KR100436309B1 - Paenibacillus polymyxa cby having enhanced productivity of colistin and process for preparing same - Google Patents

Abstract

The present invention relates to an improved productivity of colistin, Paenibacillus polymyxa CBY (No. KCTC 18098P) and a method for preparing the same, UV, Nitrozoguanine (NTG) or Penivacillus polymyx CBY, prepared by mutations by treatment with ethylmethane sulfonate (EMS), exhibits colistin productivity at least 100-fold higher than wild strains.

Description

PARISBACILLUS POLYMYXA CBY HAVING ENHANCED PRODUCTIVITY OF COLISTIN AND PROCESS FOR PREPARING SAME

The present invention relates to a mutant strain Paenibacillus polymyxa (CBY) CBY and a method for producing the same, which has improved productivity of antibiotics for feed addition colistin.

Colistin is a peptide-based antibiotic produced from Bacillus polymyxa and was first reported to have antimicrobial activity against Gram-negative bacteria in 1950 (Koyama Y. et al., J. Antibiotics, 3 , 457, 1950). It was used to treat infections caused by Gram-negative bacteria such as Shigella, Escherichia coli, Pseudomonas aeruginosa, and Salmonella, and was developed in 1952 as an antibiotic for animals in Japan and industrialized (Lee Jang Lak, Animal Drug Commentary , 272 , Seoul National University Press.

Colistin has the structure of Formula 1.

As shown in Formula 1, colistin has a basic structure of 6 mol of L-α, γ-diaminebutyric acid (DAB), 2 mol of L-threonine, and 1 mol of L-leucine and D-leucine, respectively. 6-methyloctanoic acid or isooctanoic acid binds to DAB at the terminal of the basic structure to form colistin A and colistin B, respectively.

Research on colistin is the most active in Japan. In 1963 the structure of colistin was revealed and in 1969 the biosynthetic pathway of colistin was partially identified (Suzuki T. et al ., J. Biochem ., 54 , 25 (1963); Suzuki, T. et al ., J. Biochem . , 54 , 412 (1963); Suzuki, T. and Fujikawa, K., B, J. Biochem ., 56 , 2, 182-189 (1964)). In 1962, optimal culture conditions for colistin production were established (Morito, T., Nippon Nogeikagaku Kaishi, 36 , 18 (1962); Morito, T., Nippon Nogeikagaku Kaishi, 36 , 24 (1962)). , Bacillus colistinus Koyama and Bacillus polymyxa var. Colistin was produced using KY-7584 (Morito, T., Nippon Nogeikagaku Kaishi, 36 , 18 (1962); Suzuki, T., J. Ferment. Technol, 60. 60. 603-606 (1982)) .

Currently, all of the colistin products distributed in Korea are imported from Japan and China, and they are commercialized. As the demand for colistin increases, the price burden on raw material imports increases. In addition, although the market size of colistin is increasing year by year, there is no domestic production of colistin.

Therefore, the inventors of the present invention have phenomena which improve the productivity of colistin by mutating the penivacillus polymix ATAT 21830 alone or in combination with mutants such as UV, NTG, and EMS in order to obtain a mutant with improved productivity of colistin. The present invention was completed by screening Bacillus polymyc CBY and using this to establish a method for mass production of colistin.

It is an object of the present invention to provide mutant Paenibacillus polymyxa CBY with improved productivity of colistin.

It is another object of the present invention to provide a process for producing Phenibacillus polymix yarn CBY.

Still another object of the present invention is to provide a method for mass production of colistin using a Penivacillus polymix CBY strain.

Figure 1 is a schematic of the development process of Penicillus polymyc CBY with improved productivity of colistin,

Figure 2 shows the results of the high performance liquid chromatography of the Penivacillus polymix CBY culture medium,

Figure 3 shows the results of high performance liquid chromatography of colistin standard solution,

Figure 4 shows the results of the analysis of the culture pattern in a 5 L stirring fermenter of the penivacillus polymix CBY by time,

Figure 5 shows the results of time-dependent analysis of the culture pattern in a 5 L stirred fermenter of Penibacillus polymix ATAT 21830.

The Paenibacillus polymyxa CBY of the present invention is obtained by treating and mutating a penicillus polymyx ATCC 21830 with a mutagen. What can be used as a mutagen in the present invention can be exemplified UV, NTG and EMS, these are treated alone or in combination so that the killing rate of the strain is 99% or more.

When treated with mutagens alone, mutations by UV irradiation irradiated with 254 nm ultraviolet rays for 25 seconds at a distance of 35 cm, and mutations by NTG or EMS resulted in NTG or EMS with a final concentration of 0.02 mg / ml, respectively. Then, the mixture was shaken at 30 ° C. for 0.5 to 1.5 hours, and then washed several times with brine.

The combined treatment was a combination of NTG, EMS or ultraviolet irradiation. NTG or EMS was shaken for 0.5 to 1 hour with the final concentration of 0.02 to 0.1 mg / ml, preferably 0.05 mg / ml, and the UV treatment was performed for 10 to 15 seconds, preferably in the same manner as the single treatment. Irradiate for 12 seconds.

Bacteria treated with the mutagen are plated in Bennett agar medium and incubated for 48 hours to obtain mutant strains that have formed colonies. At the same time, the parent strain, Penivacillus polymyx ATCC 21830, is cultured in the same medium. Then, 0.2 ml of the cultured E. coli (ATCC 8739) cultured in Bennett agar medium for 24 hours at 37 ℃ sterilized and diluted with 5 ml of agar medium maintained at 50 ℃ and stratified for 24 hours at 37 ℃ Incubate for a while. By comparing the size of the low-ring ring formed by the mutant strain and the parent strain, all colonies with a large size of the low-ring ring are selected.

To examine the colistin productivity of the selected mutant strains, each mutant strain is inoculated into a liquid medium and incubated at 28 to 32 ° C., preferably at 30 ° C. for 24 hours. Take 0.1 to 0.5 ml, preferably 0.2 ml of the culture broth, and take 30 ml of the main fermentation medium (2-4% jade, 5-8% starch, 1% soybean oil, 1-3% ammonium sulfate) in a 500 ml flask. 0.1-0.5% potassium dihydrogen phosphate, 0.01-0.05% iron sulfate, 0.5-2% calcium carbonate, 0.05-0.8% antifoam, pH 7.2) and incubator with stirring at 200-300 rpm, preferably 250 rpm Shake culture for 3 days at 28 to 32 ℃, preferably 30 ℃. After diluting 1 ml of the culture and centrifuging, HPLC was performed on a C18 column with 0.05 M Na ₂ SO ₄ : CH ₃ CN mixed solution (76:24 (v / v)) to measure colistin content. All strains with colistin productivity greater than 4 g / l are selected.

Subsequently, in order to select the most mutant strains with the highest productivity of colistin in a stirred fermenter among the mutated strains, penivacillus polymyx mutant strains were respectively inoculated in the same main fermentation medium as the culture medium in the flask culture, and the culture solution The starch is incubated with the addition of starch so that the sugar concentration in the mixture is maintained at 1%. The pH of the culture is 5.5-6.0, the aeration rate is maintained at 1.0-1.5 vvm, and the stirring speed is gradually increased starting at 500 rpm to maintain the dissolved oxygen concentration at 40%. The culture temperature is initially maintained at 30 ± 0.2 ℃, when the strain concentration is maximized to 22 to 25 ℃ to maximize the productivity of colistin.

After diluting the culture solution, the supernatant obtained by centrifugation was filtered and subjected to high performance liquid chromatography (HPLC) to measure the amount of colistin contained in the sample. Bacillus polymix was named CBY. The colistin productivity by this strain is about 10 g / l, which is about a 100-fold increase compared to the productivity of about 0.1 g / l of the parent strain Penivacillus polymyx ATCC 21830.

Colistin produced in large quantities by the penivacillus polymyx CBY of the present invention can be used as an antibiotic for feed addition.

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

Reference Example 1: Determination of total sugar concentration in culture

To measure the starch concentration in the culture, 1 ml of culture and 10 ml of 18% HCl solution were added to the glass tube, boiled at 100 ° C. for 15 minutes, and cooled at room temperature. 10 ml of 6N NaOH was added thereto to neutralize the mixture, and distilled water was added to make a final volume of 50 ml and mixed well. The mixed solution was dispensed into 1.5 ml microtubes by 1 ml and centrifuged at 13,000 rpm for 5 minutes. The supernatant was taken and transferred to a small glass tube, to which 1 ml of 1% DNS reagent was added and boiled at 100 ° C. for 5 minutes. Cool with cold water and dilute with distilled water 10 ml.

The absorbance of this mixture was measured at 540 nm using a spectrophotometer, and glucose solutions of 0, 0.2, 0.4, 0.8, and 1.2 g / l were used as standard samples.

Reference Example 2: Determination of Colistin Content

In order to measure the content of colistin in the culture, 1 ml of the culture was diluted 5 to 40 times with distilled water and centrifuged, and the supernatant was filtered using a 0.45 μm syringe filter (Satorius). Prepared. After injecting the sample into HPLC (Waters alliance 2690. PDA 996) equipped with a C18 column (ODS-BP, DAISO), 0.05 M Na ₂ SO ₄ : CH ₃ CN mixture (76:24 (v / v)) was flowed at a flow rate of 1.0 ml / min, and the eluate was detected at 215 nm to measure the colistin content contained in the sample. The standard sample used colistin sulfate (Meiji, Japan). It was.

Example 1: Selection of Mutant

Step 1

Penibacillus polymix ATAT 21830 was inoculated in a liquid medium (0.2% yeast extract, 0.7% beep extract, 1% peptone, 0.3% NaCl) and incubated for 24 hours at 30 ℃. The cultures were plated in Bennet agar medium (0.1% yeast extract, 0.1% beef extract, 0.2% NZ amine type A, 1% glucose, 1.8% agar) and incubated for 24 hours at 30 ° C. Strain CS-2 with excellent growth potential of 0.2 mm in diameter was selected.

Step 2

The CS-2 strain was cultured in liquid as in step 1, and the cells obtained by centrifugation of 1 ml of this culture were suspended in 9 ml of 0.1 M phosphate buffer (pH 7.0), and then the ultraviolet light, which is a mutagen, in the suspension as described below. NTG and EMS, respectively, were treated alone to kill more than 99% of CS-2 strains.

Mutations caused by ultraviolet radiation were irradiated with 254 nm ultraviolet rays for 25 seconds at a distance of 35 cm, and mutations caused by NTG or EMS were shaken at 30 ° C. for 60 minutes by adding NTG or EMS at a final concentration of 0.02 mg / ml, respectively. After incubation, the solution was washed several times with saline. In order to prevent reverse mutations due to photoactivation of the mutant strains, the treatment of UV-treated samples was carried out in the dark room using red light.

The bacterial solution obtained by treating each mutagen alone was plated in Bennett agar medium and incubated at 37 ° C. for 48 hours to obtain colonies. Escherichia coli (ATCC 8739) was added to this medium in a liquid medium (0.15% beef extract, 0.15% yeast extract, 5% peptone, 0.1% glucose, 0.4% NaCl, 0.4% K ₂ HPO ₄ , 0.1% KH ₂ PO ₄ ). Sterilize 0.2 ml of the culture solution obtained by incubating for 24 hours at ℃, and dilute with 5 ml of agar medium (0.15% beef extract, 0.3% yeast extract, 0.6% peptone, 1.5% agar) maintained at 50 ° C. The strain CS-3 having a diameter of about 8 mm was obtained by incubating for 24 hours at.

Step 3

CS-3 strains were treated with NTG or EMS to a final concentration of 0.05 mg / ml and shaken for 0.5 to 1 hour, and UV treatment was carried out with a double combination of mutagens by irradiation for 12 seconds in the same manner as single treatment. After the culture in the same manner as in step 2 to obtain a strain CS-4 strain of about 10 mm in diameter of E. coli.

Step 4

CSG strains were treated with NTG and EMS to the final concentration of 0.05 mg / ml in the same manner as single treatment and shaken incubated for 0.5 to 1 hour. Was treated in combination in triplicate, and CBY was selected from among the strains having an E. coli low-ring ring diameter of about 14 mm.

Example 2: Determination of Colistin Production in Selected Mutant strains

In order to investigate the colistin productivity of all the mutant strains selected in Example 1, the mutated strains were respectively inoculated in a liquid medium (0.2% yeast extract, 0.7% beef extract, 1% peptone, 0.3% NaCl) and at 30 ° C. Incubated for 24 hours. Take 0.2 ml of the culture broth, add 30 ml of main fermentation medium (4% corn meal, 5% starch, 1% soybean oil, 3% (NH ₄ ) ₂ SO ₄ , 0.2% dihydrogen phosphate in 500 ml flask). Potassium, 0.02% iron sulfate, 2% calcium carbonate, 0.8% antifoaming agent (Konix-pp, Polyol Korea Co., Ltd.), pH 7.2) and incubated for 3 days at 30 ℃ while stirring at 250 rpm in the incubator. 1 ml of the culture was used to measure colistin content as in Reference Example 2 to select all strains having colistin productivity of 4 g / l or more.

Example 3: Establishment of strain selection and fermentation conditions by agitated fermentor culture

Colistin productivity in stirred fermenters of mutant strains excellent in colistin productivity obtained in Example 2 was measured as follows. 0.2 ml of the cultures of the selected mutants were inoculated in 30 ml of liquid medium (0.2% yeast extract, 0.7% beef extract, 1% peptone, 0.3% NaCl) and incubated at 28 ° C. for 20 hours to activate the bacteria. 1 ml of each culture was taken and inoculated in a 5 L fermenter containing 3 L of the main fermentation medium, and cultured with starch added in a fed-batch manner to maintain the sugar concentration in the culture at 1%. The pH of the culture was maintained at 5.5 to 6.0 by adding 25% NH ₄ OH, the aeration amount was 1.0 to 1.5 vvm, and the stirring speed was maintained to maintain the dissolved oxygen (DO) concentration at 40% or more. Gradually increased starting at 500 rpm. The incubation temperature was initially maintained at 30 ± 0.2 ℃, and when the strain concentration reached the highest, that is, when the cell count was 10 billion / ml when directly examined using a hemocytometer, the culture temperature was lowered to 22 ℃ to increase the productivity of colistin. Maximized. A certain amount of sample was taken during the culture to measure the total sugar concentration and the content of colistin in the fermentation broth according to the methods of Reference Examples 1 and 2 ( FIG. 4 ).

Through this experiment, the optimum culture conditions were established, and the best strain of colistin productivity was finally selected and named as Penivacillus polymixa CBY. In 2001, the Korea Biotechnology Research Institute, Gene Collection Bank (KCTC) Deposited on November 29, Accession No. KCTC 18098P.

Figure 4 shows the change of pH, DO, starch and colistin content with the incubation time of the penivacillus polymix CBY. As can be seen in Figure 4 , the strain of the present invention has a maximum productivity of 10 g / l significantly improved productivity compared to 0.1 g / l of the penicillus polymix yarn ATCC 21830 of FIG .

The present invention relates to the penivacillus polymix C CBY and a method for manufacturing the same, and the use of the penivacillus polymix c CBY strain can produce large amounts of colistin, which will greatly contribute to the localization of colistin and development of animal husbandry.

Claims (6)

Paenibacillus polymyxa mutant CBY (KCTC 18098P) with enhanced productivity of colistin. A method for producing Penibacillus polymix CBY (KCTC 18098P), characterized in that the phenibacillus polymix ATAT 21830 is mutated alone or in combination with a mutation selected from the group consisting of ultraviolet, NTG and EMS. The method of claim 2, And culturing the mutant strain obtained by mutating by the mutagen in agar medium, and then screening for E. coli to select strains having excellent low-ring production. A method for producing colistin, characterized by culturing Penibacillus polymyc CBY (KCTC 18098P) in a suitable medium. The method of claim 4, wherein The medium production method of colistin, characterized in that the medium comprises jade, starch, vegetable oil, ammonium sulfate, potassium dihydrogen phosphate, iron sulfate, calcium carbonate and antifoaming agent. The method according to claim 4 or 5, Inoculate Phenibacillus polymyx CBY into the fermentation broth, add starch to the culture medium in a fed-batch formula and maintain the sugar concentration at 1%, while maintaining the pH of the culture medium at 5.5 to 6.0 and the aeration rate at 1.0 to 1.5 vvm, After culturing at 30 ± 0.2 ℃ while maintaining the concentration at 40%, the mass production method of colistin characterized in that the culture temperature is lowered to 22 to 25 ℃ when the concentration of the cells is maximum.