KR100777488B1 - Effective and Selective Method of [?-HyMeLeu4]Cyclosporin A Separation from Sebekia benihana Culture Broth - Google Patents

Abstract

The present invention is a method for effectively and selectively recovering [gamma-hydroxymethylleucine 4] cyclosporin A bioconverted from cyclosporin A using a rare actinomycete Senekia benihana using a resin having hydrophobic properties. It is about.

Cyclosporin A, [gamma-hydroxymethylleucine 4] cyclosporin A, Severkia bennihana, bioconversion, recovery, purification

Description

Effective and Selective Method of [γ-HyMeLeu4] Cyclosporin A Separation from Sebekia benihana Culture Broth} from Sebekia benihana culture             

1 is a structural formula of [gamma-hydroxymethylleucine 4] cyclosporin A bioconverted from cyclosporin A by Sebekia benihana.

2 is a liquid chromatographic analysis of the concentrations of cyclosporin A and [gamma-hydroxymethylleucine 4] cyclosporin A remaining in the residue after treatment of Sebekia benihana cultures with various types of resins having hydrophobic properties. Chart of the results measured using HPLC (HPLC)

Cyclosporin A is an oligopeptide of 11 amino acids with hydrophobic properties. It is a potent immunosuppressive agent for treating rejection in organ transplant patients. It has side effects such as renal toxicity, hepatotoxicity, hypertension and periodontal tissue. It has various physiological effects such as hypertrophy and hair growth effect.

Cyclosporine A is the most abundant of the 24 structurally similar metabolites (cyclosporine A to Z) produced when incomplete fungi, Topocladium inflatum, are cultured in aerobic liquid. Cyclosporin A is biosynthesized by a multifunctional single enzyme called cyclosporin synthetase, in which 11 amino acids are sequentially linked, N-methylated, and form peptide bonds.

  When bioconversion of cyclosporine A using various bacteria and fungi, [gamma-hydroxymethylleucine4] cyclosporin A ([γ-hyMe Leu4] -cyclosporin A) was developed by the rare actinomycetes Sebekia benihana. 35% with [gamma-hydroxysilin4] cyclosporin A ([[gamma] -HyLeu4] -cyclosporin A) 4.5% with [gamma-methylleucine 4, gamma-hydroxymethylleucine 6] cyclosporin A ([[gamma] -HyMeLeu4] , γ-HyMeLeu6] -cyclosporin A) was found to convert 8.6% (J. Antibiotics, 1996; 49; 781-787). In addition, Korean Patent Nos. 10-0316604 and US Pat. No. 65,215,952 disclose that [gamma-hydroxymethylleucine 4] cyclosporin A ([[gamma] -hyMe Leu4] -cyclosporin A) obtained by bioconversion from cyclosporin A has various properties of cyclosporin A. The immunosuppressive properties and hepatotoxicity are reduced, and hair growth is maintained, so it can be used very usefully.

Bioconversion from cyclosporin A to [gamma-hydroxylmethylleucine 4] cyclosporine A using Sebecia benihana described in Kuhnt (J. Antibiotics. 1996: 49 (8): 781-787). The method of separating and purifying the material is extracted by adding 1,2-dichloroethane to the culture medium in which the mycelia are separated and concentrated by using a vacuum rotary concentrator, followed by gel filtration with Sephadex LH-20 gel. The fraction is a method of separating [gamma-hydroxylmethylleucine 4] cyclosporin A using silica gel chromatography. However, this method has a disadvantage in that industrialization is difficult because the gel filtration process is difficult to scale up.

To date, no recovery of [gamma-hydroxymethylleucine 4] cyclosporine A recovery has been reported except for the method of Kuhnt (J. Antibiotics. 1996: 49 (8): 781-787). For example, there are British Patent 1491509, US Patents 4117118 and 5256547 and 5156960. However, these separation and purification methods of cyclosporin A also include a gel filtration process similar to the method described in Kuhnt's thesis, and thus scales up the [gamma-hydroxymethylleucine 4] cyclosporine A recovery purification process. There is a problem that is not suitable for application or reference.

Therefore, the present inventors do not use the organic solvent extraction concentration method, which is a disadvantage of the conventional [gamma-hydroxymethyl leucine 4] cyclosporine A recovery and purification method, and the gel filtration process, which is difficult to apply industrially due to difficulty in scaling up. We tried to find an efficient and selective recovery and purification method of [gamma-hydroxymethyl leucine 4] cyclosporin A, and using the hydrophobic resin, unreacted cyclosporin A and the conversion [gamma-hydroxymethyl leucine 4] cyclosporin A The present invention was completed by successively adsorbing and recovering and selectively desorbing only [gamma-hydroxymethylleucine 4] cyclosporin A by changing pH, salt and alcohol concentration conditions.

For the use of [gamma-hydroxymethylleucine 4] cyclosporin A, which is bioconverted from cyclic cyclosporin A consisting of 11 amino acids having hydrophobic properties, using Sebekia benihana, for the purpose of hair regrowth or hair loss inhibitor It is an object of the present invention to provide a method for efficiently and selectively recovering and purifying [gamma-hydroxymethylleucine 4] cyclosporin A from a culture by applying hydrophobic resin.

Example 1: Bioconversion from cyclosporin A to [gamma-hydroxymethylleucine 4] cyclosporin A using Sebekia benihana

Sebekia benihana strain used for the bioconversion from cyclosporin A to [gamma-hydroxymethylleucine 4] cyclosporin A is strain KCTC 9173 stored in the Biotechnology Center of the Biotechnology Research Institute, and the yeast extract for its culture (Yeast Extract) 1%, Soybean Powder 0.5%, Glucose 0.7%, Soluble Starch 1%, and calcium carbonate (CaCO3) 0.005% medium was used, 30 ℃ After 36 hours of incubation, cyclosporin A dissolved in DMSO (Dimethyl Sulfoxide) was added at a concentration of 500 mg / L, and further 36 hours were incubated to convert biosporin A to [gamma-hydroxymethylleucine 4] cyclosporin A. Completed.

Example 2: Bioconverted Culture Pretreatment

After bioconversion of [gamma-hydroxylmethylleucine 4] cyclosporin A from cyclosporin A as in Example 1, alcohol was added to the culture to a final concentration of 33% and centrifuged to remove the Severkia venihana hyphae. Then, a solution containing residual cyclosporin A and [gamma-hydroxymethylleucine 4] cyclosporin A was obtained and used for the adsorption recovery process using hydrophobic resin. [Gamma-hydroxymethylleucine 4] cyclosporin A content analysis was performed using liquid chromatography (HPLC), and the analysis conditions were 3: 7 of water and acetonitrile (Acetonitril), and the column temperature was 55 ° C.

Example 3: Screening of Hydrophobic Resin Adsorbing [Gamma-Hydroxymethylleucine 4] Cyclosporin A

Dilute resins with the same hydrophobic properties were selected to effectively adsorb cyclosporin A consisting of 11 amino acids with hydrophobic properties and hydroxylated but also highly hydrophobic [gamma-hydroxymethylleucine 4] cyclosporine A ( Table 1), these resins were added to the liquid obtained in Example 2 at concentrations of 1, 2, 5, 10% and treated for 2 hours at room temperature. The residue was taken up after the adsorption treatment, and the concentrations of [gamma-hydroxylmethylleucine 4] cyclosporin A and cyclosporin A were analyzed by liquid chromatography (HPLC), and the results are shown in FIG. 2. Experiments included Amberlite XAD-16, Amberber XAD-7, Sufelco Diaion HP2MG, Amberlite XAD-1180. ) Hydrophobic resins were used. Amberlite XAD-16 and Amberlite XAD-1180 resin showed better adsorption than other hydrophobic resins and decided to use relatively low cost Amberlite XAD-16 to reduce costs in mass production. It was.

Table 1. Hydrophobic resins used for adsorption of [gamma-hydroxylmethylleucine 4] cyclosporine A

Resin Amberlite X-ED-16 Amberlite XAD-16 Amberlite XAD-7 Amberlite XAD-7 SUPELCO DIION HP2MG (Supelco Diaion HP2MG) Amberlite XAD-1180 (Amberlite XAD-1180) Price (Won / 100g) 47,100 41,700 43,500 65,800 Characteristic Hydrophobic Polyaromatic Resin Moderately Polar Acrylic Resin Moderately Polar Acrylic Resin Hydrophobic Polyaromatic Resin Surface area 800 m2 / g 450 m2 / g 500 m2 / g 600 m2 / g Average pore diameter 100 Å 90 Å 200-800 Å 800 Å Pore volume 1.82 ml / g 1.14 ml / g 1.2 ml / g 1.68 ml / g Mesh size 20-60 20-60 25-45 20-60

Example 4 Selective Desorption Conditions of Amberlite XED-16 Resin

Ethanol to confirm the difference according to pH conditions, salt concentration conditions, and alcohol concentration conditions when desorption of [gamma-hydroxylmethylleucine 4] cyclosporin A and cyclosporin A adsorbed on Amberlite XAD-16 selected in Example 3 Desorption of cyclosporin A and [gamma-hydroxymethylleucine 4] cyclosporin A attached to the resin at a concentration of 40 to 50%, a NaCl concentration of 0 to 1 M, and a pH of 4 to 10 was performed by liquid chromatography (HPLC). Analyzed.

The results of the analysis are shown in Tables 2, 3, and 4, and the higher the alcohol concentration, the higher the amount of [gamma-hydroxymethylleucine 4] cyclosporin A desorbed, but the purity was lowered. -The amount of hydroxylmethyl leucine 4] cyclosporin A was found to increase. Therefore, the optimum desorption condition of [gamma-hydroxymethyl leucine 4] cyclosporin A was found to be 40% alcohol concentration, pH 4, NaCl 1 M, and [gamma-hydroxymethyl leucine 4] cyclosporine A desorption under such conditions. Since the ratio of is larger than the ratio of cyclosporin A, it was found that [gamma-hydroxymethyl leucine 4] cyclosporin A can be obtained more selectively.

Table 2. Change analysis of [gamma-hydroxymethylleucine 4] cyclosporin A desorbed at pH and NaCl concentrations in 40% ethanol

 NaCl concentration (M) PH 4 PH 7 PH 10 water*(%) Desorption amount † (%) water*(%) Desorption amount † (%) water*(%) Desorption amount † (%) 0 100 6.2 91.8 20.0 82.3 8.2 0.1 100 10.5 86.5 16.0 79.8 10.4 0.5 100 16.3 80.3 22.7 78.7 10.5 One 100 22.1 82.0 26.5 77.6 24.7

* Purity = purity of [gamma-hydroxymethylleucine 4] cyclosporin A measured by HPLC after desorption

† Desorption amount = percentage of [gamma-hydroxymethylleucine 4] cyclosporine A obtained after desorption of [gamma-hydroxymethylleucine 4] cyclosporin A of the culture medium before the resin adsorption

    Table 3. Change analysis of [gamma-hydroxymethylleucine 4] cyclosporin A desorbed at pH and NaCl concentrations in 45% ethanol

 NaCl concentration (M) PH 4 PH 7 PH 10 water*(%) Desorption amount † (%) water*(%) Desorption amount † (%) water*(%) Desorption amount † (%) 0 65.0 26.4 60.1 22.4 74.7 20.4 0.1 67.1 28.8 72.2 22.5 75.3 24.9 0.5 66.4 28.9 67.6 26.1 75.1 31.6 One 69.5 32.3 67.1 30.4 77.6 54.5

* Purity = purity of [gamma-hydroxymethylleucine 4] cyclosporin A measured by HPLC after desorption

† Desorption amount = percentage of [gamma-hydroxymethylleucine 4] cyclosporine A obtained after desorption of [gamma-hydroxymethylleucine 4] cyclosporin A of the culture medium before the resin adsorption

Table 4. Change analysis of [gamma-hydroxymethylleucine 4] cyclosporin A desorbed at pH and NaCl concentrations in 50% ethanol

 NaCl concentration (M) PH 4 PH 7 PH 10 water*(%) Desorption amount † (%) water*(%) Desorption amount † (%) water*(%) Desorption amount † (%) 0 76.2 40.2 64.8 38.5 58.1 28.1 0.1 71.8 46.3 70.8 48.9 51.6 32.4 0.5 68.1 60.2 66.9 46.9 65.6 61.8 One 66.4 64.3 59.7 44.6 64.7 67.7

* Purity = purity of [gamma-hydroxymethylleucine 4] cyclosporin A measured by HPLC after desorption

† Desorption amount = percentage of [gamma-hydroxymethylleucine 4] cyclosporine A obtained after desorption of [gamma-hydroxymethylleucine 4] cyclosporin A of the culture medium before the resin adsorption

The present invention provides a hydrophobic mixture of cyclosporin A and [gamma-hydroxymethylleucine 4] cyclosporin A remaining in the culture when obtaining [gamma-hydroxymethylleucine 4] cyclosporine A from cyclosporine A by a method for bioconversion. The present invention relates to a method of selectively desorbing and recovering only [gamma-hydroxymethylleucine 4] cyclosporine A by adsorption using resin, and compared to a recovery purification method using conventional Sephadex gel and silica gel. Because A and [gamma-hydroxymethyl leucine 4] cyclosporin A can be effectively concentrated and recovered, and only [gamma-hydroxymethyl leucine 4] cyclosporin A can be purified when desorption under appropriate alcohol, salt and pH conditions. [Gamma-hydroxymethylleucine 4] cyclosporin A is used for industrially useful purposes such as hair repellents. May be used for the manufacturing process is very effective in establishing order group.

Claims (5)

In preparing [gamma-hydroxymethylleucine 4] cyclosporin A from cyclosporin A by a bioconversion method, a mixture of cyclosporin A and [gamma-hydroxymethylleucine 4] cyclosporin A remaining in the culture has hydrophobic properties. After adsorption using a resin having, a gamma-hydroxymethylleucine 4 cyclosporin A was used therefrom with a desorption solvent having a pH of 3.5 to 6, an alcohol concentration of 40% to 50% and a NaCl concentration of 0.001M to 1M. Purification method of [gamma-hydroxymethyl leucine 4] cyclosporine A, characterized by selectively desorbing and recovering bays The method of claim 1, wherein the hydrophobic resin is characterized in that the Amberlite XAD-16 (Amberlite XAD-16) or Amberlite XAD-1180 (Amberlite XAD-16) The method according to claim 2, wherein the hydrophobic resin Amberlite XED-16 is used at 1 to 10%. delete The method of claim 1, wherein the desorption condition is characterized in that the desorption solvent has a pH of 4, an ethanol concentration of 40% and a NaCl concentration of 1M.

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