KR20040076836A - Hyper-productive mutant strain of Actinoplanes teichomyceticus producing teicoplanin - Google Patents

Abstract

PURPOSE: A teicoplanin producing variant of Actinoplanes teichomyceticus is provided, thereby significantly increasing the production of teicoplanin and antibiotics. CONSTITUTION: A method for isolating Actinoplanes teichomyceticus M76-35-7(KCCM-10363) comprises the steps of: inducing the mutagenesis of wild-type Actinoplanes teichomyceticus(ATCC-31121) with ultra violet rays; spreading the mutant Actinoplanes teichomyceticus(ATCC-31121) on the agar medium; culturing the microorganism and selecting a strain forming a larger clear zone than that of parental strain or others; inducing the mutagenesis of the selected strain with ultra violet rays; and selecting one strain of Actinoplanes teichomyceticus producing a large amount of teicoplanin.

Description

Hyper-productive mutant strain of Actinoplanes teichomyceticus producing teicoplanin

The present invention is to produce teicoplanin (Teicoplanin), a Gram-positive bacterium treatment agentActinoplanes teichomyceticus) The present invention relates to a mutant strain and a method for producing fermentation of teicoplanin using the mutant strain.

Teicoplanin is a bactericidal glycopeptide antibiotic produced by the fermentation of Etinoplanes teicomyceticus and inhibits the cell wall synthesis of Gram-positive bacteria. In particular, teicoplanin not only treats infection caused by penicillin resistant strains, but also treats infections caused by vancomycin resistant strains.

Teicoplanin is the last of the five amino acids bound to N-acetylmuramic acid of peptidoglycan, consisting of N-acetylmuramic acid and N-acetylglucosamin. Inhibits transglycosylation and transpeptidation during cell wall synthesis of Gram-positive bacteria by binding to a dipeptide of D-alanyl-D-alanine that binds to (Mackay, JP et al. , J. Am. Chem. Soc , 116 : 4581-4590 (1994)). Therefore, teicoplanin is more effective in the treatment of Gram-positive bacteria infection than Gram-negative bacteria, and especially, Staphylococcus and Streptococcus have higher therapeutic effects. Teicoplanin is also used to treat infections caused by methicillin resistance Staphylococcus aureus (MRSA), which is resistant to methicillin (Malabarba, A. et al., J. Antibiotics , 37 : 988-999 (1984)).

The biological characteristics of the teicoplanin components are shown by T-A2-4 and T-A2- when 50% of pneumonia-infected rats are treated by administering the respective teicoplanin components to rats infected with pneumonia. Even the lowest dose of 5 showed a therapeutic effect. However, T-A2-1, T-A2-2 and T-A2-3 showed 50% of normal rats died when T-A2-1, T-A2-2 and T-A2-3 were administered at about 1,000 mg / kg to 2,000 mg / kg, respectively. -A2-4 and T-A2-5 showed 50% mortality even when only 500mg / kg to 1,000mg / kg were administered (Borghi, A. et al. 1984, J. Antibiotics, 37: 615-620).

Research and reporting on Actinoplanes teichomyceticus is not yet sufficient , and reports on teicoplanin biosynthetic pathways and regulatory mechanisms are extremely limited. Therefore, the potency improvement of teicoplanin producing strains is mainly through artificial mutations, and studies to increase production titers using teicoplanin precursors have been reported (Borghi, A. et al. J. General Microbiology) , 137 : 587-592 (1991)). In addition, the productivity of T-A2-2, which accounts for the largest proportion of the five components of teicoplanin, is increased, or the teicoplanin derivative is produced by organic synthesis to reveal the biochemical properties of the derivative. (Malabarba, A et al. J. Antibiotics , 37 : 989-999 (1984), Malabarba, A et al. J. Antibiotics , 39 : 1430-1442 (1986)).

The technical problem to be achieved by the present invention is to isolate and develop the mutant strains of Etinoplanes teichomy- ceticus microorganism producing high yields of Tecoplanin , a Gram-positive bacteria treatment, by optimizing the culture conditions The purpose is to improve the productivity of teicoplanin.

Figure 1 is a graph showing the change in the concentration of sugar and the production of teicoplanin over time in the 7L fermentor.

Therefore, an object of the present invention is artificially mutated wild-type Etinoplanes Teicomyceticus ATCC-31121 using ultraviolet light, and then smeared and grown cultured mutant strains generated by staphylococcus aureus did not grow around the mutant strains Among strains in which the growth zone is larger than the parent strain or the growth inhibition ring is significantly larger than the size of the fungi, the strains that produce the most teicoplanin are selected and mutated by using ultraviolet rays. Isolation method of teicoplanin-producing strain Etinonones teicomyceticus M76-35-7 (Accession No. KCCM-10363) characterized by screening for strains producing excessive amounts of coplanin and isolated tei Coplanin production mutant Etinoplanes teicomyceticus M76-35-7 (Accession No. KCCM-10363).

In addition, after species culture of Etinoplanes teicomyceticus M76-35-7 (Accession No. KCCM-10363), growth was carried out containing 10-30 g / L of glucose, 2-6 g / L of yeast extract, and inorganic salts. After growing in a medium, the method for fermentation and production of teicoplanin in high yield by growing in a production medium containing 50-150 g / L dextrin, 3-7 g / L yeast extract, potato protein, soy flour and inorganic salts. to provide.

At this time, the species culture is characterized in that the initial concentration is 30 to 50g / L, the concentration of glucose is maintained at 10 to 15g / L, the composition of the production medium is 80 ~ 150g / L dextrin, 3 ~ yeast extract It is characterized by containing nitrogen sources and inorganic salts such as 7g / L, potato protein 12-24g / L, soy flour 12-24g / L, corn steep liquor, cotton seed flour.

Hereinafter, the present invention will be described in more detail.

The present invention provides Teicoplanin overproduction strain Etinonones teicomyceticus M76-35-7 (Accession No. KCCM-10363). Meanwhile, the mutant strains were mutated by wild-type Etinoplanes teicomyceticus ATCC-31121 using UV to isolate the mutant strains, and the mutant strains were selected by artificial mutants using UV again. . In addition, the strain Etinonones Teicomyceticus M76-35-7 (Accession No. KCCM-10363) cultured in a growth medium was cultured and fermented by adjusting the type of nitrogen source, type and concentration of carbon source. It is characterized in that the production of flan, wherein the growth medium is a medium containing glucose 20g / L, yeast extract 4g / L, peptone 4g / L, potassium monophosphate 2g / L, potassium diphosphate 4g / L and glucose in the medium Shake culture until a concentration of about 10 g / L remained. Production medium includes 80g / L dextrin, 18g / L soy flour, 18g / L potato protein, 5g / L yeast extract, 0.5g / L magnesium sulfate, 1.2g / L sodium chloride, 0.1g / L calcium chloride, 5g / L calcium carbonate. Containing medium.

UV, which is a mutagen to the parental Etinoplanes teicomyceticus strain, was treated for 40-60 seconds to the spores of the strain. After 2 hours of cancer reaction, the mutant strains were grown on screened agar medium for screening strains that produce higher teicoplanin than wild strains, and cultured until spores were grown at 30 ° C.

Ingredient Scheme of the Selection Medium ingredient density(%) dextrin 2.0 Yeast extract 0.5 Magnesium Sulfate (MgSO ₄ · 7H ₂ O) 0.05 Sodium Chloride (NaCl) 0.01 Calcium chloride (CaCl ₂ ) 0.01 Agar 2

In order to select strains that overproduce teicoplanin among the strains cultured in the selection medium, Staphylococcus aureus was cultured in a liquid medium and plated on the grown mutant strain, and then grown at 37 ° C to grow Staphylococcus bacteria around the mutant strain. Failed to select the strains produced growth inhibition ring (clear zone) is larger than the parent strain or significantly larger growth inhibition ring compared to the size of the colony. After mutating the selected strain to UV three times, the strain that overproduces teicoplanin was finally selected.

Culture method and analysis method of mutated strain isolated in the present invention is as follows.

Cultivation of the mutant strain of the present invention was incubated for 7-8 days at 30 ℃, 200rpm. The teicoplanin concentration of the fermentation broth was calibrated to pH 11 with sodium hydroxide (5M NaOH) solution to remove the cells, and then filtered with a 0.45㎛ filtration membrane followed by a reversed phase column (C ₁₈ , Zorbox, Japan) and UV detector. Measurement was performed using HPLC (Hewlett Packard 1100 series, USA) equipped with a detector (254 nm). In this case, the mobile phase is a solution in which 0.2% formic acid and acetonitrile are mixed in a 9: 1 solution (A solution) and 0.2% formic acid and acetonitrile in a 3: 7 solution (B solution). Was used in combination, and the flow rate was 1 mL / min.

Morphological, Physiological and Biochemical Properties of the Mutant Etinoplanes teichomyceticus ATCC 31121 and Inventive Variation Etinonones teicoomyceticus M76-35-7 Showing High Teicoplanin Productivity The characteristics are shown in Table 2 below.

Morphological, Physiological and Biochemical Properties of the Inventive Mutant Etinoplanes Teicomyceticus M76-35-7 characteristic Kinju (Etino Planes teikomaisetikkusu ATCC31121) Variation strain of the present invention (Etinoplanes teicomyceticus M76-35-7) Cell shape Mycelium (filamentous) Mycelium (filamentous) Color of fungi orange color orange color Soluble pigment - - Oxygen requirements Aerobic Aerobic Growth of bacteria by temperature 20 ℃ + + 30 ℃ + + 33 ℃ + + 40 ℃ - - Sulfuric acid formation + + Melanin formation + + Tyrosine digestion - - Starch decomposition + +

Use of the carbon source of the present invention mutant Etinoplanes teicomyceticus M76-35-7 Carbon source Proliferation Chinju (Etinoplanes Teikomaisetikkusu ATCC31121) Variant strain (Etinoplanes teikomaisetikkusu M76-35-7) glucose + + Glycerol + + Galactose + + Fructose ± ± Sucrose + + Lactose ± + Maltose + + Sorbitol + + Inositol - - Xylose + + dextrin + ++ Mannos + + Mannitol + +

Hereinafter, the present invention will be described in detail as follows.

(Example 1) Teicoplanin productivity comparison experiment of the parent strain and the present invention mutant strain in the Erlenmeyer flask

Teicoplanin productivity of the selected strains was compared under the following conditions. Each of the selected strains was inoculated into a 100 ml Erlenmeyer flask containing 10 ml of production medium (Table 4) and incubated at 200 rpm, 30 ° C. for 7-8 days. The culture solution was calibrated to pH 11 by adding sodium hydroxide, and the cells were removed and analyzed by HPLC to investigate the yield of teicoplanin. The results are summarized in the table below.

Ingredient composition table of production medium ingredient Concentration (g / L) dextrin 80 Yeast extract 5 Soy flour 18 Potato protein 18 Magnesium Sulfate (MgSO ₄ · 7H ₂ O) 0.5 Sodium Chloride (NaCl) 1.2 Calcium chloride (CaCl ₂ ) 0.1 Calcium Carbonate (CaCO ₃ ) 5

Teicoplanin Productivity of Parent and Mutant of the Invention Strain Teicoplanin (mg / L) Parent (ATCC31121) 50 Variant of the invention M76-35-7 514

Example 2 Teicoplanin Fermentation Production Test (Teicoplanin Productivity Experiment on Carbon Source Type)

The culturing method is the same as in Example 1, and the teicoplanin production experiment of the present invention mutant Etinoplanes teicomyceticus M76-35-7 according to the type of carbon source in a 100 mL Erlenmeyer flask. Several kinds of carbon sources were added and incubated at 30 ° C. and 200 rpm for 7 to 8 days. Experimental results of different types of carbon sources were shown in Table 6, and showed the optimum productivity when dextrin was used as the carbon source.

Effect of Carbon Source on Teicoplanin Production Using Mutant Etinoplanes Teicomyceticus M76-35-7 Carbon source Teicoplanin (mg / L) dextrin 514 Fructose 337 Galactose <125 glucose <125 Lactose 167 Maltose 447 Mannos 145 Sorbitol <125 Starch 167 Sucrose 192

Example 3 Teicoplanin Fermentation Production Test (Teicoplanin Productivity Test According to Dextrin Concentration)

Culture method is the same as in Example 1, the initial concentration of dextrin concentrations of 80g / L, 100g / L, 120g / L, 150g / L and incubated as shown in Table 7 and the optimum productivity at 150g / L dextrin concentration appear.

Effect of Initial Dextrin Concentration on Teicoplanin Production Using Etinonones Teicomyceticus Mutants Dextrin concentration (g / L) Teicoplanin (mg / L) 80 490 100 564 120 538 150 655

Example 4 Teicoplanin Fermentation Production Test According to Various Nitrogen Sources Using Etinonoless Teicomyceticus Mutant

Teicoplanin production experiments of the present invention mutant strains were performed according to the type of nitrogen source. The culture method is the same as in Example 1, the production medium (dextrin 80g / L, calcium chloride 0.1g / L, sodium chloride 1.2g / L, magnesium sulfate 0.5g / L, calcium carbonate 5g / L, soy flour 18g / L, potato protein 18g / L, yeast extract 5g / L) was added to 0.5% of various types of nitrogen sources, and the results were as shown in Table 8, and the highest teicoplanin production was obtained when incubated with the addition of cotton seed flour and corn steep liquor. .

Effect of Nitrogen Sources on Teicoplanin Production Using Etinoplanes Teicomyceticus Mutants Nitrogen source Teicoplanin (mg / L) percentage(%) T-A2-2 T-A2-3 T-A2-4 T-A2-5 not-added 555 64.6 10.6 13.0 11.9 corn steep liquor 968 61.3 10.9 13.8 14.1 Corn steep solid 384 54.5 17.5 18.4 9.6 Cotton seed flour 1051 66.3 10.3 11.2 12.1 Fishmeal 702 64.5 17.6 9.4 8.5 Gluten meal 629 53.7 19.6 14.1 12.6 HVP 929 59.2 17.3 12.0 11.4 NH ₄ Cl ^-a - - - - (NH ₄ ) ₂ SO ₄ ^-a - - - - Wheat meal 898 62.3 11.6 11.6 14.4

^{-a not} detected

Example 5 Teicoplanin Production Test According to Temperature Change

Teicoplanin production experiments of the mutant Etinoplanes teicomyceticus M76-35-7 according to various culture temperatures in 7L fermenter were performed.

Species culture: A single colony of Etinoplanes teicomyceticus was inoculated into a 500 ml flask containing 25 ml of preculture medium, incubated for 48-60 hours at 250 rpm and 30 ° C in a shaker, followed by 12.5 ml of the culture solution. Inoculated into a 5L fermenter containing 2.5L species culture medium was incubated at 28 ℃, 900rpm for 60 to 72 hours. When 10 g / L of glucose remained in the seed culture, 230 ml of the seed culture solution was inoculated into a 7 L fermenter containing 4.6 L of the production medium and incubated at 900 rpm for 7 to 8 days.

Ingredient Scheme of Pre-Culture Medium ingredient Concentration (g / L) glucose 20 Yeast extract 4 peptone 4 Potassium monophosphate (KH ₂ PO ₄ ) 2 Potassium Diphosphate (K ₂ HPO ₄ ) 4 Magnesium sulfide (MgSO ₄ .7H ₂ O) 0.5

Component scheme of species culture medium ingredient Concentration (g / L) glucose 30 Yeast extract 5 Soy flour 18 Potato protein 18 Sodium Chloride (NaCl) 1.2 Calcium chloride (CaCl ₂ ) 0.1 Magnesium sulfide (MgSO ₄ .7H ₂ O) 0.5 Calcium Carbonate (CaCO ₃ ) 5

Main culture: The main culture was performed in a 7L fermenter (Kobiotech Co., Korea). In culture, the production medium containing sugar and yeast extract was 4.6L, and the initial stirring speed and aeration rate were adjusted to 900rpm and 1vvm, respectively. pH was not adjusted during the entire fermentation process. Experimental results of different incubation temperatures were shown in Table 12 and showed optimum production when incubated at 33 ° C.

Ingredient composition table of production medium ingredient Concentration (g / L) glucose 20 dextrin 60 Yeast extract 5 Soy flour 18 Potato protein 18 Sodium Chloride (NaCl) 1.2 Calcium chloride (CaCl ₂ ) 0.1 Magnesium sulfide (MgSO ₄ .7H ₂ O) 0.5 Calcium Carbonate (CaCO ₃ ) 5

Effect of Culture Temperature on Teicoplanin Production Using Etinonones Teicomyceticus Mutants Temperature (℃) Cell (%) Teicoplanin (mg / L) 25 39 584 28 40 1347 33 35 1549

Example 6 Teicoplanin Production Test under Optimized Conditions

Teicoplanin production experiments of the mutant Etinoplanes teicomyceticus M76-35-7 of the present invention were carried out in a medium and temperature optimized in a 7L fermentor. Pre-culture and species culture were the same as in Example 5 and carried out the main culture in 7L fermenter (Kobiotech Co., Korea). The composition of the production medium was added to 15 ml / L corn steep liquor in Table 11 (Table 13). In the culture, the production medium containing sugar and yeast extract was 4.6L, and the initial stirring speed and aeration rate were adjusted to 900rpm and 1vvm, respectively, and cultured at 33 ° C. pH was not adjusted during the entire fermentation process. Experiments in the optimized medium and temperature were as shown in Figure 1 and produced 2.5g / L of teicoplanin.

Composition Chart of Optimal Production Medium ingredient Concentration (g / L) glucose 20 dextrin 60 Yeast extract 5 Soy flour 18 Potato protein 18 Sodium Chloride (NaCl) 1.2 Corn steep liquor 15 ml Calcium chloride (CaCl ₂ ) 0.1 Magnesium sulfide (MgSO ₄ .7H ₂ O) 0.5 Calcium Carbonate (CaCO ₃ ) 5

The effect of the present invention is to develop a strain and a fermentation process that significantly increased the production of antibiotic teicoplanin, a Gram-positive bacterium therapeutic agent, which is dependent on the total amount of imports, and can be used industrially to ferment and produce teicoplanin at low cost and high yield. It can have a comparative advantage in import substitution effect and international competitiveness.

Claims (2)

After artificially mutating the wild strain Etinoplanes teicomyceticus ATCC-31121 using ultraviolet rays, smearing the grown mutant strains to prevent growth of staphylococci around the mutant strains (clear zone) Strains that produce the largest amount of teicoplanin among the strains that are larger than the parent strain or the growth inhibitory ring is significantly larger than the size of the parent strain, and then mutated using UV light to overproduce teicoplanin Isolation method of teicoplanin-producing strain Etinonones teicomyceticus M76-35-7 (Accession No. KCCM-10363) characterized by selecting the strain to be Teicoplanin-producing variant strain Etinoplanes teicomyceticus M76-35-7 isolated according to the method of claim 1 (Accession No. KCCM-10363)