KR100450562B1 - Process for preparing pravastatin precursor and its derivatives - Google Patents

Abstract

The present invention relates to a method for producing pravastatin precursor, and more particularly, to obtain a strain having excellent ability to produce pravastatin precursor by mutating known penicillium citrinum ( Penicillium citrinum ), and aerobic culturing this to hyperlipidemia A method of producing provastatin precursors useful as therapeutic agents.

Description

Process for preparing pravastatin precursor and its derivatives

The present invention relates to a method for producing pravastatin precursor, and more particularly, to obtain a strain having excellent ability to produce pravastatin precursor by mutating known penicillium citrinum ( Penicillium citrinum ), and aerobic culturing this to hyperlipidemia A method of producing provastatin precursors useful as therapeutic agents.

Hyperlipidemia, especially hyperlipidemia, is known to be one of the main causes of heart disease such as myocardial infarction or arteriosclerosis. Therefore, we have tried to find compounds that can lower cholesterol levels. As a result, penicillium sp. ML-236B, a substance capable of lowering cholesterol concentration, was isolated (US Pat. No. 3,983,140 and UK Pat. No. 1,453,425).

This ML-236B is called comppactin as a pravastatin precursor and is known to be produced using soil microorganisms or through chemical conversion. Specifically, Compactin is Penicillium brevicompactin ( Penicillium brevicompactin ), Penicilmyses sp. , Tricoderma longibraiatum , Tricodedma pseudokoning , It is disclosed in Japanese Patent Publication No. 4-349034, which can be produced by culturing Hyphomyces chrisopomus and Penicillium citrinum , Gliocladium sp. It is disclosed in Korean Patent No. 186758 that YJ-9515 (KCTC 02528BP) can be produced by aerobic culture.

However, the production method of Compactin using the above-mentioned microorganisms has a long main culture time and a very low production rate per unit liquid due to the characteristics of the strain, thereby lowering economic efficiency.

The present inventors have found that this is obtained by a mutation treatment to a result Penny room Solarium sheet rinum ATCC 18199 research to find a way to solve the above problems strain to produce comb paktin at a high concentration, the present invention It was completed.

Accordingly, it is an object of the present invention to provide a mutant strain capable of producing high concentrations of gompaktin.

Another object of the present invention is to provide a method for producing the comvatin and the derivatives of pravastatin precursor using the above mutant strain.

In order to achieve the above object, characterized in that the present invention provides a comb paktin and mutant master penny chamber penny room Solarium sheet rinum producing derivatives thereof Solarium sheet rinum KBT 1100 (accession number: KCCM-10316) with a high productivity do.

In order to achieve the other object, the present invention is characterized by providing a method for producing a compantin and derivatives thereof by aerobic culture of the mutant strain.

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

The strain of the present invention is a mutant strain having a high productivity of Compactin of about 3,850 mg / L, and is produced by inducing mutations in the known penicillium citrinum ATCC 18199.

In other words, the parent strain Penicillium citrinum ATCC 18199 was irradiated with UV light, followed by mutagenesis of the selection medium (bacto peptone 10g / l, soybean powder 10g / l, sodium nitrate 2g / l, magnesium salt 1g / l). , Glucose 30g / L, Glycerol 70g / L, Compactin 2g, Agar 15g / L), cultured to give preferentially 25 colonies resistant to Compactin, and then incubated these liquids to high concentrations of Compactin Strains to produce were selected.

The strain was named Penicillium citrinum KBT-1100 strain and was deposited with the Korean spawn association on September 20, 2001 and was given accession number KCCM-10316.

Using the penicillium citrinum KBT-1100 strain (KCCM-10316) of the present invention is a method for producing compactin.

Penicillium citrinum KBT-1100 strain (KCCM-10316) in a preculture medium having a composition of 10 g / l bacterium peptone, 20 g / l soybean powder, 2 g / l sodium nitrate, 1 g / l magnesium salt and 100 g / l sucrose ), And incubated for 1 to 2 days in a rotary shaker at 150 to 200 rpm, 25 to 28 ° C., and then cultured strains were 10 g / l bactopeptone, 30 g / l soybean powder, and 3 g / l sodium nitrate. , Aseptically inoculated into this culture medium (pH 6.0) having a composition of 1 g / l magnesium salt and 100 g / l sucrose, incubated for 3 days under the same conditions as the species culture, and further incubated for 9 days with the addition of 50% glycerin solution. To produce compactin.

Compactin can be obtained from the culture medium described above by filtration and purification using a conventional method.

Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to these examples.

That is, Streptomyces acid to bopil Russ FERM by the BP-1145 mutation treatment with UV screening the improved strain productive of pravastatin sodium, which in naming and Korea Culture Center of Microorganisms as Streptomyces carboxylic bopil Russ (Streptomyces carbophilus) KBT229 Deposited September 20, 2001, the instrument was assigned accession number KCCM-10317.

The parent strain Penicillium citrinum ATCC 18199 was incubated to the growing log phase and centrifuged under conditions of 4,000 rpm and 15 minutes to obtain supernatant cells. 8 ml of 0.85% saline was added to the cells to suspend the cells. The cells were added to sterile glass petri dishes and irradiated with UV to induce mutations. At this time, UV was irradiated for 20 seconds, UV-protected petri dishes wrapped with foil to prevent photoreaction after UV irradiation, and then dark reaction in the refrigerator for 2 hours.

Then, after taking a sample from the petri dish, the selection medium (bakto peptone 10g / l, soybean powder 10g / l, sodium nitrate 2g / l, magnesium salt 1g / l, glucose 30g / l, glycerol 70g / l, 2 g of compactin, agar 15 g / l) was plated with platinum, and cultured to give priority to the 25 strains showing compactin resistance.

Example 2 Isolation of High Potency Production Strains

250 ml Baffle was used to prepare 50 ml of preculture medium (pH 6.0) having a composition of 10 g / l bactopeptone, 20 g / l soybean powder, 2 g / l sodium nitrate, 1 g / l magnesium salt and 100 g / l sucrose. After injection into a flask, which was autoclaved at 121 ° C. for 30 minutes, inoculated with the 25 compaktin resistant strains isolated in Example 1, and the inoculation flask was rotated at 25 to 28 ° C. at 150 to 200 rpm. Incubated for 1-2 days at.

Next, a 500 ml baffle flask with 100 ml of this culture medium (pH 6.0) having a composition of 10 g / l bactopeptone, 30 g / l soybean powder, 3 g / l sodium nitrate, 1 g / l magnesium salt and 100 g / l sucrose It was inoculated in, and sterilized for 30 minutes at 121 ℃ sterilized inoculated to 5% of each strain cultured in the whole culture medium, and then incubated for 3 days under the same conditions as the species culture. Then, 50% glycerin solution was added to 20% (v / v) of the culture medium and further incubated for 9 days.

The yield of compactin and derivatives contained in the culture of each mutant strain was quantified using HPLC, and the results are shown in Table 1. HPLC quantification was performed using C ₁₈ (Symetric) at 25 ° C. with solvent conditions of 77% methanol, 0.1% acetic acid, 0.1% trimethylamine, 22.8% water, and analyzed at a flow rate of 1 ml / min and a wavelength of 238 nm. It was.

Strain Compactin Production (mg / ℓ) pH PMV (%) Mutant strain 1 3850 6.30 36 Mutant 2 1280 6.77 34 Mutant 3 993 7.14 32 Mutant strain 4 1061 6.22 33 Mutant 5 1183 5.98 37 Mutant strain 6 1404 5.64 32 Mutant strain 7 2653 6.50 32 Mutant strain 8 2300 6.10 34 Mutant 9 1864 5.84 30 Mutant 10 2103 5.90 32 Mutant strain 11 2862 6.30 34 Mutant strain 12 3216 6.87 36 Mutant 13 864 6.40 28 Mutant strain 14 2740 6.54 32 Mutant 15 1957 5.97 32 Mutant 16 3510 6.87 34 Mutant 17 2600 6.32 30 Mutant 18 3760 6.20 32 Mutant strain 19 3597 6.74 36 Mutant 20 2156 6.10 32 Mutant 21 3625 6.45 36 Mutant strain 22 3720 6.25 34 Mutant 23 2950 6.47 36 Mutant 24 2735 6.32 32 Mutant 25 3254 6.57 30

As a result, mutant strain 1, which has the highest productivity for Compactin, was selected and named as Penicillium citrinum KBT-1100 strain, and was deposited with the Korean spawn association on September 20, 2001 and deposited no. KCCM-10316. Was granted

Experimental Example 1 Compactin Productivity Comparison

Compactin productivity of the parent strain Penicillium citrinum ATCC 18199 was measured in the same manner as in Example 1. As a result, the compaktin productivity of the parent strain was 40 mg / L, which indicates that the penicillium citrineum KBT-1100 (KCCM-10316) according to the present invention is an excellent strain having more than 95 times the productivity of the compactin than the parent strain. Able to know.

Experimental Example 2 Comparison of Compactin Production in Fermenter

100 ml of the spawn medium (bacto peptone 10 g / l, soybean powder 20 g / l, sodium nitrate 2 g / l, magnesium sulfate 1 g / l, sucrose 100 g / l) was placed in a 1000 ml flask and sterilized for 30 minutes at 120 ° C. Next, inoculated with penicillium citrineum KBT-1100 (KCCM-10316) incubated in agar medium and cultured by rotating shaking at 25 ℃, 150rpm was used as a spawn.

Then, main culture medium (10 g / l bacterium peptone, 30 g / l soybean powder, 3 g / l sodium nitrate, 1 g / l magnesium sulfate, 100 g / l sucrose) was put into 7 L, 30 L and 500 L fermenters, respectively. Next, 5% (v / v) of the seed culture incubated in the flask was incubated for 9 days at 25 ° C. and 250 rpm. On the other hand, during the fermentation, the sugar was added to the container containing the acid alone or other nutrients in the container containing the acid, the pH was automatically added using a automatic control device while maintaining the pH of 4.5 ~ 5.0 and fermented. The yield of Compactin and its derivatives in the fermentation broth was quantified in the same manner as in Example 2. The results are shown in Table 2.

Fermenter Production amount (mg / ℓ) 500ml baffle flask 3,850 7ℓ 4,900 30ℓ 5,850 500ℓ 5,100

From Table 2, it can be seen that the use of the strain of the present invention is a useful strain for industrialization since it can produce compactin at high titer even in a fermenter.

As described above, if the production of the comvatin, the pravastatin precursor using the strain of the present invention can be significantly increased compared to the prior art, it is possible to produce in high titer in 7L, 30L, 500L fermenter also immediately industrially There is an advantage available.

Claims (2)

Penicillium citrinum KBT-1100 (KCCM-10316), which is a mutant strain of Penicillium citrinum with the ability to produce pravastatin precursors and derivatives thereof. A method for producing pravastatin precursors and derivatives thereof by culturing the penicillium citrinum KBT-1100 (KCCM-10316) strain of claim 1.