KR20030076027A - Microorganisms hydroxylating vitamin D3 and manufacturing process of calsitriol by using of them - Google Patents

Abstract

The present invention relates to Pseudonocardia autotrophica ID9302, which introduces a hydroxyl group to a precursor of vitamin D3 to produce active vitamin D3, wherein the wild strain Pseudonocardia autotroph isolated from soil. It is characterized by high production of 25-hydroxyvitamin D3 and calcitriol by mutating the mutants with mutations such as UV, NTG, EtBr, and optimization of various carbon sources, nitrogen sources, and other media sources, and improving the cultivation and bioconversion processes. .

Description

Microorganisms hydroxylating vitamin D3 and manufacturing process of calsitriol by using of them

The present invention uses a strain Pseudonocardia autotrophica ID9302, which produces calcitriol, a therapeutic agent for osteoporosis, and introduces a hydroxyl group at positions 1 and 25 of the carbon source of vitamin D3 using a bioconversion technique. A method of producing high dihydroxyvitamin D3 (calcitriol) was also developed and a method of high producing calcitriol (Fig. 1) by introducing a hydroxyl group at a carbon source 1α position using 25-hydroxyvitamin D3 as a precursor. will be.

Osteoporosis is a balance between bone formation and bone resorption in normal people to maintain healthy bones, but in post-menopausal women and the elderly, bone formation is slow and bone resorption is rapidly weakening the bone tissue. Bone formation and bone resorption are controlled by a variety of complex physiological mechanisms, and in postmenopausal women, the production of the female hormone estrogen decreases, resulting in less and less bone formation and subsequent bone resorption resulting in osteoporosis. In the elderly, as the metabolic function of both bone formation and bone resorption decreases, bone resorption becomes more prevalent than bone formation, resulting in osteoporosis. For the prevention or treatment of such osteoporosis, bone resorption inhibitors, bone formation promoters and calcium adjuvant are used. Particularly, calcitonin, which is an estrogen or Ca-binding protein, or calcidiol or calcitriol, an active vitamin D3, is administered to promote Ca absorption in bone. Estrogen is currently used as a hormone, but it can prevent the rapid decrease in bone mass, but it is limited to use because it can cause serious side effects such as breast and uterine cancer (Yoo HJ, 2001, Estrogen metabolism as a risk factor for head and neck cancer, Otolaryngol Head Neck Surg 124 (3): 241-7). Calcitonin can be administered only by subcutaneous injection, which is inconvenient to administer and decomposes in the body due to protein properties. In contrast, active vitamin D3 calcitriol is an in vivo substance produced by the conversion of vitamin D3 in the liver and kidney in normal people with fewer side effects, simple administration by oral administration, and physiological absorption of Ca into bone. It is known to promote osteoporosis with an excellent therapeutic effect. In addition to clinically used for the treatment and prevention of osteoporosis, calcitriol has been reported to treat parathyroid hypoplasia, chronic kidney failure, dry skin, tumors, etc. and recently, it has been reported to inhibit cell proliferation (Jensen SS et al., 2001). , Inhibitory effects of 1alpha, 25-dihydroxyvitamin D3 on the G (1) -S phase-controlling machinery.Mol.Endocrinol . , 15 (8): 1370-80).

Calcitriol, the active vitamin D3, is currently produced by organic synthesis, and therefore must undergo a multi-step organic synthesis process, taking into account the stereospecificity and regiospecificity of the chemical structure and selectively introducing a hydroxyl group at position 1 or 25. Thus, the reactor operation is complex and expensive (Kametani et al., 1987, Synthesis of vitamin D3 and related compounds, Med. Res. Rev. , 7, 147-171). Derivatives by chemical synthesis have been actively studied for the synthesis of calcitriol and the synthesis of derivatives. In addition to the introduction of hydroxyl groups of 1α and 25 carbons, it attempted hydroxylation to other carbons, iodination, bromination, phosphorylation, etc. There was a new inducer and chemical conversion of derivatives such as carboxylation, methylation and carbon chain increase.

Studies using animal organs to introduce hydroxylation into vitamin D3 have also been reported. Hydrogenation was reported at position 1α using homogenate or mitochondrial fraction of chicken kidney, hydroxylation at position 25 was also reported using mouse liver, and biosynthesis studies of calcitriol using plant tissue were reported. . Recently, studies on the conversion of 7-dihydrocholesterol to calcitriol using keratinocytes under UV conditions have been reported. However, as a result of research using these tissues or organs, productivity is so low that a large amount of organs are required, so it is difficult to separate a small amount of active material actually produced, which is inefficient and difficult to apply to actual production.

Bioconversion reactions by microorganisms have already been demonstrated to be stereospecific and regiospecific. Therefore, active-form vitamin D3 can be replaced in the production method in an economical way by biotransformation using the hydroxylation function of the microorganism from a conventional organic synthesis method (Rosazza et al., 1979, Mictobial models for drug metabolism , Adv. Appl. Microbiol., 25, 169-208). In 1990, a study on the transformation of vitamin D3 derivatives by microorganisms was carried out by Taisho Pharmaceutical of Japan, which investigated the strain of Streptomyces , a soil actinomycete, and reported the hydroxylation of vitamin D3 using this strain (USP 4,892,821). However, the bioconversion capacity of calcitriol-producing strains using vitamin D3 or 25-hydroxyvitamin D3 as precursors is a result of the triangular flask-scale culture, and the bioconversion method using buffers can increase productivity. The productivity is also low. In 1992, Taisho Pharmaceutical reported the bioconversion of vitamin D3 to calcitriol using Amycolata sp. With 25-hydroxyvitamin D3 and 8.3 mg / l for calcitriol and 0.17 mg / l. It is judged to be remarkably low compared to the productivity of the invention.

Therefore, by separating and improving microorganisms having bioconversion ability as in the present invention, by introducing a hydroxyl group by bioconversion by using vitamin D3 as a precursor, by developing a bioconversion process capable of high production of calcitriol, an active vitamin D3, It can be lowered dramatically.

An object of the present invention is to isolate the strain of cholecalciferol producing 25-hydroxyvitamin D3, an active vitamin D3 by bioconversion, in the soil and then mutating the strain, and also media, culture, bioconversion process By maximizing the ability to introduce hydroxy groups into vitamin D3 by optimizing and the like, high yield of 25-hydroxyvitamin D3 and calcitriol is also provided, and a method of producing calcitriol by bioconversion using 25-hydroxyvitamin D3 as a precursor It is.

1 shows the structure of calcitriol produced by the bioconversion of Pseudocardia autotrophica ID9302

Figure 2 shows the shape of the Pseudonocardia autotrophica ID9302 by Scanning Electron Microscope (× 12.5K).

Figure 3 shows the productivity of calcitriol and 25-hydroxyvitamin D3 according to the incubation time during mass fermentation

Hereinafter, the present invention will be described in detail.

Vitamin D3 is involved in the metabolism of calcium and phosphorus in the living body, and is also produced in trace amounts by UV irradiation in the skin using ergosterol or 7-dihydrocholesterol as precursors. The resulting vitamin D3 is converted into 25-hydroxyvitamin D3 by the introduction of a hydroxy group on carbon number 25 in the liver, which in turn is introduced into the 1α carbon by the kidney, thereby introducing 1α, 25-dihydroxyvitamin D3, 25-hydroxyvitamin and calcitriol, which are converted to calcitriol and thus converted, are known as active vitamin D3 with Ca absorption and metabolic activity in vivo (Hector et al., 1983, Vitamin D: Recent advances, Ann. Rev. Biochem . 52: 411-439), among which calcitriol is found to be the most active (Joji et al., 1991, Applied and environmental microbiology , 2841-2846). Thus, in order to search for strains with the ability to introduce biotransformation, that is, hydroxyl groups into vitamin D3, soil was collected from various places to separate microorganisms. Among them, wild strain producing 25-hydroxyvitamin D3 was isolated by bioconversion of precursor vitamin D3 from soil collected from the rear of the institute. In detail, the actinomycetes were screened and isolated from the collected soil to obtain only the cells, and then suspended in 20mM potassium phosphate buffer (pH6.8) and dissolved in vitamin D3 in ethanol at a concentration of 100 mg / ml. ㎖) was added and mixed, followed by reaction for 3 days in a shaker (200rpm, 28 ℃). After adding 3 ml of the extraction solvent (methylene chloride / methanol = 1/2) to 3 ml of the reaction mixture, the methylene chloride layer was taken and concentrated under reduced pressure at 40 ° C. or lower. Was found, and wild strains were selected that produced substances exhibiting UV patterns and RTs consistent with 25-hydroxyvitamin D3 pure products. The productivity of 25-hydroxyvitamin D3 was low at 0.82 mg / l.

After understanding the nutritional composition of various media sources for the selected strains, we confirmed the productivity of active vitamin D3 according to media sources such as carbon source, nitrogen source, and metal source, and based on this, multi-stage of UV, NTG, EtBr mutants Variation increased the productivity of the active vitamin D3, and also confirmed the difference in productivity according to the spawn culture medium and incubation period during the cultivation process. To maximize the productivity of 25-hydroxyvitamin and calcitriol.

The identification of strains is described by Shirling and Gottlieb's method (Shirling et al., 1966, Methods for characterization of Streptomyces species, Int. J. System. Bacteriol ., 16, 313-340) and Bergey's manual of systematic bacteriology (Williams et al., 1989, Bergey's Manual of systematic bacteriology Vol. 4, Williams & Wilkins et al.

The morphological characteristics of the strains are that the mycelia form irregular rectiflexible branches, and the spore chains form many spores in glycerol-asparagine agar, inorganic salts-starch agar, oatmeal agar, etc. In the dead phase, there was a segmentation phenomenon and the spore surface was smooth, cylindrical form (Fig. 2).

The cultivation characteristics were that the solid medium was grown in various media sources, the initial growth was late, and the cell mass increased rapidly after 4-5 days of cultivation. The wrinkles were present on the colony surface. Pigmented, colony reverse side was light brown. In the liquid culture, the core continued to grow until 7 days of culture, and then decreased, and mycelia were extended from the core.

The physiological characteristics are described in Table 1 and the wall chemotype is type VI and the whole-cell sugar pattern is type A. Therefore, according to the above results, it was confirmed that it is a related strain of Pseudonocardia autotrophica, and it was named Pseudonocardia autotrophica ID9302. Was given a deposit number

.Characteristics ID9302 Arabinose + Fructose + Glucose + Inositol + Mannitol + Raffinose + Rhamnose + Sucrose + Xylose + 10 ℃ + 45 ℃ - Adenine + Casein - Hypoxanthine + Tyrosine + Urea + Esulin + Starch +

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1

As a method of improving the strain, strain improvement using a multi-stage mutagen of UV irradiation, NTG treatment, and EtBr treatment was performed. First, spread the strain on the SYM medium at 28 ℃ for 7 days

Incubated sufficiently. After enough spores were formed, the spores were suspended in 20% glycerol, sonicated for 10 minutes, filtered through sterile cotton wool, collected in spores, stored in a deep freezer for 3 days, and thawed to determine the number of surviving spores. 10 ⁴ / ml) was used for the test aliquot.

In UV, mutations are most likely to occur at 99.9% mortality. The time point was about 7-10 minutes, and the colonies were selected under conditions showing a mortality rate of 99.99% after 15 minutes of treatment. In detail, 1ml per shale smeared and dried and incubated for 1 hour in a 28 ℃ incubator. The treated spores were irradiated with UV (254 nm) at 0, 1, 1.5, 2, 2.5, 3, 4, 5, 7, 10, 12, 15 and 30 minutes, respectively, wrapped in a curry foil and placed in a 4 ℃ refrigerator for 2 hours. After storage, the colonies were selected by culturing for 4 days at 28 ℃ incubator.

In the NTG treatment, the mutation rate is known to be the best at 40-50% killing. This strain is very sensitive to NTG and treated at 10 minute intervals showed 50% mortality between 10 and 20 minutes, but it was 99% after 45 minutes. Colonies were selected under abnormally killed conditions.

In EtBr treatment, EtBr was treated in 37 ° C. culture to induce the mutation of the bacteria. According to the paper, 10 or 20ug / ml should be treated, but the initial growth of the strain was slowed down to less than 5ug / ml to induce mutations of 60% mortality. As a result of culturing the mutated strain (ID9302) according to the culturing process, the productivity of 25-hydroxy vitamin D3 was increased by 5 times compared to wild strain 4 mg / L.

Example 2

ID9302 was incubated in solid medium of 1% starch, 0.4% yeast extract, 1% malt extract, and 2% agar for 4 days and cut into 1 cm 2 area x glucose 1.5%, soyton 1.5%, sodium chloride 0.5%, 50 ml (in 300 ml Erlenmeyer flask) containing sterile liquid medium (pH 7.0) containing 0.2% calcium carbonate was used as the primary seed medium. ID9302 inoculated in the primary seed medium was shaken at 28 rpm at 28 ° C. for 1-3 days and used as a secondary seed culture medium. The pH of the secondary seed culture should be maintained between 6.8-7.8. The culture medium was used as a dry yeast 0.4%, glucose 1%, starch 3%, fish meat 1%, sodium chloride 0.2%, phosphate 0.01%, calcium carbonate 0.2%, pH 7.0 sterile liquid medium 50ml in a 300ml Erlenmeyer flask Shaking was incubated at 28 ° C. at 200 rpm for 6-10 days. The dose of the precursor vitamin D3 was 5 mg dissolved in 50 μl of ethanol, and 6 ml of extraction solvent (methylene chloride / methanol = 1/2) was added to 3 ml of the culture medium, followed by mixing. Concentration was confirmed by productivity by comparing the area of 25- hydroxyvitamin D3 and calcitriol showing the UV pattern and RT consistent with the pure 25-hydroxyvitamin D3 and calcitriol pure by HPLC analysis. The sphere ODS-H80 (150 × 4.6mmID), mobile phase was 85% -100% methanol (in H2O), flow rate was 1ml / min, and detection was performed using a photodiode array detector. The maximum productivity of 25-hydroxyvitamin D3 was 10 mg / l and the maximum productivity of calcitriol was 1 mg / l. This is the result showing the highest productivity among the production test of active vitamin D3 using the bioconversion capacity of microorganisms published to date.

Example 3

In addition to the conditions of the main culture medium of Example 2, the production of active vitamins is possible even by adding or replacing carbon sources such as sucrose, trehalose, maltose, and nitrogen sources such as soy flour, peanut, corn flour, and peptone. In addition, the productivity of the active vitamin D3 also increases and decreases in culture by varying the medium concentration of Example 2.

Example 4

When the dose of vitamin D3, the precursor of Example 2, was increased to 20 mg, the productivity of 25-hydroxyvitamin D3 was 40 mg / l and the productivity of calcitriol was 5.5 mg / l, which was proportional to the concentration of the precursor. The productivity of active vitamins is believed to increase.

Example 5

Instead of the vitamin D3 precursor of Example 2, 1 mg of 25-hydroxyvitamin D3 was dissolved in 50 µl of ethanol, and the same method as in Example 2 yielded 1 mg / L of calcitriol productivity. When bioconversion was increased to 4, 5 mg, calcitriol productivity was also increased proportionally to 2, 3.2, 4.1, and 5.4 mg / l.

Example 6

In addition to ethanol used as a precursor solubilizer, surfactants cremophore, propylene glycol, dipropylene glycol, tween 85, tween 80, detaine 96, and PEG 300 were mixed in the same ratio with ethanol to administer 5 mg of vitamin D3 to 50 ml. When the same process as in the 25-hydroxyvitamin D3 and calcitriol productivity was confirmed that the increase of the use of cremophore, tween 80, tween 85, 2.5 times higher than the ethanol alone, 2.5 mg / ℓ of calcitriol productivity It was. When 20 mg of vitamin D3 was administered under the same conditions, the productivity of 25-hydroxyvitamin D3 was 50 mg / l and the productivity of calcitriol was 9.7 mg / l.

Example 7

The main culture using the medium and the conditions of the Example was carried out 20L culture in a 30L fermenter and the culture conditions of the fermenter were incubated for 10 days at 28 ℃, aeration rate 1VVM, agitation speed 500rpm. Since vitamin D3 dose was 2g and the initial growth of the strain was slow, the mixture was stirred at 500rpm, and then cultured by lowering the stirring rate to 400rpm on the 3rd day of culture to prevent the hyphae and to control the growth rate. After 5 days of culture, the aeration rate is lowered to 0.5VVM to control the growth of the strain so that the pH of the culture solution does not exceed 8.0. Maximum productivity was shown on day 8 of culture, and the productivity of 25-hydroxyvitamin D3 was 10.4 mg / l and calcitriol was 3.86 mg / l (Fig. 3). This is because the hydroxylation by the p450 system is known to be controlled by oxygen, and thus the oxygen transfer capacity by the fermenter is considered to be a better result. These results also indicate that higher calcitriol productivity can be predicted with higher vitamin concentrations.

Example 8

In order to optimize the bioconversion capacity of vitamin D3 to 25-hydroxyvitamin D3 and calcitriol using ID9302, the culture process was established. In detail, ID9302 was grown in solid medium at 28 ° C for 4 days and then seed cultured. Incubate for 3 days in the medium. Since the stationary phase is maintained for 3 days, the medium is cultured after 3 days of preculture. The medium composition is the same as the preculture medium, and 5% of the medium culture was used as the inoculum, and the medium culture was carried out for 3 days, and the pH was not to exceed 8. Based on the main culture solution, the main culture is inoculated with 5% of the intermediate culture solution. Since the p450 hydroxylase system is activated at the beginning of the culture, the biotransformation reaction is carried out by inoculating the precursor at the same time as the main culture. Because of this phenomenon, the growth rate of the strain is maintained slowly so that the pH does not exceed 8 while adjusting the aeration amount and the stirring speed on the third day of the culture.

Example 9

After centrifugation of 20 L of the bioconversion reaction, the cells were extracted with 10 L of acetone and concentrated under reduced pressure at 40 ° C. or lower. The culture filtrate was adsorbed onto HP20 and eluted with methanol to separate the active substance. It was mixed with the cell extract. The mixture was re-extracted with hexane and dichloromethane (2/1) and concentrated under reduced pressure at 40 ℃ or less. The concentrated solution was dissolved in hexane and dichlormethane (3/7), and left at -20 ° C for 1 hour. The supernatant was collected and applied to a Sephadex LH20 column. The mobile phases were hexane and dichlormethane (3/7), followed by vitamin D3, 25-hydroxyvitamin D3 and calcitriol. The purity of vitamin D3 and 25-hydroxyvitamin D3 was more than 90%, and vitamin D3 and 25-hydroxyvitamin D3 were isolated 1.2g and 110mg, respectively, which could be reused as precursors for calcitriol production. Calcitriol aliquots were separated under conditions of YMC J'sphere ODS column, 55% CH 3 CN, 265 nm, 5 ml / min to give 52 mg of pure white crystals of calcitriol.

According to the present invention, the production cost of calcitriol can be greatly reduced by producing stereospecific and regiospecific materials produced by organic synthesis by bioconversion of microorganisms. Calcitriol, an osteoporosis treatment, can be produced stably, economically and environmentally.

Claims (2)

Pseudocardia autotrophica ID9302 with bioconversion ability to introduce hydroxyl groups into vitamin D3 Method for producing calcitriol by introducing a hydroxyl group to precursor vitamin D3 or 25-hydroxyvitamin D3 using Pseudonocardia autotrophica ID9302 having bioconversion ability