) and the elution was given a linear gradient of 0-80% acetonitrile in 0.1% TFA for 1 mL / The pure peptide was separated and purified by min flow rate. As a result, as shown in Figures 1a and 1b, several peaks were observed in reverse phase HPLC analysis of the wounded catfish epithelial mucosa extract, and no peaks were observed in reverse phase HPLC analysis of the unwounded catfish extract. At this time, peaks A, B and C of FIG. 1B were collected separately and used for antimicrobial activity analysis. In the three peaks induced, peaks A and C showed antimicrobial activity, and peak A showed antimicrobial activity 200 times stronger than peak C. Thus peak A showed strong antimicrobial activity and was specifically purified using C18 reversed phase HPLC and fully characterized. Purification of the antimicrobial peptide of peak A was confirmed to be more than 95% homogeneous by reversed phase HPLC (FIG. 1B) and MALDI-MS.

The total amount of antimicrobial peptide purified at this stage was about 0.1 μg / g mucus and the purified antimicrobial peptide was named paracin I (derived from the genetic name of catfish parasilurus).

Second Step: Molecular Weight and Amino Acid Sequencing of the Novel Antibacterial Peptide Paracin I

The molecular weight of the antimicrobial peptide paracin I was determined by MALDI-MS. Approximately 20 nmol of the affinity peptide was dissolved in 50% acetonitrile containing 7% (w / v) sinapinic acid and adsorbed onto a Pt probe. After removing the solvent by hot air, the peptide adsorbed on the Pt probe was applied to a vacuum chamber and analyzed. Amino acid sequencing was performed by an automated Edman assay using Biosystems gas phase sequencer Model 477A (Foster City, CA, USA). Amino acid sequence homology studies were performed by computerized query of GenBank ^TM / EMBL databank. As a result, as shown in FIG. 2, the molecular weight of the antimicrobial peptide paracin I was measured by MALDI-MS at 2000.4 Da, and amino acid sequencing showed 19 residues containing 3 arginine and 5 lysines. (FIG. 3) and a net charge of +8. The complete amino acid sequence of the antibacterial peptide paracin I was Lys-Gly-Arg-Gly-Lys-Gln-Gly-Gly-Lys-Val-Arg-Ala-Lys-Ala-Lys-Thr-Arg-Ser-Ser. The molecular weight of paracin I obtained by MALDI-MS was almost identical to the amount calculated from the amino acid sequence. This indicates that no late-gene translation translation occurred in Paracin I. In the amino acid sequence homology study, paracin I was very identical to the N-terminal region of histone H2A, a replication-dependent protein. As shown in FIG. 3, the antimicrobial peptide paracin I showed 95% identity since 18 of 19 residues were identical to the N-terminal of buporin I. The antibacterial peptide paracin I also showed 89% identity with the N-terminal of human histone H2A.5.

Step 3: Preparation of Derivatives of the Antibacterial Peptide Paracin I

Using an automated peptide synthesizer, SEQ NO. 1, SEQ NO. 2, and SEQ NO. 3 peptides were synthesized, and these synthesized derivative peptides were purified purely using C18 reversed phase high performance liquid chromatography (Waters Associates, USA).

Amino Acid Sequences of Peptide Derivatives of the Invention Peptide Amino acid sequence SEQ NO.1 K G R G K Q G G K V R A K A K T R SEQ NO.2 K G R G K Q G G K V R A K A K SEQ NO.3 S S R T K A K A R V K G G Q K G R G K

Experimental Example 1: Investigation of antimicrobial and hemolytic activity of novel antimicrobial peptide paracin I and its derivatives

Each fraction of the antimicrobial peptide paracin I and its derivatives obtained in Example 1 was affinity, resuspended in water and analyzed for antimicrobial activity against B. subtilis ATCC 62037. Purity of the isolated peptide was measured by reverse phase HPLC and matrix-associated laser desorption ionization mass spectrometry (MALDI-MS) (Kratos Kompact MALDI, Manchester, England). The antimicrobial activity of the samples was determined in each purification step by a radial diffusion assay using B. subtilis ATCC 62037 as in the method of Lehrer et al. That is, 20 mL cultures of B. subtilis ATCC 62037 cells in an intermediate logarithmic phase were washed with cold 10 mM sodium phosphate buffer (NAPB), pH 7.4 and resuspended in 10 mL cold NAPB. Cell suspension containing 1 × 10 ⁶ microbial colony forming units (CFU) was added to the lower layer agar (10 mM sodium phosphate, 1% (v / v) trypticase soy broth (TSB), 1% (w / v). Agarose, pH 6.5) was added to 6 mL and the mixture was poured into Petri dish. 3 mm wells were made on solidified lower layer agar and treated with 5 μL of sample. After incubation at 37 ° C. for 3 hours, the lower layer agar was covered with a nutritious top agar cover layer (6% (w / v) TSB, 1% (w / v) agarose) and incubated at 37 ° C. overnight. Antimicrobial activity was determined by observing the microbial growth inhibition zone around the 3mm well. Minimum inhibitor concentrations of peptides isolated against microorganisms were determined according to Moore et al. In other words, 100 μL (10 ⁵ CFU / mL) of the microbial suspension in 3% (w / v) TSB was mixed with serial double dilutions of peptides isolated in sterile 96-well microtite plates (Denmark). It was. The final concentration of the peptide was in the range of 0.5 to 200 μg / mL. 96 well plates were incubated overnight at 37 ° C. and growth inhibition was determined by measuring absorbance at 620 nm with a model 550 microplate reader (Bio-Rad, CA, USA). Minimal inhibitory concentraction (MIC) was defined as the lowest concentration of growth inhibited peptide. Hemolytic activity of the antimicrobial peptide was analyzed according to the method of Park et al. The antimicrobial activity of the antimicrobial peptide Paracin I and its derivatives was compared to 10 other microorganisms, including gram negative, gram positive microorganisms and fungi. As shown in Table 2, the novel antimicrobial peptide Paracin I and its derivatives have strong antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi. In particular, Bacillus subtilis, Streptococcus mutans and Gram-negative bacteria Escherichia coli The antimicrobial activity against the fungus Candida albicans was very good. Derivative peptides also showed stronger antimicrobial activity than antimicrobial peptides. On the other hand, in the MICs of Table 2, the antimicrobial peptide paracin I showed about 12-100 hours more potency than the merginin 2 purified from Xenopus laevis and up to 8 hours more than buforin I isolated from Bufo bufo gagarizans. In the hemolysis test for human erythrocytes, the antimicrobial peptide paracin I (200 μg / mL) showed only 0.2% hemolysis, while the hemolytic peptide melittin had an effect of 99.2% at the same concentration.

Minimum Inhibitory Concentrations of Paracin I, Buporin I and Merginine 2 microbe Minimum inhibitory concentration (㎍ / mL) Paracin I Buporin I Merginine 2 SEQ NO. One SEQ NO. 2 SEQ NO. 3 Gram-positive Bacillus subtilis Staphylococcus aures Streptococcus mutans Pseudomonas putida 1212 4484 505010050 0.510.52 0.510.52 1212 Gram-negative Escherichia coli Salmonella typhimurium Serratia sp. 124 848 1002550 0.514 0.512 124 Fungus Cryptococcus neoformans Sacctharomyces cerevisiae Candida albicans 222 444 122525 111 121 222

Experimental Example 2: Structure Determination of Antimicrobial Peptide Paracin I

In this experiment, the secondary structure of the antimicrobial peptide paracin I was measured using a spectrometer when TFE was present or absent. For Circular dichroism (CD), the antimicrobial peptide paracin I samples were dissolved in either 50 mM NAPB or 50% (v / v) TFE in 50 mM NAPB. CD spectra were recorded at room temperature on a Jasco model J-715 spectrophotometer with a cell path length of 1 mm. Five scans were performed on the samples and averaged wavelengths in the 200-250 nm range. The ellipse value represents the mean value of the residue ellipse [θ] (degrees cm ² dmol ^-1 ), and the content of α-helix, β-sheet and random coil was measured according to Greenfiled and Fasman's method. As a result, the CD spectrum was evenly processed and the baseline was determined by the calculation provided by the manufacturer. As shown in FIG. 4, when analyzed according to Greenfield and Fasman's method, the CD spectrum of paracin I in 50 mM NAPB showed 11% α-helix, 33% β-sheet, and 56% random coil. However, the addition of the solvent TFE, which induces α-helix, slightly increased the β-sheet content in paracin I. As a result, the antimicrobial peptide Paracin I formed β-strands in both hydrophobic and hydrophilic environments instead of the linear amphoteric α-helices common to many other antimicrobial peptides. The axial projection diagram of paracin I is shown as shown in FIG. 5 at 170 ° rotation / residue to determine whether paracin I of the amphoteric β-strand type is possible. Atrophy of the antimicrobial peptide paracin I formed an almost complete amphoteric β-strand with 7-8 cationic residues on the hydrophilic site of the β-sheet plan.

As described in detail in the above embodiment, the antimicrobial peptide paracin I and its derivatives isolated from the catfish of the present invention exhibit strong antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria and fungi, and strongly inhibit microbial expression in the wound and hemolysis. Since there is little activity, it is a very useful invention in the biopharmaceutical industry because it has an excellent effect that can be used as a new antibacterial agent such as wound healing accelerator, trauma treatment, mouthwash and eye drops.

Claims (3)

Novel antimicrobial peptide paracin I comprising all or part of the amino acid sequence represented by the following (I) isolated from catfish I. K G R G K Q G G K V R A K A K T R S S (I) A derivative of a novel antimicrobial peptide isolated from catfish, characterized in that the amino acid sequence of the novel antimicrobial peptide paracin I according to claim 1 is resynthesized as shown in (II). K G R G K Q G G K V R A K A K T R Or (II) K G R G K Q G G K V R A K A K or S S R T K A K A R V K G G Q K G R G K Use of a novel antimicrobial peptide Paracin I and its derivatives isolated from catfish as an antimicrobial agent.