TWI519643B - A novel nonomuraea sp. env1 and a process for producing pravastatin using the same - Google Patents

Description

Novelnobacteria (Nonomuraea sp. ENV1) and method for producing pravastatin by using the same

The present invention relates to a method for preparing pravastatin sodium salt, and more particularly to a method for converting compstatin sodium salt to pravastatin sodium salt by a novel Narania.

In recent years, with the rapid development of the economy, the main cause of death of human beings has changed from infectious diseases to cancer, cerebrovascular diseases, heart disease and diabetes. Among them, cerebrovascular disease, heart disease and its complications are caused by atherosclerosis, and dyslipidemia is the most important deterioration factor. Among them, "hyperlipidemia" refers to the main fat in the blood - cholesterol and triacid. The glyceride (neutral fat) is too high. According to IMS statistics, global drug sales in 2011 will reach 890 billion US dollars, of which the second-ranked drug is Cholesterol & Triglyceride Reducers.

Today's hypolipidemic drugs can be broadly classified into the following four categories: the first category of HMG-CoA reductase inhibitors series: Pravastatin (Pravastatin) / Lovastatin / Simvastatin / Atorvastatin; the second type of Isobutyric acid products: Gemfibrozil / Clofibrate, etc. The third type of Bile acid series products: Probucol/Cholestyramine, etc.; and the fourth type of Nicotinic acid series products: Nicofuranose/Nicomol. HMG-CoA reductase inhibitors are among the most effective and widely used drugs.

Among the HMG-CoA reductase inhibitor drugs, pravastatin (Pravachol and Mevalotin, etc.) is the third-ranked hypolipidemic drug in current use, second only to Atorvastatin and simvastatin. Pravastatin is synthesized from compstatin. However, when it enters the animal, it will first circulate to the liver. The P450 enzyme of the liver cell will reduce it to a metabolite with a hydroxy group, which is the source of hepatic toxicity. Pravastatin has a more hydroxy group than compstatin, and pravastatin after liver metabolism no longer requires hydroxylation, so it has lower hepatotoxicity than compstatin, resulting in annual sales of pravastatin of more than $3 billion, and Growing year by year.

Pravastatin, with a hydroxy group, is a highly hydrophilic, active compound that selectively acts on the major organs of cholesterol synthesis (liver, small intestine), blocks the synthesis of cholesterol, lowers liver cholesterol, and makes LDL The activity of the receptor is enhanced, resulting in an increase in LDL uptake from the blood to the liver, so that LDL-cholesterol in the serum is lowered, which can rapidly and strongly lower serum cholesterol and improve serum lipids. In addition, because of its organ selectivity, it has a very weak effect on other organs other than the liver and small intestine (including organs produced by hormones), and thus has fewer side effects. At the same time, it has been found that the efficacy of anti-atherosclerosis or cardiovascular disease is far more than other cholesterol-lowering drugs. It is safe for long-term use and does not increase the incidence of cancer. It is easy to use and has few side effects.

In fact, the new use of pravastatin is still being discovered. For example, according to a report in the journal Neurology, it is now common in elderly people with dementia, which can be reduced by pravastatin by 73%. In addition, there are reports that pravastatin can reduce the incidence of stroke by an average of about 22%. There are also reports that pravastatin can reduce the likelihood of diabetes and is being administered to people with heart disease. When pravastatin was used to prevent cholesterol elevation, it was unexpectedly found that 30% of patients had a reduced risk of diabetes. It is thought that the pravastatin and statins series drugs can lower triglycerides in the blood, while triacids Glycerol is associated with both stroke and diabetes. Taken together, pravastatin not only lowers blood cholesterol, but also has the opportunity to prevent drugs for dementia, stroke and diabetes in the elderly.

The two-stage production method of pravastatin means that compstatin is first synthesized by the fermentation method, and then the baitcitin is hydroxylated into pravastatin by the enzyme group produced by the microorganism. The strains which have the biotransformation of compstatin to pravastatin include: Streptomyces roseochromogenu NRRL-1233, Streptomyces roseochromogenus IFO-3363, rose color chain Mold-3O (Streptomyces roseochromogenus IFO-3411) (U.S. Patent No. 4,346,227), Streptomyces carbophilus SANK-62585 (Ferm BP-1145, U.S. Patent No. 5,179,013) and Streptomyces coles (Streptomyces halstedii) (Japanese Patent No. 4-349034). However, the original strains of these microorganisms were not well tolerated by compstatin, resulting in a decrease in the yield of pravastatin. Recently, many patented strains have been published to have high tolerance to Kang Baiting, and those with a conversion rate of more than 50%, such as Streptomyces exfoliates yj-118 (US Patent No. 6,306,629), small orders Micromonospora sp. (WO Pat. No. 0103647), Actinomyces madura ATCC 55678 (Actinomadura sp. ATCC 55678) (U.S. Patent No. 6,274,360).

Taking Streptomyces carbophilus as an example, the biotransformation of compstatin to pravastatin is not accomplished by a single enzyme but by a cytochrome (Cytochrome) P450, reductase, NADH or NADPH regeneration system. Therefore, although the microorganisms that can convert compstatin into pravastatin are ubiquitous in nature, there are very few economical values for the tolerance and conversion efficiency of combidin.

In view of the above, it is a first object of the present invention to provide a novel strain which has extremely high tolerance to the Babbottin sodium salt and has a very high conversion rate for the conversion of the Babbottin sodium salt to pravastatin sodium salt.

Edge to achieve the above objects, the present invention provides a novel that satisfied absurd anthracene bacteria ENV1 (Nonomuraea angiospora ENV1), its registered number in the Republic of China Food Industry Development Research Institute Foundation for the BCRC 910540, wherein: the absurd is satisfied that anthracene The strain ENV1 is extremely resistant to compactin sodium, and the biotransformation method can be used to effectively convert the Babbottin sodium salt into pravastatin sodium.

A second object of the present invention is to provide a method for converting compstatin sodium salt into pravastatin sodium salt, which has the effect of obtaining high yield of pravastatin.

In order to achieve the above object, the present invention inoculates the above-mentioned Nattomycin ENV1 in a medium containing a betacitrine sodium salt, and then fermented in the medium, and then continues to culture in the medium to add the Babbottin sodium salt. Transform into pravud Statin sodium salt.

A third object of the present invention is to provide a medicament for lowering human cholesterol which has the effect of obtaining high-yield pravastatin.

In order to achieve the above object, the present invention provides the above-prepared pravastatin sodium salt and a pharmaceutically acceptable excipient or carrier into a drug for lowering human cholesterol.

In summary, the N. faecalis ENV1 provided by the present invention has extremely high tolerance to the Babbottin sodium salt, and can effectively convert the Babbottin sodium salt into pravastatin sodium salt to obtain a low side effect and a safer drop. Blood cholesterol drugs.

The novel strain of the present invention, ENV1, has good degradation ability to polycyclic aromatic hydrocarbon-phenanthrene, and has high tolerance to the sodium salt of bevacin, and can effectively utilize the fermentation method. The sodium salt is converted into pravastatin sodium salt, and pravastatin can be used as a cholesterol-lowering drug substance.

The following is an experiment conducted by the present invention, indicating the source of the strain used and the experimental parameters and results:

1, strain screening

A selection of phenanthrene-degrading bacteria from oil-contaminated soil in Kaohsiung, Taiwan was screened by selective medium screening. These strains were separately cultured in YMG (yeast-malt-glucose) (yeast-malt-glucose) liquid medium. (yeast extract 0.3%, malt extract 0.3%, 腖0.5%, glucose 1%, pH 6.5), after shaking at 200 rpm for 48 hours at 28 ° C, 50-1,000 μg/ml of the Babbottin sodium salt was added, and the culture was continued for 24-72 hours to determine the utilization rate of the Babbottin sodium salt and the conversion rate of pravastatin sodium salt by HPLC. The results showed that two strains had the ability to convert compstatin to pravastatin under these conditions. Among them, Neisseria gonorrhoeae ENV1 has the highest tolerance to the Babbottin sodium salt, and can effectively convert the Babbottin sodium salt into pravastatin sodium salt. This strain is a phenanthrene-degrading bacterium.

2, strain identification

The sputum ENV1 was identified by the Food Industry Development Research Institute. Based on the analysis of the cytochemical composition of the strain, the type characteristics of the strain and the physiological and biochemical characteristics of the hyphae, the strain was an ampoule. Nonomuraea angiospora , named as N. faecalis ENV1, deposited in the Food Industry Development Institute, under the strain number BCRC 910540.

(i) Analysis of cytochemical components

The cell wall amino acids and whole cell carbohydrate components are meso-DAP (meso-diaminopimelic acid), xylose (xylose), madurose (madura), glucose and galactose, respectively. According to the classification of Lechevalier et al., it belongs to Chemotype III B type.

(ii) Morphological characteristics of the strain

The vegetative hyphae of the strain on the SCY medium is yellow, the aerial strain is white, the sporulation is good, and the spore chains are mostly hook-shaped, with a length of 4 to 10 spores. Sub (see Figure 1 and Figure 2).

(iii) The use of strains for various carbohydrates and substances is shown in Table 1.

(iv) 16S rDNA partial sequence

After the strain was cultured, the Genomic DNA was extracted, and the 16S rDNA gene fragment was amplified and sequenced by a polymerase chain reaction (PCR) reaction, and the 16S rDNA partial sequence thereof is shown in Table 2.

(v) comparison results

The above results with Bergey's Manual of Determinative Bacteriology, and the ratio of the literature, and with reference to the results of 16S DNA Sequence Data GenBank alignment, this strain was identified that satisfied security Qiusibole absurd anthracene bacteria (Nonomuraea angiospora).

The above is a description of the bacterium of the present invention provided by the preferred embodiment of the present invention, and the experimental methods, steps, experimental results and discussion used in the preferred embodiment of the present invention will be described below.

3. Tolerance of Neisseria gonorrhoeae ENV1 to Kang Baiting sodium salt

(i) Inoculum culture

The inoculum was inoculated into SCY seed culture solid medium (casein hydrolysate 0.05-0.2%; yeast extract 0.05-0.2%; soluble starch 0.5-2.0%; KH ₂ PO ₄ 0.01-0.08%; MgSO ₄ .7H ₂ O 0.05- 0.2%; compstatin sodium salt 0.002-0.01% and Bacto agar 2.0%, pH 7.0), cultured at 28 ° C for 7 days to 20 days.

(ii) shake flask fermentation

The inoculum was inoculated into YGP liquid medium containing the sodium salt of carbestine (yeast extract 0.1-1.0%, glucose 1.0-3.0%, Pharmamedia 1.0-3.0%, combestine sodium salt 0.002-0.01%, pH 6.5) Medium, rotation speed 220 rpm, shaking culture at 28 °C.

(iii) Tolerance of Neisseria gonorrhoeae ENV1 to Babetin sodium salt

After 48 to 72 hours of the above shake flask culture, 200-2,500 μg/ml of the Babbottin sodium salt was added, and the culture was continued under the same conditions, and the utilization rate of the Babbottin sodium salt and the conversion rate of pravastatin sodium salt were quantified by HPLC every 24 hours. It is shown that if the amount of the Baining sodium salt added exceeds 2,000 μg/ml, the growth of the cells becomes quite sluggish, and the conversion rate of pravastatin is not more than 29%.

4. The ability of Neisseria gonorrhoeae ENV1 to convert compstatin sodium salt to pravastatin sodium salt

Inoculate 3-15% of the inoculum in YGP liquid production medium containing 0.002-0.01% of Bainbatin sodium salt (yeast extract 0.5-1.5%, glucose 0.5-2.5%, Pharmamedia 0.1-0.4%, combestine sodium salt) In 0.002-0.01%, pH 6.5), after shaking at 220 rpm for 48 to 72 hours at 28 °C, add 300-1,500 μg/ml of Babbottin sodium salt and continue the culture under the same conditions. When the pH of the culture solution is higher than 7.0. When adding 0.1-0.8% glucose, 0.05-0.5% Yeast extract and 0.05-0.5% cotton extract (Pharmamedia). The utilization of compstatin sodium salt and the conversion rate of pravastatin sodium salt were quantified by HPLC every 12 hours. The results showed that the utilization rate of compstatin sodium salt exceeded 93% within 36-72 hours, and the conversion rate of pravastatin was about 40. -66%.

The present invention also provides a biotransformation method for converting the Babbottin sodium salt into pravastatin sodium salt by using the above-described N. sphaeroides ENV1, wherein the above-mentioned fermentation method is used to effectively convert the Babbottin sodium salt into pravastatin sodium salt. The conversion rate can be 40-66%.

5. Gene sequence of the cytochrome P450 of Neisseria gonorrhoeae ENV1

Next, the experimental conditions and data for converting the Babbitt sodium salt to pravastatin sodium salt using Nattobacterium ENV1 according to a preferred embodiment of the present invention are provided, but are not intended to limit the scope of the present invention.

Example 1: Adding a low concentration of bevacin sodium salt

The inoculum was inoculated into the inoculum medium (casein hydrolysate 0.1%; yeast extract 0.15%; soluble starch 1%; KH ₂ PO ₄ 0.05%; MgSO ₄ .7H ₂ O 0.1%; compstatin sodium salt 0.005% and Bacto agar 2.0 %, pH 7.0), cultured at 28 °C for 7 days to 20 days. The 500 ml flask contains 50 ml of YGP liquid production medium (yeast extract 0.5-1.5%, glucose 0.5-2.5%, Pharmamedia 0.1-0.4%, combestine sodium 0.002-0.01%, pH 6.5) Inoculate 3-12% of the inoculum, rotate at 220 rpm, shake culture at 28 °C for 48 to 72 hours, add 500 μg/ml of Babbottin sodium salt, continue the culture under the same conditions, and quantify the utilization of the Babetin sodium salt by HPLC every 12 hours. Rate and conversion rate of pravastatin sodium salt. The results are shown in Table 3.

The HPLC analysis conditions are as follows:

Column: C18, 4.6 × 250 mm

Detector: UV 238 nm (nm)

Flow rate: 0.8 ml / min

Mobile phase: methanol: triethylamine (TEA): acetic acid: H ₂ O = 70: 0.1: 0.1: 30

Oven temperature: 35 ° C

Example 2: Adding high concentration of betridinate sodium salt

As in the case of Example 1, but adding 1,000 μg/ml of valdecin sodium salt, the results are shown in Table 4.

Example 3: Maintaining pH

As in the case of Example 1, firstly inoculate 5-15% of the inoculum in a YGP liquid production medium containing 0.005% of Bainbatin sodium salt (yeast extract 0.5-1.5%, glucose 0.5-2.5%, cotton abundance (Pharmamedia) 0.1-0.4 %, Kang Baiting sodium salt 0.002-0.01%, pH 6.5), at 220 rpm, and cultured at 28 ° C for 48 to 72 hours, 750 μg/ml of Babbottin sodium salt was added, and the cultivation was continued under the same conditions. When the pH of the culture solution was higher than 7.0, 0.1-0.8% glucose, 0.05-0.5% yeast extract and 0.05-0.5% cotton extract (Pharmamedia) were added. Quantification of the use of betacitine sodium salt by HPLC every 12 hours The rate and conversion rate of pravastatin sodium salt are shown in Table 5.

Example 4: Continuously adding Babetin sodium salt and maintaining the pH value

According to the conditions of the third embodiment, the 10 liter fermentation tank is used as a production tank, and the plastatin is converted into a fed-batch fermentation method, and the final operation volume is 5 liters, and the ventilation volume is 0.5 to 2.0 vvm. Feeding speed averaged 750 μg/ml of Babbottin sodium salt every 12-48 hours, continue to culture under the same conditions, when the pH of the culture solution is higher than 7.0, 0.1-0.8% glucose, 0.05-0.5% yeast extract and 0.05-0.5% cotton extract (Pharmamedia). After 11 days, the total addition amount of carbestine sodium salt was 6,000 μg/ml, the utilization rate of carbestine sodium salt was about 88.5%, and the conversion rate of pravastatin was about 54.6%. The results are shown in Table 6.

It is known from the above Experiment 4 that the strain N. faecium ENV1 ( Nonomuraea angiospora ENV1) has extremely high tolerance to the Babbottin sodium salt in the environment in which the Bainbin sodium salt is continuously added. From the results of Experiment 1 to Experiment 4, the conversion rate of the Babbitt sodium salt to pravastatin sodium salt was more than 50%, and the strain Nattobacterium ENV1 could prepare a high yield of pravastatin sodium salt. It is known from Experiments 3 and 4 that a higher yield of pravastatin sodium salt can be obtained when the pH of the culture solution is controlled to be less than 7. The pravastatin sodium salt is then mixed with other pharmaceutically acceptable excipients or carriers to produce a drug that lowers human cholesterol.

The above description is only a preferred embodiment of the present invention, and the application of the present invention is applicable. Equivalent changes in the specification and the scope of the patent application are intended to be included in the scope of the invention.

Figure 1 is an electron micrograph of the aerial hyphae and spore chains of the genus ENV1; and Figure 2 shows the spores of the bacterium ENV1.

Claims (8)

A method for converting a Babbitt sodium salt into pravastatin sodium salt using N. faecium ENV1 ( Nonomuraea angiospora ENV1), the accession number of the Nanabacterium ENV1 is BCRC 910540, which comprises the following steps: The sputum ENV1 is inoculated in a medium containing compactin sodium to culture and ferment; then the saponin sodium salt is added to continue the culture in the medium to convert the carbestine sodium salt into pravastatin sodium (pravastatin sodium). Wherein the medium is a YGP liquid medium comprising 0.5-1.5% of yeast extract, 0.5-2.5% of glucose, 0.1-0.4% of pharmamedia and 0.002-0.01% of benzalkonium sodium salt, and the medium maintains pH. 6.5. The method of claim 1, wherein the re-added concentration of the becamin sodium salt is from 1 to 2000 μg/ml. The method according to claim 2, wherein the conversion of the sodium sulphate ENV1 to the pravastatin sodium salt is 20-70%. The method of claim 1, wherein the medium is maintained at a pH of 7.0 or less. The method of claim 1, wherein the re-added concentration of the becamin sodium salt is from 1 to 60 μg/ml. The method of claim 5, wherein the conversion of the Kamivudine sodium salt to pravastatin sodium salt by the N. faecalis ENV1 is 20-70%. The method of claim 1, wherein the tolerance of the natsin ENV1 to the valdecin sodium salt is in the medium of the bacterium of the genus ENV1 Kang Batin sodium salt 1-2000 μg / ml. A phenanthrene-degrading ENV1 (Nonomuraea angiospora ENV1), which is Nonomuraea angiospora, the accession number of the genus ENV1 is BCRC 910540, The method is characterized in that the strain is tolerant to 500 μg/ml of compactin sodium, and the biotransformation method can effectively convert the Babbottin sodium salt into pravastatin sodium (pravastatin sodium). The genus ENV1 has the following microbiological properties: (i) cytochemical composition analysis of cell wall amino acids and whole cell saccharide components are meso-DAP (meso-diaminopimelic acid) (medium-diaminopimelic acid) , xylose (xylose), maduose (madura), glucose and galactose, belonging to Chemotype III B; (ii) morphological characteristics of the strain. The vegetative hyphae are yellow on the SCY medium, and the aerial strain is white. The sporulation is good, the spore chain is mostly hook-shaped, and the length is 4 to 10 spores; (iii) the growth temperature of the strain is 20-50 ° C; (iv) the utilization of various carbohydrates and substances by the strain is as follows: The strain on starch The hydrolysis of casein, L-tyrosine, esculin, and hypoxanthine has a positive reaction, and has a negative reaction to the hydrolysis of xanthine, adenine, and gelatin, and has a positive reaction to nitrate reductase production. .