KR100474111B1 - Streptomyces sp. AMLK-335 strain and use of compound produced therefrom - Google Patents

Abstract

The present invention relates to the use of the compounds produced by Streptomyces sp. AMLK-335 ( Streptomyces sp. AMLK-335) isolated from the soil, and the strain produced by the present invention, the cyclo (proline-phenylalanine) and cyclo ( Leucine-proline) shows not only strong antimicrobial activity against microorganisms resistant to conventional antibiotics, especially vancomycin-resistance enterococci (VRE), but also excellent antitumor activity.

Description

Use of AML-335 strain of Streptomyces sp. And its production compound {Streptomyces sp. AMLK-335 strain and use of compound produced therefrom}

The present invention relates to a composition comprising Streptomyces sp. AMLK-335 ( Streptomyces sp. AMLK-335) isolated into soil and a compound having anti-VRE and anti-tumor activity produced by the strain as an active ingredient.

Until now, many kinds of antibiotics have been discovered and developed and widely used in the treatment of diseases. However, as the pathogenic microorganisms become increasingly resistant, the current medical community suffers from severe soreness. In 1929, Penicillin was discovered by Fleming in England, which helped the treatment of microbial infections, but soon became resistant and a new antibiotic, meticillin, was discovered. It plays a big role in the treatment of the Phycococcus aureus . However, the overuse of these antibiotics has also led to the emergence of MRSA (methicillin-resistant Staphylococcus aureus ), which is also resistant to most antibiotics, sometimes referred to as 'superbugs'. Since then, the first antibiotic in the glycopeptide family has used vancomycin (vancomycin; van is derived from vanquish) and teicoplanin family of antibiotics (Griffith, RS). (1984) J. Antimicrob. Chemother . 14: 1-5], these antibiotics have been used most effectively in the treatment of penicillin and methicillin antibiotic resistant strains. Vancomycin in the United States is the only antibiotic approved for the treatment of MRSA infections, while Europe has allowed the use of Teicoplanin, an antibiotic of the same glycopeptide family, and in developing countries, as well as MRSA, In particular, the use of vancomycin has continued to increase over the past decade for the treatment of highly resistant strains of methicillin-resistant Staphylococcus epidemidis (MRSE). In other words, these Gram-positive bacteria are the main pathogenic bacteria, and vancomycin has been the only antibiotic that has been effective against all major Gram-positive pathogenic bacteria until recently as a potent antibiotic against multi-resistant Gram-positive bacteria for many years. Thalia I. Nicas, Michael L. Zeckel and Daniel K. Braun. (1997) Trends In Microbiology. However, since 1980, more and more resistant strains have been generated due to excessive use of antibiotics. Especially, resistance to glycopeptide antibiotics has been used as a food additive in avopacin (avoparcin) for animal weight gain in Europe for over 20 years. As the gram-positive pathogenic bacterium resistant to vancomycin, which has been called as the last antibiotic since 1986, enterococci strains has been continuously found in rapid growth [Kim, Chang-ho, (1998)]. 11: 2]. Vancomycin-resistant enterococci strains are communicable and anaerobic Gram-positive cocci that form part of the normal flora of the intestine, and in a minority are relatively toxic pathogens isolated from the oral cavity or female genitalia. Therefore, although it is generally not easy to cause disease in normal people, it has emerged as an important hospital infection bacterium after being known to cause various opportunistic infections in patients with chronic diseases or in hospitals. Since vancomycin-resistant enterococci (VRE) were first discovered and reported in feces of leukemia patients in Europe in 1986 [Leclercq, R., Derlot, E., Duval, J. and Courvalin, P. ( 1988) N. Engl. J. Med. 319: 157-161], the rapid increase in VRE around US hospitals, which has emerged as a major new pathogen, and the U.S. Centers for Diseases Control and Prevention (CCP) reports that the isolation rate of VRE pathogenic bacteria In the United States, it increased about 20 times from 0.3% to 7.0% from 1989 to 1993, especially 34 times in the intensive care unit (Centers for Disease Control and Prevention. (1993) MMWR 42: 597-599). ). Seven types of enterococci reported so far include E. faecalis, E. faecium, E. faecium, and E. casseliflavus. Reported [Centers for Disease Control and Prevention. (1993) MMWR 42: 597-599] Since 1992, Park et al. First reported E. durans , a highly resistant to vancomycin, from a specimen of leukemia patients as a VRE [Park Ji-won, Kim Yang-ri] . , Shin Wan-sik, Kang Moon-won, Han Kyung-ja, Shim Sangin (1992) 24: 133-137], and in 1995, Kim et al. Reported that four strains of E. casseliflavus and 100 strains of vancomycin-resistant enterococcus were isolated and reported as the flora of the colon. Sungmin Kim, Namyong Lee, Jaehoon Song (1996) Infection. 28: 245 ~ 251], in Korea, few studies have been conducted to investigate the incidence of VRE in hospitals or to identify VRE by genetic methods. Vancomycin resistance has four phenotypes, Van A, Van B, Van C, and Van D, depending on the degree of resistance and the acquisition of genes. Van A type is vancomycin (MIC;> 256 µg / ml), teicoplanin ( E. faecium) , which is highly resistant to (MIC;> 16 [mu] g / ml), is mainly present [Willey, B., McGeer, AJ., Ostrowski, MA., Kreiswirth, BM. and Low, DE. (1994) Infect, Control Hosp. Epidemiol. 15: 548-556] The genotype of Van A can be expressed by transferring to enterococci, streptococci, plasmids or chromosomes of Listeriae [Courvalin, P (1990) Antimicrob. Agents Chemother. 34: 2291-2296., 11.Murray, BE. (1991) J. Infect. Dis. 163: 1185-1194]. Van B type is mainly found in Enterococcus faecium, Enterococcus faeclis , and has varying degrees of resistance to vancomycin (MIC; 4-1000 μg / ml). , Susceptibility to teicoplanin (MIC; <2 μg / ml), but not in leclercq R, Derlot E, Weber M, Duval J, Courvalin P (1989) Antimicrob. Agents chemother 33: 10-15] Some Van B-type enterococci can also acquire resistance to teicoplanin, so the sensitivity test cannot distinguish them from Van A type. Quintiniliani, R. Jr., Evers, S. and Courvalin, P. (1993) J. Infect. Dis. 167: 1220-1223. Van C is a phenotype present in Enterococcus gallinarium, Enterococcus casseliflavus and Enterococcus flavessen (MIC; 4-32) . Μg / ml) and low levels of innate resistance and susceptibility to teicoplanin (MIC; <2 μg / ml) [Arthur, M. and Courvalin, P. (1993) Antimicrob. Agents Chemother. 37: 1563-1571., Gold, HS., Unal, S., Cercenado, E., Thauvin-Eliopoulos, C., Elipoulos, GM and Wennerstein, CB., Et al. (1993) Antimicrob. Agents Chemother. 37: 1604-1609., 16. Baptista, M., Depardieu, F., Courvalin, P. and Arthur, M. (1996) Antimicrob. Agents Chemother. 40: 2291-2295.14-16]

Van A type and B type can acquire the gene and can transfer resistance to other strains, but Van C type has inherent resistance gene. Van D type has a low degree of resistance to vancomycin (MIC; 16 to 64 µg / ml), and is susceptible to teicoplanin (MIC; 2 to 4 µg / ml), but is resistant to other species due to acquired resistance. Metastasis is possible [Leclercq, R. and Courvalin, P. (1997) Clin. Infect. Dis. 24: 545-556]. Vancomycin inhibits cell wall synthesis by forming a complex of carboxy-terminal D-alanine and antibiotic, a peptidoglycan precursor of cell wall components. When the resistance gene is expressed, the D-alanine-D-alanine portion of the cell wall precursor is inhibited by D-alanine-D-lactate. Cross-linkage reduces the ability of vancomycin to bind to the cell wall [Reynolds, PE (1989) Eur. J. Clin. Microbiol. Infect. Dis. 8, 943-95] A representative example of such resistance mechanism is Van A operon, which appears in TN1546 transposon and consists of seven different genes [Leclercq, R. and Courvalin, P]. (1997) Clin. Infect. Dis. 24: 545-556]. Enterococci's bencomycin resistance mechanism is converted to van RP by phosphorylation of van R gene by the regulatory gene van S when exposed to bencomycin, activating van X, van A, van H. Later, the van X gene encodes D, D-dipeptidase to determine the terminal D-alanine-D-alanine portion of the peptidoglycan precursor. Van H, which hydrolyzes and encodes dehydrogenase, converts pyruvate to D-lactate to ligase encoded by van A gene. ligase binds D-lactate to the distal end of the peptidoglycan precursor and decreases its ability to bind to vancomycin on the cell surface, resulting in resistance to vancomycin [Leclercq , R. and Courvalin, P. (1997) Clin. Infect. Dis. 24: 545-556]

With the advent of glycopeptide resistance, many researchers have noted the importance of research and development of vancomycin as an alternative antibiotic. Some VREs are rather susceptible to conventional antibiotics such as penicillin, ampicillin, quinolone, aminoglycoside, chloroamphenicol, and doxycyclin. Treatment with either single or combined therapies is also possible, but these are all experimental results in vitro , and the same effect could not be expected in clinical practice [Wright, GD et al. (1993) Biochemistry 32: 5057-5063. Recently, various antibiotics, namely vancomycin derivatives [Cooper, R, DG et al. (1996) J. Antibiot. 49: 575-581, Nicas, TI et al. (1996) Antimicrob. Agents Chemother. 40: 2194-2199], quinupristin-dalfopristin (Synercid) and oxazolidinone as novel streptogramin antibiotics among teicoplanin derivatives and non-glucopeptide antibiotics. Antibiotics, etc. have been developed as synthetics and have effects on Gram-positive bacteria and Mycobacteria, but show bacteriostatic effects on Gram-positive pathogenic bacteria [Chow, JW et al. (1997) Clin. Infect. Dis. 24: 91 ~ 92] U-100592 and everninomycin antibiotics, SCH 27899, are currently under development, but likewise have bacteriostatic effects against VRE and MRSA [Brickner, SJ (1996) Curr. Pharm. Des. 2: 175-194]. Glycylcyclines, fluoroquinolone, and some of them have been introduced and used in the clinic, and their effects are noted. However, it is expected that it will take some time before they are commercialized. It only has a bacteriostatic effect, so it is likely to recur after treatment, and there are still limitations in the treatment of serious Gram-positive bacterial infections such as endocarditis [Willey, B., McGeer, AJ., Ostrowski, MA., Kreiswirth, BM. and Low, DE. (1994) Infect. Control Hosp. Epidemiol. 15: 548-556]. Vancomycin is by far the most potent antibiotic for the treatment of endocarditis, sepsis, MRSE or other methicillin-resistant coagulase-negative Staphylococci, particularly severe infections caused by prosthetics. It is a substance. Therefore, as the related bacteria such as MRSA and MRSE increase frequently, the use of vancomycin and teicoplanin in the hospital will continue to increase in the future, but once infected with VRE, it can be effectively treated. Antibiotics are not yet available [Thalia I. Nicas, Michael L. Zeckel and Daniel K. Braun. (1997) Trends In Microbiology vol. 5, No. 6.]. Therefore, much research has been conducted on the prevention of VRE infection rather than treatment. The Hospital Infection Control Practice Advisory Committee (HICPAC) proposes a number of recommendations for the restriction of vancomycin use and for prevention of transmission through the establishment of a VRE surveillance system [Control Practice Advisory Committee (1995) Infect. Control Hosp. Epidemiol. 16, 105-113] Continuous spreading of vancomycin-resistant strains and vancomycin-resistant strains attempt to prevent the spread of other strains. HICPAC suggests the use of selective antibiotics, which include serious infections with β-lactam-resistant gram-positive strains and severe allergies to β-lactam antibiotics. Patients infected with Gram-positive strains with an allergy reaction, prevention of endocarditis, and use of vancomycin for the prevention of infection by methicillin-resistant Staphylococci of prosthetics during surgery It was. On the other hand, domestic hospitals use the same glycopeptide antibiotic teicoplanin, which has relatively few side effects, instead of vancomycin for clinical treatment, and limits the use of teicoplanin. It is not supposed to.

At present, the range of glycopeptide antibiotic resistant strains in the world includes not only enterococci, but also tei including Staphylococcus haemolyticus , Staphylococcus epidermidis . Coplanin-resistant coagulase negative Staphylococci has also been shown. Recently, the vancomycin-resistant Enterococcus faecium (VREF) and the vancomycin-resistant Staphylococcus aureus (VRSA), called superbacteria, have been separated, which is the strongest. In addition to being found throughout the country under the name of being resistant to antibiotics, strains with resistance to other species and multi-drug resistance to antibiotics have emerged. I'm nervous. Superbacteria are known as malignant bacteria that are parasitic on the skin, infect through wounds, penetrate bones and joints, and cause pneumonia, reaching a 40% mortality rate. This highly resistant Staphylococcus aureus was first discovered in Japan in 1996, and has also developed in 1997 in Oregon, Michigan, and New Jersey, USA. Of these patients, Oregon lives despite vancomycin. It is believed to have died in Hong Kong in February 1999.

At present, there is no special alternative for the effective treatment of vancomycin-resistant bacteria, and there is no special alternative. Also, the emergence of new resistant strains continues the current medical community. The development of anti-tolerant antibiotics is focused on the continuous development of resistance to enterobacteriaceae and staphylococci so far, but the screening research is ongoing. This is the point where intensive measures for VRE need to be studied globally. Although the measures recommended by the HICPAC should be carried out, the transition to other species is rapidly progressing from the fact that it is still in the prevention. Therefore, it is urgent to develop new antibiotics that show strong bactericidal effect against resistant pathogens. It is feed.

The present inventors have completed the present invention by dividing and purifying cyclo (proline-phenylalanine) and cyclo (leucine-proline) of dipeptide family which have bactericidal effect against vancomycin resistant bacteria and revealing its structure by actinomycetes. It was.

Accordingly, an object of the present invention is to isolate and identify a strain of Streptomyces sp. That exhibits strong antimicrobial activity against vancomycin-resistant bacteria and produces anti-tumor substances from soil. Another object of the present invention is to provide an anti-VRE and anti-tumor composition comprising cyclo (proline-phenylalanine) and cyclo (leucine-proline) produced by the strain as an active ingredient.

The object of the present invention is to isolate the strain after the pure strain from the soil to isolate the antibiotic producing strains of the isolated strains to identify and culture and measure the anti-VRE and anti-tumor activity of the material produced by the strain And achieved by analyzing the structure.

Hereinafter, the configuration of the present invention will be described.

In the present invention, the strain Streptomyces genus from the soil was isolated and identified and named AMLK-335 ( Streptomyces sp. AMLK-335).

The present invention Streptomyces genus AMLK-335 strains produce dipeptide-based cyclo (proline-phenylalanine) and cyclo (leucine-proline), the compounds are excellent antibacterial and anti-tumor activity, in particular vancomycin-resistant enterococci ( vancomycin-resistance enterococci (hereinafter abbreviated as 'VRE') is characterized by strong antimicrobial activity.

Cyclo (proline-phenylalanine) and cyclo (leucine-proline) isolated and purified in the present invention are units for tablets, powders, granules, capsules, suspensions, emulsions or parenteral administration using pharmaceutically acceptable carriers or excipients. It can be formulated into a dosage form or several dosage forms.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Production of Substances Showing Anti-VRE Activity and Anti-tumor Activity by Isolation and Identification of AMLK-335 Genus Streptomyces and Culture of the Strain

First step: isolation of soil actinomycetes

Take 1 g of soil at a depth of about 10 cm from the surface of uncultivated soils around the country, air-dried it for 2 to 3 weeks in a well-ventilated shade, and then crush it finely. Part of it is placed in a crucible for ash analysis. Dry heat treatment was performed for 1 hour in a drying oven, and the rest was used as a source for separating soil microorganisms. 1 g of each heated and unheated soil sample was added to 1 mL of sterile distilled water and stirred for about 1 minute. The supernatant was diluted with sterile distilled water by a 10-fold dilution method, and each 1 mL of the diluted solution was sterilized. Plates were prepared by dispensing 20 ml of S agar medium, an actinomycetes isolated medium and AV agar medium containing antibiotics, vitamins, and trace elements, respectively, in a petri-dish, and then incubating in a 27 ° C. incubator. Incubated for 14 days. Actinomycetes colonies (colony) shown on the plate was plated on the same separation medium, and the AMLK-335 strain was purely separated, and the actinomycetes strain AMLK-335 was purified from yeast extract-malt extract agar. After implanted in and incubated at 27 ° C. for about 7 days, cultured so that the mycelia and spores were sufficiently developed and stored at room temperature and used for the experiment.

Second Step: Strain Identification

The colony morphology of the isolated strain AMLK-335, morphology and spore formation of trophic mycelia and mycelium, morphological characteristics such as the shape of the spore surface, DAP isomer composition and amino acid composition of the cell wall, etc. The genome was identified by comparison with the suprageneric classification of Lucci (1989) and the classification method of actinomycetes and its flexible bacteria. The species identification was carried out by the TAXON program developed by Dr. Ward of the Department of Microbiology, University of Newcastle upon Tyne, UK. 1993). The TAXON program is a program that can be easily identified by comparing the characteristics of the unknown probability with the characteristics of the identification probability matrix prepared based on the results of the classification, by simply inputting the unit trait characteristics with + and-. . In the TAXON program, 50 unit traits were compared in major clusters, 39 unit traits were compared, and 39 such as Willcox probability, taxonomic distance, 95% taxon radius, and% probability of strain further away. The type of identification score shows the result of identification. The 95% taxon radius represents the radius of the sphere containing 95% of the taxons identified as a result of the four identification scores, and the% probability of strain further away is outside the identified unknown test strain. Shows what percentage of the strain is stochastic. In addition, the inoculator PCII medium for the examination of the production conditions of antibiotics was pre-cultured as the pre-culture medium and used as the inoculum. Antibiotic activity was assayed using the K-98 Enterococcus faecium 642 and K-98 Enterococcus faecium 637 assays using the disk diffusion method, and the diameter of the transparent ring formed around the disk (mm) was measured. It is shown as antimicrobial activity.

The effect of carbon source is to prepare a medium by adding 2.5% of various carbon sources to PY medium excluding carbon source, and inoculate 2% of pre-culture solution to each medium and incubate at 27 ℃ for 4 days to produce antibiotics. The impact of each carbon source was examined. In addition, by selecting the best antibiotic material among the carbon sources, the optimum concentration of the carbon source was determined by examining the antibiotic productivity according to the concentration of the carbon source. The effect of nitrogen source is to add 1.1% of various nitrogen sources to PY medium except nitrogen source to prepare medium, inoculate 2% of whole culture solution to each medium and incubate at 27 ℃ for 4 days to produce antibiotics. The effects of each nitrogen source were examined. In addition, by selecting the best antibiotic material among nitrogen sources, the optimum concentration of nitrogen sources was determined by investigating the productivity of antibiotics according to the concentration of nitrogen source. Experimental results show that the isolate, AMLK-335, has a DAP isomer of the cell wall LL-DAP, and the peptide of the cell wall is a glycine-linked peptidoglycan when the amino acids are compared with the actinomycetes wall chemotype and peptidoglycan type (Lechevalier et al., 1970). type A3γ, corresponds to wall chemotype I, and actinomycetes The genus belonging to wall chemotype I in the group classification is Arachnia and Pimelobacter genus, Nocardiodes genus and Streptomycetes among actinobacteria (Goodfellow, 1989). AMLK-335 was classified as Streptomyces through comparative analysis of morphological characteristics and culture characteristics, and morphological characteristics, such as classification of genus belonging to Streptomycetes of Locci (1989) and colony form of isolated strain AMLK-335, and culture In consideration of the chemical properties such as DAP isomers and amino acids of the cell wall, the isolate AMLK-335 was classified as Streptomyces (Table 1).

For species identification, the types of unit traits necessary for the hydraulic identification of major clusters and the characteristics of the unit traits of the isolated strain AMLK-335 are shown in Table 2. The hydraulic identification using the TAXON program is ideal for higher Willcox probabilities of 0.85 or higher, Taxon distance within 95% Taxon radius, shorter and higher% probability of strain furthur away. According to the TAXON program, the isolated strain showed the highest Willcox probability of 0.983458 in major cluster 18 ( Streptomyces cyaneus ), but it did not fall within 95% Taxon radius 0.4497 and the% probability of strain furthur away value was low as 0.1756. It was judged that it was the outermost strain of cluster 18, ie, Streptomyces cyaneus species group, and only 37 unit traits among 50 taxon items showed the same result when compared with centrotype strains as shown in Tables 3a and 3b. Therefore, no identification was made in major clusters. The results of hydraulic identification in the minor cluster are shown in Table 4, and the results of the Taxon program analysis using the database of the strains with the completed hydraulic classification are shown in Table 5. Willcox probability of minor cluster 54 ( Streptomyces aldoflavus ) Was highest at 0.876289, but it was far from 95% Taxon radius 0.3776, and the% probability of strain furthur away was low at 0.0000, compared to the nearest outer-most member of the 54 clusters. Since only 27 of the unit traits showed the same result, it was determined that the strain was present at the outermost of cluster 54, ie, Streptomyces aldoflavus species group. As a result of the above hydraulic identification, there was no best match strain in the major cluster and the minor cluster. Therefore, it is considered as a new species of Streptomyces genus because of the large difference in the results of the various unit types.

In addition, several genes have been used for the purpose of molecular classification and identification. Among them, the most commonly used gene is 16S rRNA, and their sequencing is useful for identifying and identifying microorganisms and their evolutionary relationships due to various useful features. In addition, certain 16S rRNAs are very slow in evolution and exhibit a conserved sequence and secondary structure common to many organisms, allowing for the comparison of various taxa. As shown in FIGS. 1 and 2, nucleotide sequences of two variable regions were determined, and phylogenetic similarities were compared with those of 16S rRNA base sequences registered in GenBank. In Figure 1 (set 1), AMLK-335 showed 97% similarity to Streptomyces galbus , and in Figure 2 (set 2), it showed 98% similarity to Streptomyces platensis . In conclusion, AMLK-335 strain was found to be closest to Streptomyces platensis in comparison with numerical data and 16S rRNA sequence.

The strains isolated and identified as described above were named Streptomyces sp. , The present inventors named Streptomyces sp. AMLK-335, and deposited on July 6, 2002 at the Microbial Deposit Center of the Biotechnology Research Institute. The accession number is KCTC 10300BP.

Comparison of Characteristic Diagnosis of Streptomyces, Streptobetacillium, Kineosporia, Sporticaya, and AMLK-335 Strains characteristics Strepto-myces Strepto-vericillium Kineosporia Sporichthya AMLK-335 Colony size Discrete + Discrete- small + Microscopic- Discrete + Substratemycelium Spheres + - - - + Sporangia - - + - - Motile spores - - + - - Aerial mycelium chains of arthros- pores + + - + + arthros- pores in verticils - + - - - Spore surface smoth + + - + + Spore surface hairy, spiny, or warty + - - - - Motile spore - - - - - Wall chemotypes I I I I I Wall chemotype: I, LL-DAP and Glassine

Identification of AMLK-335 Strains against Streptomyces Main Group by TAXON Program TAXONmajor cluster (centrotype member) TAXONdistance 95% TAXONradius % prob. ofstrain furtheraway WillcoxProbability 33 ( Streptomyces chromogenes ) 0.4750 0.3955 0.0035 0.000281 6 ( Streptomyces violaceus ) 0.4979 0.4126 0.0026 0.005567 18 ( Streptomyces cyaneus ) 0.4991 0.4497 0.1756 0.983458 5 ( Streptomyces exfoliatus ) 0.5076 0.4455 0.0558 0.002443 10 ( Streptomyces fulvissimus ) 0.5091 0.4036 0.0001 0.000000 19 ( Streptomyces diastaticus ) 0.5102 0.4508 0.0756 0.008165 1B ( Streptomyces anulatus ) 0.5535 0.4404 0.0002 0.000032 23 ( Streptomyces microflavus ) 0.5539 0.3931 0.0000 0.000000 30 ( Streptomyces filipinensis ) 0.5574 0.3845 0.0000 0.000000 61 ( Streptomyces lavendulae ) 0.5598 0.4118 0.0000 0.000001 17 ( Streptomyces griseoviridis ) 0.5625 0.3943 0.0000 0.000000 15 ( Streptomyces chromofuscus ) 0.5673 0.4271 0.0000 0.000053 65 ( Kitasatoa spp.) 0.5674 0.3374 0.0000 0.000000 29 ( Streptomyces lydicus ) 0.5687 0.3831 0.0000 0.000000 32 ( Streptomyces violaceoniger ) 0.5773 0.3854 0.0000 0.000000 3 ( Streptomyces atroolivaceus ) 0.5802 0.3631 0.0000 0.000000 21 ( Streptomyces griseoruber ) 0.5823 0.3709 0.0000 0.000000 31 ( Streptomyces antibioticus ) 0.5886 0.4131 0.0000 0.000000 42 ( Streptomyces rimosus ) 0.5960 0.3507 0.0000 0.000000 40 ( Streptomyces phaeochromogenes ) 0.6001 0.3805 0.0000 0.000000 37 ( Streptomyces griseovus ) 0.6095 0.3658 0.0000 0.000000 16 ( Streptomyces albus ) 0.6144 0.3347 0.0000 0.000000 12 ( Streptomyces rochei ) 0.6187 0.4173 0.0000 0.000000 20 ( Streptomyces olivaceovirdis ) 0.6215 0.3720 0.0000 0.000000 1A ( Streptomyces albidoflavus ) 0.6355 0.3782 0.0000 0.000000 1C ( Streptomyces halstedii ) 0.6439 0.3883 0.0000 0.000000

Comparison of Taxonomic unit characters between Streptomyces 18 group and AMLK-335 strain of the present invention Taxonomic unit chara-cters (TAXON code) % value incluster 18 HNO incluster Centype incluster 18 StrainAMLK-335 Outer-mostin cluster18 RFS 5 - - + - SPI 82 + + - + RED 32 - - - + GRY 29 - - - - ROS 21 - - - - PIG 34 - - + - YBP 13 - - + - MPI 97 + + + - MTY 84 + + + - BUT 32 - - - - CYS 74 + + - + VAL 71 + + + - PHE 66 + + + - HIS 84 + + + + HYD 29 - - + - LEC 11 - - + - LIP 50 + + - + PEC 55 + - - + NO3 37 - - + + H2S 89 + + + + SUB 45 - - + + LUT 34 - - + + ALB 3 - - - - CER 5 - - - - MUR 63 + + - - NIG 11 - - - - HIP 3 - - - - ELA 42 - + - + XAN 82 + + - + ARB 55 + + + - NEO One - - - - RIF 45 - + + + OLE 13 - - - - PEN 63 + + + + 45C 42 - + - + 7NA 18 - - - - 01Z 16 - - - - PHN 63 + + - + 01T 45 - - + +

Comparison of Taxonomic unit characters between Streptomyces 18 group and AMLK-335 strain of the present invention Taxonomic unit chara-cters (TAXON code) % value incluster 18 HNO incluster Centype incluster 18 StrainAMLK-335 Outer-mostin cluster18 T01 One - - - - SUC 92 + + - - INO 95 + + + - MAN 97 + + + + RHA 92 + + - + RAF 99 + + + + MEZ 76 + + - + ADO 84 + + + + MEB 97 + + + + DEX 61 + + - + XYT 21 - - - +

Classification using TAXON program Morphology and pigmentation     Spore chain morphology: rectiflexible (RFS): + spiral (SPI):-     Color of spore mass: gray (GRY):-     Diffusible pigment: Production (PIG): + yellow / brown (YBP): + 2. Antimicrobial activity Bacillus subtilis (SUB): + Micrococcus luteus (LUT): + Streptomyces murimu s (MUR):- 3. Biochemical test     Pectin hydrolysis (PEC):- 4. Degrative tests     Guanine (GUA):-Xylan (XYN):-Elastin (ELA):-     Urea (URE): + Xanthine (XAN):-Allantoin (ALL): + 5. Antibiotic resistance     Rifampicin (RIF): + Penicillin (PEN): + 6. Growth test     45 ° C (45C):-NaCl (7NA):-Phenol (PEN): +     Potassium tellurite (01T): + Sodium azide (01Z):- 7.Compounds as sole source of nitrogen (1%, w / v)     Potassium nitrate (POT):-L-Phenylalanine (PHE): +     L-Histidine (HIS): + 8.Organic compounds as sole source of carbon (1%, w / v)     L-Arabinose (ARA):-Sucrose (SUC):-myo-Inositol (INO): +     Mannitol (MAN): + L-Rhamnose (RHA):-Raffinose (RAF): +     D-Melibiose (MEB): + Xylose (XYL): + Salicin (SAL):-     Sodium acetate (ACE):-D-Fructose (FRU): + D-Lactose (LAC): +     Sodium citrate (CIT):-Sodium propionate (PRO):-Sodium pyruvate (PYR):-

Identification of AMLK-335 Strains for Streptomyces Subgroup by TAXON Program TAXONminor cluster (centrotype menber) TAXON 95% distance TAXON% radius prob. of strainfurther away Willcox Probability 35 ( Streptomyces chattanoagensis ) 0.5328 0.3015 0.0000 0.081289 8 ( Streptomyces alni ) 0.5388 0.3154 0.0000 0.041055 54 ( Streptomyces alboflavus ) 0.5489 0.3776 0.0000 0.876289 11 ( Streptomyces prunicolor ) 0.5559 0.3015 0.0000 0.000821 39 ( Streptomyces longisporoflavus ) 0.5881 0.3570 0.0000 0.000197 9 ( Streptomyces californicus ) 0.5992 0.3462 0.0000 0.000000 63 ( Streptomyces xanthochromogenes ) 0.6005 0.4066 0.0000 0.000338 44 ( Streptomyces pactum ) 0.6044 0.3675 0.0000 0.000003 45 ( Streptomyces aurantiacus ) 0.6087 0.3570 0.0000 0.000000 70 ( Streptomyces viridoflavus ) 0.6091 0.3875 0.0000 0.000001

Third step: culturing AMLK-335 strain of Streptomyces to produce antimicrobial substances

PC Ⅱ medium (glucose 5.0g / L, made by inserting 4-5 glass beads into the test tube (20 Ø × 175 mm) in the actinomycetes Streptomyces isolated in step 1 and identified in step 2 Polypeptone 3.0g / L, yeast extract 2.0g / L, meat extract 5 / 0g / L, asparagine 0.5g / L, thiamine · HCl 0.1g -31, Yamato, Co., Japan) was precultured for 48 hours at 280 strokes / min. Subsequently, antibiotic production medium PY medium (glucose 5.0g / L, polypeptone 3.0g / L, yeast extract 2.0g / L, meat extract 5 / 0g / L, soluble starch 10.0g / L, glucerol 10.0g / L, Sterilize 100 ml of casein 1.0 g / L, CaCO ₃ 2.0 g / L, Thiamine and HCL 0.01 g / L in a 500 mL Erlenmeyer flask and sterilize 2% (v / v) of the preculture. Inoculation was carried out for 96 hours at 250 rpm using a rotary shaker (Model: TGR 1-D, IWASHIA, Japan) at 27 ℃.

Fourth Step: Preparation of Anti-VRE Activity and Anti-Tumor Activity Samples

After culturing AMLK-335 in the strain Streptomyces isolated in the first step, centrifuged (2,000 × g, 20 min.) To obtain the supernatant, the cells were extracted with antibiotics in absolute acetone and dried under reduced pressure to obtain antibacterial activity. It was used as a sample for measuring activity and antitumor activity.

Step 5: Determining Anti VRE Activity

Van A type (vancomycin, teicoplanin MIC> 256 ㎍ / ml) isolated from Korea University Guro Hospital's Infectious Diseases to measure the antimicrobial activity of AMLK-335 from Streptomyces genus, especially against vancomycin-resistant enterococci. ) K-98 Enterococcus faecium 642 strain and Van B (vancomycin MIC> 256 μg / ml, teicoplanin MIC> 0.75 μg / ml) and K-98 Enterococcus faecium 642 ( Enterococcus faecium 637) Six strains of different origins and origins were used for the distribution and antimicrobial activity was investigated for Gram-positive bacteria, Gram-negative bacteria, yeast and fungal strains. At this time, the medium used to prepare the assay plate was Brain Heart Infusion medium, and the preculture was added to the test tube in a sterilized medium by adding 5 mL of Brain Heart Infusion broth to the test tube. Colonies were inoculated and incubated at 37 ° C. for 18-24 hours. Assay method was used to prepare a middle layer, 20 mL of Brain Hert Infusion medium was poured into sterile petri-dish and coagulated to form a lower layer, and the same medium containing the precultured bacteria solution 5 The mL was used as a middle layer on the lower medium. Experimental results showed that E. faecium , VanA (MIC> 200 μg / ml), K-98-642 for VRE (vancomycin resistant enterococci) species; E. faecium , VanA (MIC> 200 μg / ml), K-99-137; E. faecium , VanA (MIC> 200 μg / ml), K-99-158; E. faecium , VanA (MIC> 200 μg / ml), K-99-229; E. faecium , VanA (MIC> 200 μg / ml), K-99-38; E. faecalis , Van A (MIC> 200 μg / ml), K-99-266: E. fa ecium , VanA (MIC> 200 μg / ml), K-99-243: E. faecium , VanA (MIC> 200 μg / ml), K-99-34, E. faecium , VanA (MIC> 200 μg / ml) Ml), K-99-17; E. faecium , VanA (MIC> 200 μg / ml), K-99-14; E. faecium , VanA (MIC> 200 μg / ml), K-00-221, E. faecium , VanA (MIC> 200 μg / ml), K-00-227 Showed strong activity

Step 6: Antitumor Activity Measurement

Antitumor activity was measured using the same sample that measured the antimicrobial activity in the fifth step. Each tumor cell line was inoculated with cells of each inoculation concentration and cultured for 24 hours, followed by treatment with tumor cell proliferation inhibitors, and then cultured for 48 hours. Four hours before incubation, tumor cells, wells containing cyclo (pro-phe) and cyclo (leu-pro), and 50 μl of MTT (0.5mg / ml) solution were added to wells (drunk blank wells). After 96wellplate was incubated for 4 hours at 37 ℃ further to induce formazan formation, the supernatant was removed by centrifugation after the additional incubation. In order to dissolve the blue formazan generated after centrifugation, 100 ul of DMSO was added to each well to completely dissolve the formazan, and then the absorbance (540 nm) of each well was measured. The MTT assay calculated [1- (OD of control cells) x100 and expressed it as% inhibition, and it was determined that the tumor cell proliferation inhibitory effect was higher than 50%. Human, stomach), KB (human, granular epithelial cells), HL60 (human, leukemia), K562 (human, leukemia), ME-180 (human, peritoneal metastatic cells), WiDr (human, malignant tumor cells), 3LL ( 14 tumor cell lines and 1 normal cell line (MDBK) showed strong antitumor activity against several tumor cell lines.

Example 2: Purification and Structural Analysis of Cyclo (Proline-Phenylalanine)

Antimicrobial and anti-tumor actives produced from actinomycetes Streptomyces genus AMLK-335 were purified and analyzed.

First, the sample obtained by acetone extraction of the AMLK-335 culture of Streptomyces genus in Example 1 was adsorbed by Diaion HP-20, washed with 25% methanol and eluted with absolute methanol. The eluted was extracted with ethyl acetate (pH 7.0) to take an ethyl acetate layer and extracted with diethyl acetate. The ether layer obtained was concentrated under reduced pressure, suspended in methanol, passed through Sephadex LH-20, and eluted with absolute methanol. The obtained active fractions were concentrated in vacuo and subjected to thin-layer chromatography using a solvent mixture of chloroform: methol: acetic acid (80: 15: 5), and finally methanol: water (70:30). High-performance liquid chromatography (HPLC; Hewlett-Packard 1100, μ-bondapak C18 column (10 μm, 150 × 3.9 mm, flow rate 0.5 ml / min) using a phosphorus developing solvent was used to obtain a single peak at 3.016 minutes of retention time). The compound represented was isolated.

The purified compounds were examined for the physicochemical evaluation and chemical structure of the purely purified active substance by various instrumental analysis.

As a result of the physicochemical properties, the melting point was measured at 134 ° C. In the UV spectrum analysis, the antibiotic showed the maximum absorbance at 232.0 nm and 258.7 nm. Elemental analysis revealed C (69.09%), O (12.88%), N (11.39%), and H (6.61%). No S was found.

Molecular weight 244 was obtained from the mass spectrum for the peak of 22.79 minutes in the gas chromatogram, ¹ H-NMR (CDCl ₃ ); The peaks of six protons were observed from δ1.89 to 2.82, and the peaks of four protons were observed from δ3.57 to 4.59, and the peaks of one proton were observed from δ5.76, and δ7.25 At ~ 7.37, peaks estimated to be five protons were observed. Therefore, the total number of protons was 16, and 14 carbon signals appeared in the ¹³ C-NMR spectrum, which turned out to be 14 carbons.

As a result of the measurement of the IR spectrum, the absorption wavelength was shown as NH group at 3043 cm-1 and 3266 cm-1 and Amide group at 1750-1730 cm-1, and C = benzene nucleus at 1467 cm-1 and 1569 cm-1. Absorption wavelength resulting from internal vibration of C was observed.

More GC / MS, NMR, the results determined by the instrument analyzed and comprehensive chemical structural analysis and the structural formula of IR and UV, Streptomyces sp. The antibiotic produced from AMLK-335 has a molecular weight of 244 and has been identified as cyclo (pro-Phenalanine) [cyclo (Pro-Phe)] with the formula (C ₁₄ H ₁₆ N ₂ O ₂ ) (formula I) .

Example 3: Purification and Structural Analysis of Cyclo (Leucine-Proline)

First, the sample obtained by acetone extraction of the AMLK-335 culture of Streptomyces genus in Example 1 was adsorbed by Diaion HP-20, washed twice with 60% methanol, and eluted with absolute methanol. The eluted was extracted with ethyl acetate (pH 7.0) to obtain an ethyl acetate layer and extracted twice with diethyl ether. The ether layer thus obtained was concentrated under reduced pressure, suspended in 80% methanol, passed through Sephadex LH-20, and eluted with absolute methanol. The obtained active fractions were concentrated in vacuo and subjected to thin-layer chromatography using a solvent mixture of chloroform: methol: acetic acid (70:20:10), and finally methanol: water (80:20). High-performance liquid chromatography (HPLC; Hewlett-Packard 1100, μ-bondapak C18 column (10 μm, 150 × 3.9 mm, flow rate 0.5 ml / min) using phosphorus developing solvent) was used to obtain a single peak at 5.023 minutes of retention time. The compound represented was isolated.

The purified compounds were examined for the physicochemical evaluation and chemical structure of the purely purified active substance by various instrumental analysis.

The purified compound had the property of white powder, solubility was well dissolved in methanol, and the results of elemental analysis of C, H, N showed C (62.61%), H (8.50%) and N (13.35%). The molecular formula was determined by NMR analysis C ₁₁ H ₁₈ N ₂ O ₂ .

Based on the above comprehensive chemical analysis and structural formula, Streptomyces sp. Antibiotics produced from AMLK-335 have been identified as cyclo (Leu-Pro) [Formula II].

Example 4 Determination of Anti-VRE and Anti-Tumor Activity of Cyclo (Proline-Phenylalanine) and Cyclo (Leucine-Proline)

Antimicrobial activity and antitumor activity were measured in the same manner as in Steps 3 and 4 of Example 1.

Anti-VRE activity of Cyclo (pro-phe) and Cyclo (leu-pro) was measured in E. faecium K-99-158, 229 at 25ug / ml. It was found that high anti-VRE activity was observed, and high anti-VRE activity was observed in E. faecium K-99-243, 34, K-00-221, and 227 at 12.5ug / ml of Cyclo (leu-phe). there was.

Anti-tumor activity of Cyclo (pro-phe) and Cyclo (leu-pro) was measured in WiDr, Hep-2, K562, 3LL at 500ug / ml of Cyclo (pro-phe) as shown in Table 7,9. , 98,98,96% high anti-tumor activity, 97,97,98,96% higher than KB, HL60, U937, THP-1 at 500ug / ml of Cyclo (leu-pro) It was found to exhibit antitumor activity. However, the normal cell line, MDBK, was 42,48%, respectively.

Cyclo (pro-phe) anti-VRE activity measurement results       Species MIC (g / ml) Genotype VANa TECb Cyclo (pro-phe) E.faecium K-98-642 512 > 512 50 Van a E.faecalis K-99-137 512 64 > 100 Van a E.faecium K-99-158 512 64 25 Van a E.faecium K-99-229 512 512 25 Van a E.faecium K-99-38 512 256 > 100 Van a E.faecalis K-99-266 512 32 50 Van b a: vancomycin, b: teicoplanin

Antitumor Activity of Cyclo (pro-phe) Bell Cell type Concentration of cyclo (pro-phe) (µg / ml) 500 100 50 10 5 One SNU-1 Human stomach 85 * 81 * 56 * 0 0 0 WiDr Human colon 90 * 79 * 8 0 0 0 KB Human mouth epidermoid 94 * 80 * 76 * 64 * 56 * 0 Caki-2 Human kidney 45 44 10 0 0 0 Hep-2 Human larynx 98 * 88 34 0 0 0 HEC-1B Human uterus 40 22 0 0 0 0 SF-188 Human brain 52 * 38 10 0 0 0 K562 Human leukemia 98 * 75 * 55 * 0 0 0 3LL Mouse lung 96 * 62 * 51 * 11 0 0 P388 Mouse leukemia 89 * 80 * 67 * 0 0 0 MDBK Biovine kidney 42 21 0 0 0 0 * Sensitive (i.e.,% inhibition ?? 50) experimental data are 5 repetitive measurements.

Determination of anti-VRE activity for each strain of the composition of the invention species Minimum inhibitory concentration (MIC ㎍ / ml) Genotype VAN TEC Cyclo (leu-pro) E.faecium K-98-642 256 32 50 vanB E.faecium K-98-229 256 32 25 vanB E.faecium K-99-38 512 512 > 100 vanA E.faecium K-99-137 512 32 25 vanA E.faecium K-99-158 256 32 25 vanB E.faecium K-99-266 512 512 25 vanA E.faecium K-99-243 256 16 12.5 vanB E.faecium K-99-34 256 16 12.5 vanB E.faecium K-99-17 512 512 50 vanA E.faecium K-99-14 512 32 50 vanB E.faecium K-00-221 512 64 12.5 vanA E.faecium K-00-277 512 32 12.5 vanB ^a genotype of each strain was determined by PCR primers specific for vancomycin ^b teicoplanin ^{c, d} vanA, vanB.

Antitumor Activity of Cyclo (pro-phe) and Cyclo (leu-pro) Bell Cell type Concentration of cyclo (leu-pro) (µg / ml) 500 100 50 10 5 One SNU-1 Human stomach 85 * 74 * 56 * 0 0 0 WiDr Human colon 50 * 42 6 0 0 0 KB Human mouth epidermoid 97 * 88 * 76 * 64 * 56 * 0 Caki-2 Human kidney 56 * 44 10 0 0 0 Hep-2 Human larynx 58 * 45 34 0 0 0 HEC-1B Human uterus 32 11 0 21 0 0 SF-188 Human brain 52 * 40 10 0 0 0 K562 Human leukemia 64 * 43 25 0 0 0 HL60 Human leukemia 97 * 91 * 84 * 0 0 51 U937 Human leukemia 98 * 93 * 88 * 75 * 70 * 52 * THP-1 Human leukemia 96 * 61 * 48 31 0 0 Farrow Human melanoma 54 * 40 0 0 0 0 3LL Mouse lung 75 * 52 * 35 11 0 0 P388 Mouse leukemia 89 * 80 * 67 * 0 0 0 MDBK Biovine kidney 48 24 12 0 0 0 * Sensitive (i.e.,% inhibition ≥ 50) experimental data are 5 repeated measurements.

As described above, the present invention, as described in the present invention, AMLK-335 in the actinomycetes Streptomyces genus isolated from soil not only exhibits strong antimicrobial activity against microorganisms, particularly vancomycin-resistant enterococci, which are resistant to conventional antibiotics. It is an extremely useful invention for the biopharmaceutical industry because it has an excellent effect of showing antitumor activity.

1 is a result of comparing the nucleotide sequence of 16S ribosomal DNA of Streptomyces galbus and Streptomyces genus AMLK-335 strain of the present invention.

Figure 2 is a comparison of the nucleotide sequence of 16S ribosomal DNA of Streptomyces platensis and AMLK-335 strain of Streptomyces genus of the present invention.

<110> RHEE, KI HYEONG <120> Streptomyces sp. AMLK-335 strain and use of compound produced therefrom <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 233 <212> DNA <213> Streptomyces sp. <220> <221> gene (222) (1) .. (233) <223> Nucletide sequences of streptomycete 16S ribosomal DNA <400> 1 cttaacacat gcaagtcgaa cgatgaacct ccttcgggag gggattagtg gcgaacgggt 60 gagtaacacg tgggcaatct gcccttcact ctgggacaag ccctggaaac ggggtctaat 120 accggatacg acctccgacc gcatggtctg gtggtggaaa gctccggcgg tgaaggatga 180 gcccgcggcc tatcagcttg ttggtgggta tggcctacca aggcgacgac ggg 233 <210> 2 <211> 319 <212> DNA <213> Streptomyces sp. <220> <221> gene (222) (1) .. (319) <223> Nucleotide sequences of streptomycete 16S ribosomal DNA <400> 2 gttccccgcc tggggagtac ggccgcaagg ctaaactcaa aggaattgac ggggcccgca 60 caagcagcgg agcatgtggc ttaattcgac gcaacgcgaa gaaccttacc aaggcttgac 120 atacaccgga aacctctgga gacaggcgcc cccttgtggt cggtgtacag gtggtgcatg 180 gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg 240 ttctgtgttg ccagcatgcc ttcggggtga tggggactca caggagaccg ccggggtcaa 300 ctcggaggaa ggtggggac 319

Claims (3)

Streptomyces sp. AMLK-335 (KCTC10300BP), a genus of Streptomyces that produces anti-VRE and anti-tumor actives isolated from soil. An antimicrobial and antitumor composition comprising cyclo (proline-phenylalanine) represented by Structural Formula (I), which is purified by culturing AMLK-335 (KCTC10300BP) of Streptomyces sp. An antimicrobial and antitumor composition comprising cyclo (leucine-proline) represented by the following structural formula (II), which is cultured and purified by the genus Streptomyces genus AMLK-335 (KCTC10300BP).