KR100630817B1 - Mutant Strain Having Hyperproductivity of Pravastatin Sodium and the Method for Producing Pravastatin Sodium Using Thereof - Google Patents

Abstract

The present invention relates to a pravastatin sodium high-producing mutant strain and a method for preparing pravastatin sodium using the same, and more specifically, Streptomyces exfoliatus var. Strain strain KBT2125 (KCCM 10570) and the pravastatin precursor in the culture medium The present invention relates to a method for producing pravastatin sodium, which comprises culturing the mutant strain while maintaining a constant concentration.

Variation strain Streptomyces exfoliatus KBT2125 (KCCM 10570) according to the present invention is excellent in the ability to convert the pravastatin sodium from the precursor to significantly higher pravastatin sodium productivity, and also to continuously add the optimum concentration of compactin to the culture medium Fermentation has the effect of maximizing the production of pravastatin sodium.

Pravastatin Sodium, High Potency Mutant, Streptomyces Exporiatus, Compactin, Precursor, Continuous Culture

Description

Muta Strain Having Hyperproductivity of Pravastatin Sodium and the Method for Producing Pravastatin Sodium Using Thereof}             

Figure 1 shows a process for obtaining a variant strain KBT2125 according to the present invention by UV treatment.

Field of invention

The present invention relates to a pravastatin sodium high-producing mutant strain and a method for preparing pravastatin sodium using the same, and more specifically, Streptomyces exfoliatus var. Strain strain KBT2125 (KCCM 10570) and the pravastatin precursor in the culture medium The present invention relates to a method for producing pravastatin sodium, which comprises culturing the mutant strain while maintaining a constant concentration.

Background of the Invention

There are four types of lipids in the blood: cholesterol, triglycerides (triglycerides), phospholipids and free fatty acids. Hyperlipemia refers to a condition in which lipids in the blood are increased, that is, the total cholesterol in the blood is more than 200 mg / dL or the triglyceride is more than 180 mg / dL. In fact, clinically problematic is an increase in cholesterol, an increase in triglycerides and an increase in both, these three conditions are generally considered hyperlipidemia.

Hyperlipidemia is not usually a problem in itself, but if there is an excess of fat in the blood, the fat is likely to stick to the walls of the blood vessels, which leads to arteriosclerosis, which is associated with coronary heart disease, cerebrovascular disease, and peripheral blood vessels. It can cause closure.

Recently, in our country, diet and urban life are rapidly westernized, increasing serum cholesterol, and ischemic heart disease is rapidly increasing.

Accordingly, studies to lower cholesterol levels in the blood are being actively conducted. In particular, pravastatin sodium inhibits the activity of the hydroxylmethyl glutaryl Co-A (HMG Co-A) enzyme, which acts in the process of cholesterol biosynthesis. It is known that it can inhibit cholesterol biosynthesis, making it a popular treatment for lowering cholesterol levels.

Therefore, the method of producing pravastatin by the fermentation method using microorganisms has been actively studied and the research on the high production method thereof is ongoing.

High-efficiency fermentation production of pravastatin is usually accomplished by isolating high-pravastatin sodium-producing strains or finding optimum conditions to increase the efficiency of the fermentation process.

In order to screen for high production of pravastatin sodium, resistant strains were isolated from medium containing high concentration of pravastatin precursor, compactin (ML-236B), through mutagenic treatment such as UV, and high concentration of compactin was added during fermentation. In the past, a method of selecting strains having excellent conversion ability of pravastatin sodium from compactin has been used.

In addition, in the production of pravastatin sodium, a method of converting compactin to pravastatin sodium has been generally employed by incubating by adding a pravastatin precursor, compactin (ML-236B), to the microbial culture by enzymatic-hydroxylation.

Several microorganisms have been used to produce pravastatin sodium by the enzymatic-hydroxylation method of microorganisms, and US Pat. No. 4,346,227 to Streptomyces roseochromogenes NRRL-1233, Streptomyces roseochromogenes IFO-3363, Streptomyces roseochromogenes IFO-3411 is disclosed, and Korean Patent No. 210,482 discloses Streptomyces roseochromogenes ( FO ) Streptomyces exfoliatus ) YJ-118, in Korean Patent Publication No. 1996-41369, Streptomyces roseochromogenes KCTC 12385 was used for the production of pravastatin sodium.

The present inventors have studied and applied for a method for producing pravastatin sodium using Streptomyces carbophilus KBT229 (KCCM 10370) (Korean Patent Publication No. 2003-0027571).

However, when preparing pravastatin sodium in the culture medium of the microorganisms, the compactin added in the culture medium is converted into pravastatin sodium as the fermentation progresses, and as a result, the growth and conversion of the microorganisms occurs as the pravastatin sodium accumulates at a high concentration in the fermentation broth. There was a problem that the rate is inhibited to inhibit the production of pravastatin sodium.

Therefore, the present inventors screened a novel mutant strain Streptomyces exfoliatus var. KBT2125 (KCCM 10570) having excellent conversion from the precursor to pravastatin sodium, and confirmed that the productivity of pravastatin sodium was increased as a result of culturing it. The present invention has been completed.

After all, it is an object of the present invention to provide a mutant having excellent conversion ability from pravastatin sodium precursor to pravastatin sodium.

Another object of the present invention is to provide a fermentation method for increasing the productivity of pravastatin sodium.

In order to achieve the above object, the present invention provides a variant strain KBT2125 (KCCM 10570) having excellent pravastatin sodium-producing ability ( Streptomyces exfoliatus var.).

The present invention also provides a method for producing pravastatin sodium, wherein the Streptomyces exfoliatus var. Strain KBT2125 (KCCM 10570) is cultured in a medium containing pravastatin sodium precursor.

In the method of producing pravastatin sodium, a) inoculating the strain in a pre-culture medium shaking culture; b) inoculating the preculture into the main culture medium, and adding pravastatin sodium precursor continuously to incubate while maintaining the concentration at 5 to 80 µg / ml; And c) obtaining converted pravastatin sodium from the precursor.

In the present invention, the pre-culture medium contains 0.3-5.0% glucose, yeast extract 0.05-2%, soy flour 0.3-3.0%, sodium nitrate 0.15-0.5% and calcium carbonate 0.05-1.0%, the main culture The medium may be characterized by containing 0.1 to 1.0% peptone, 0.1 to 5.0% glucose, 0.05 to 2.0% yeast extract, 0.5 to 3.0% soy flour, 0.05 to 1.0% sodium chloride and 0.05 to 0.5% calcium carbonate, In the main culture, the sugar solution may be added continuously to adjust the pH of the fermentation broth to 7.0 ± 0.5.

The present invention also comprises a) irradiating UV to a strain having a pravastatin sodium producing ability; b) a first screening step of separating the strains of colonies growing in the medium by smearing the UV-irradiated strain on agar medium containing high concentration (1-100 mg / ㎖) pravastatin sodium; c) the primary selected strain was inoculated into a flask containing a preculture medium and shaken, and the shaken culture was inoculated into a flask containing the main culture medium, and the pravastatin sodium precursor compactin was not more than 0.02% Culturing while adding at a concentration of; And d) selecting the pravastatin sodium high-performance mutant strain by analyzing the culture solution, and selecting the pravastatin sodium high-performance mutant strain.

In the screening method, the strain having a pravastatin sodium producing ability may be characterized in that the Streptomyces exfoliatus KCCM 40606T, the pravastatin sodium high-performance mutant strain Streptomyces exfoliatus var ( Streptomyces exfoliatus var .) It may be characterized as a variant strain KBT2125 (KCCM 10570).

In the present invention, in the production of pravastatin sodium using the selected mutant strain (KCCM 10570), the fermentation method was improved to increase the accumulation amount.

In other words, the conventional method of producing pravastatin sodium was a method of periodically adding the pravastatin precursor compactin to the microbial culture, and when the compound is periodically added, such as adding the compactin every 6 hours or 12 hours, the accumulation of compactin in the fermentation broth Increasing the concentration of the substrate inhibited the growth and conversion of the bacteria had a problem that is slow. If such a problem occurs, the accumulation of the product pravastatin sodium is reduced and the efficiency of the fermentation process is reduced.

In the present invention, in the conversion from compactin to pravastatin sodium in the fermentation process, the optimum concentration of the compactin that does not affect the growth rate and the conversion rate of the fermentation strains, and to determine the optimum concentration of compactin in the fermentation broth continuously The method of addition was proved to be more efficient than the addition of compactin in batch or high concentration.

In the present invention, high production mutant strains of pravastatin sodium were selected using UV. Streptomyces exfoliatus KCCM 40606T was used as a parent strain and UV-treated as a mutagen to select high productivity mutant strains. It was named Streptomyces exfoliatus var. KBT2125 and deposited with KCCM 10570 on April 28, 2004 at Korea Microbiological Conservation Center.

In the present invention, to prepare pravastatin sodium using the Streptomyces exfoliatus var. KBT2125 First, Streptomyces exfoliatus var. KBT2125 is inoculated in a pre-culture medium 1 After shaking for 3 days at 25 ~ 30 ℃, 100 ~ 200rpm shaking culture, inoculated into the main culture medium and shaking culture at 25 ~ 30 ℃, 100 ~ 200rpm while the concentration of pravastatin precursor compactin 0.02% or less The productivity of pravastatin sodium in the culture was checked while adding. The components of the preculture medium and the main culture medium are as follows:

Pre-culture medium-glucose 0.3 ~ 5.0%, yeast extract 0.05 ~ 2%, soy flour 0.3 ~ 3.0%, sodium nitrate 0.15 ~ 0.5% and calcium carbonate 0.05 ~ 1.0%

Main culture medium- peptone 0.1-1.0%, glucose 0.1-5.0%, yeast extract 0.05-2.0%, soy flour 0.5-3.0%, sodium chloride 0.05-1.0% and calcium carbonate 0.05-0.5%.

In the present invention, by mutating Streptomyces exfoliatus KCCM 40606T, the microorganisms grown in a medium containing compactin 100-500 µg / ml and Pravastatin sodium 10-100 mg / ml are selected first, and the liquid Through culture, strains with high yield of pravastatin sodium were selected secondarily (FIG. 1).

 Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Primary Screening of Mutant with High Productivity of Pravastatin Sodium

The mother strain Streptomyces exfoliatus KCCM 40606T was incubated in KCCM for at least 5 days, and the cells were suspended by adding sterile saline solution, irradiated with UV light for 10 to 60 seconds, and then Pectin-containing agar medium (peptone 10g / L, glucose 20g / L, agar 20g / L, yeast extract 1g / L, compactin 100µg / ml, pravastatin sodium 50mg / ml, pH 7.0 ~ 7.2, 27 ~ 29 ℃) Strains that generate colonies were first screened.

Example 2: Secondary Selection of Mutant Wine through Liquid Culture

In order to select pravastatin sodium productive strain among strains formed in agar medium after UV treatment, preculture medium (1.5% glucose, 0.1% yeast extract, 1.5% soy flour, 0.5% sodium nitrate and 0.1% calcium carbonate, pH7 .0-7.2) Each colony selected first in Example 1 was inoculated into a 500 ml baffled flask containing 50 ml.

The inoculated Erlenmeyer flask was shaken for 2 to 3 days at 27 to 29 ° C and 150 rpm, followed by main culture medium (peptone 0.5%, glucose 3.0%, yeast extract 0.1%, soy flour 2.0%, sodium chloride 0.5% and calcium carbonate 0.1%). 20 ml each of the cultures were inoculated in a 2L baffle Erlenmeyer flask containing 200 ml of pH 7.0-7.2), followed by incubation at 27-29 ° C. and 100-200 rpm for 1 day, followed by comb as a pravastatin precursor. Incubation was made with the addition of factin at a concentration of 0.02% or less.

After culturing for 6 days with shaking at 150 rpm at 27-29 ° C., the mutants were selected to produce high titers of pravastatin sodium by HPLC analysis. Figure 1 shows a process for selecting a variant strain KBT2125 according to the present invention by UV treatment.

Example 3: Confirmation of Pravastatin Sodium Productivity of Selected Mutants

The productivity of the pravastatin sodium of the parent strain Streptomyces exfoliatus KCCM 40606T and the variant strain Streptomyces exfoliatus KBT2125 was measured by HPLC. HPLC was analyzed under the conditions of Symmetric C18 column, column temperature 40 ℃, mobile phase methanol: triethylamine (acetic acid): water. As a result, the production amount of pravastatin sodium of the parent strain was 23 µg / ml, and the selected mutant strain produced 2,527 µg / ml (flask), which was about 110 times improved compared to the parent strain (Table 1).

                         Name Final pH PMV (%) Culture titer (mg / l) Mother strain Streptomyces exfoliatus (KCCM 40606T) 6.52 23 23 Mutant Streptomyces exfoliatus var.KBT2125 (KCCM 10570) 6.74 22 2,527

Example 4: Titration with Continuous Precursor (Compactin) Addition for Pravastatin Production

In order to compare the method of adding compactin and concentration, 21L of medium was added to a 30L fermenter, sterilized and cooled, and then inoculated with a pre-cultivated seed (secondarily selected strain in Example 2) and cultured at 27-29 ° C for 1 day. Compactin was added every six hours in a batch method and incubated for seven days with different concentrations of compactin at the time of addition. In another fermentation tank, compactin was continuously added and incubated for 7 days while maintaining each concentration of compactin in the fermentation broth, and the productivity by the continuous addition method of compactin was compared with the batch addition method.

The compactin solution was added every 6 hours of fermentation and added by batch method so that the concentration of compactin in the fermentation broth was 50 µg / ml, 100 µg / ml, 200 µg / ml, 300 µg / ml and 500 µg / ml, respectively. As a result of culturing, when the concentration of compactin was 200 µg / ml, the content of pravastatin in the fermentation broth was 2,520 µg / ml, and the content was 100% (controlled) and compared with other examples. At concentrations of 50 µg / ml and 500 µg / ml, the concentrations of pravastatin sodium in the fermentation broth were 1,050 µg / ml and 1,120 µg / ml, respectively.

In addition, the content of pravastatin sodium produced in the fermentation broth was 3,750 µg / ml or more when fermentation was continued while adding the compactin solution continuously to keep the contents of the compactin 5-30 µg / ml and 30-60 µg / ml. It was about 49% higher than the control. In the case of fermentation while maintaining the concentration of compactin at 60 to 80 µg / ml, the concentration of pravastatin sodium was 2,851 µg / ml at 13% compared to the control, and 2,630 µg / ml at the concentration of 0 to 5 µg / ml. It was 4% higher, and the content of pravastatin was 920µg / ml, which was lower than that of the control when fermented while maintaining 100-200µg / ml, respectively. In the case of fermentation while keeping the compactin at a concentration of 200 to 500 µg / ml, almost no pravastatin sodium concentration was produced at only 120 µg / ml.

Therefore, in order to produce pravastatin sodium in a fermenter, it was found that a method of converting pravastatin sodium to pravastatin sodium by continuously adding pravastatin precursor to the fermentation broth while maintaining the concentration of compactin in the fermentation broth in the range of 5 to 80 µg / ml is preferable. . Table 2 compares the production of pravastatin sodium when compactin was added at different concentrations in batch and continuous addition.

Compactin addition method         Batch addition method          Continuous addition  Compactin concentration added every 6 hours    Maintain compactin concentration in fermentation broth  Compactin concentration (µg / ml)   50  100  200   300   500  0 to 5  5-30 30 to 60 60 to 80 100-200 200 to 500 Pravastatin Sodium Production (µg / ml)  1,050  1,876  2,520  2,352  1,120 2,630 3,750 3,963  2,851  920  120    compare(%)   42   74  100   93   44  104  149  157  113   37   5

Example 5: pH adjustment by adding sugar solution

During the production of pravastatin sodium, 30% glucose solution was continuously added instead of the acid or caustic soda solution, and the fermentation broth was adjusted to 7.0 ± 0.5. As a result, the amount of pravastatin sodium was produced using the acid or caustic soda solution. 5-10% higher than that.

According to the present invention, when the production of pravastatin sodium through enzymatic hydroxylation reaction, the use of the mutant strain Streptomyces exfoliatus KBT2125 (KCCM 10570) significantly increases productivity under high concentrations of pravastatin sodium compared to the parent strain. It can be (about 110 times), and also by increasing the production of pravastatin sodium by continuously adding the optimum concentration of compactin to the culture medium, there is an advantage that can be immediately used for mass production.

As described above in detail the specific parts of the present invention, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The variant strain Streptomyces exfoliatus KBT2125 (KCCM 10570) according to the present invention is excellent in the ability to convert pravastatin sodium from a precursor, which is highly useful in pravastatin sodium production strain.

[Deposit]

Claims (7)

Streptomyces exfoliatus var. Variant strain KBT2125 (KCCM 10570) with excellent ability to produce pravastatin sodium. A method for producing pravastatin sodium, comprising culturing the Streptomyces exfoliatus var. Mutant KBT2125 (KCCM 10570) in a medium containing pravastatin sodium precursor. The method for preparing pravastatin sodium according to claim 2, comprising the following steps: a) inoculating the strain into a preculture medium, followed by shaking culture; b) inoculating the preculture into the main culture medium, and adding pravastatin sodium precursor continuously to incubate while maintaining the concentration at 5 to 80 µg / ml; And c) obtaining pravastatin sodium converted from the precursor. The method for preparing pravastatin sodium according to claim 3, wherein the sugar solution is continuously added during the main culture to adjust the pH of the fermentation broth to 7.0 ± 0.5. delete delete delete

Images