KR102015100B1 - Method for recovering or purifying gentamicin B from fermented culture medium of microorganism - Google Patents

Abstract

In the method for recovering or purifying gentamicin B according to an embodiment of the present invention, the microorganism fermentation broth containing gentamicin B and its flexible substance is passed through a cation exchange resin to adsorb gentamicin B, and gentamicin is used as an eluent. After eluting B, the gentamicin B-containing eluent is passed through an anion exchange resin and decolorized. The recovery or purification method of gentamicin B according to the present invention can overcome the economic burden, complicated process and low purification yield of the purification process generally performed because the process is simple and the recovery yield or purification purity of gentamicin B is high. It is cost-competitive and can be applied to mass production. Therefore, by using the method for recovering or purifying gentamicin B according to the present invention, it is possible to secure high-quality gentamicin B used as a precursor of an antibiotic such as isepamycin.

Description

Method for recovering or purifying gentamicin B from fermented culture medium of microorganism

The present invention relates to a method for recovering or purifying gentamicin B from a microbial fermentation broth, and more particularly, to recovering gentamicin B in high yield or purifying it with high purity from a microbial fermentation broth comprising gentamicin B and a flexible substance thereof. It is about how to.

Gentamicin is a micromonospora purprea ) or micromonospora echinospora echinospora ) is an aminoglycoside antibiotic produced by Gram-positive bacteria and E. coli, Salmonella and its modified bacteria, and Pseudomonas aeruginosa. Gentamicin is also used in the treatment of enterococcal endocarditis with penicillin or ampicillin.

Gentamicin has a number of components called flexible substances. The major constituents of gentamicin are the gentamycin C complex, and the gentamycin C complex includes gentamycin C1, gentamycin C1a, gentamicin C2 and the like. Gentamicin C complex constitutes about 80% of gentamicin and is mainly used in clinical practice. In addition, minor components constituting gentamicin include gentamycin A, gentamycin A1, gentamycin X2, gentamycin B, gentamycin B1, and the like, and the minor components constitute about 20% of gentamicin. The following Chemical Formulas 1 and 2 show the basic structures of various gentamicins or derivatives thereof, and Table 1 specifically shows the types of gentamicin and its derivatives corresponding to the structures of Chemical Formulas 1 and 2.

Gentamicin Class R1 R2 R3 R4 R5 R6 Chemical formula A H OH NHCH ₃ OH H NH ₂ One A1 OH H NHCH ₃ OH H NH ₂ One X2 CH ₃ OH NHCH ₃ OH H NH ₂ One B CH ₃ OH NHCH ₃ NH ₂ H OH One B1 CH ₃ OH NHCH ₃ NH ₂ CH ₃ OH One C1 CH ₃ OH NHCH ₃ NHCH ₃ CH ₃ NH ₂ 2 C1a CH ₃ OH NHCH ₃ NH ₂ H NH ₂ 2 C2 CH ₃ OH NHCH ₃ NH ₂ CH ₃ NH ₂ 2 G-418 CH ₃ OH NHCH ₃ OH CH ₃ NH ₂ One JI-20A CH ₃ OH NHCH ₃ NH ₂ H NH ₂ One JI-208 CH ₃ OH NHCH ₃ CH ₂ NH ₂ CH ₃ NH ₂ One

Regarding the production of gentamicin, Korean Patent No. 10-0032788 discloses magnesium chloride or manganese chloride as an inorganic salt when aerobic culture of microbial mutant strain (KFCC 10346) belonging to the genus Micromonospora A method for preparing gentamicin is disclosed, which is added at 0.2-0.4 g / l. In addition, Korean Patent No. 10-0029275 discloses a mutant micromonaspora perpuria (KFCC-10346) belonging to the genus Micro monospora and capable of growing in a medium containing high concentrations of gentamicin, by aerobic culture in a nutrient medium. A method for producing gentamicin by fermentation characterized by producing and accumulating gentamicin is disclosed. In addition, Korean Patent Publication No. 10-2009-0005436 discloses gentamicin using a recombinant strain consisting of recombining genes gntB, gntA, gntP, gntZ, gntD, btrD, kacA, and neo16 in a heterologous host that does not biosynthesize gentamicin. , A method for producing at least one substance selected from the group consisting of paromamine, 2'-N-acetyl paromamine and 2-deoxystreptamine It is. In addition, the Republic of Korea Patent No. 10-0519142 discloses a method of manufacturing a micro gentamicin mono spokes la greener Apulia (Micromonospora purpurea) KCTC 10506BP strains producing B and culturing them in gentamicin B production medium by gentamicin B is described have.

Meanwhile, isepamicin having the structure of Formula 3 is known to have much lower biotoxicity than gentamycin (ie, gentamycin C complex), and has excellent antibiotic effect against gentamycin resistant strains. Known. The ispamycin protects the 3,6 'amino group of gentamicin B with a benzyloxycarbonyl group and discloses N- (R-beta-benzyloxycarbonylamino-, as disclosed in US Pat. Nos. 4,136,254 and 4,283,528. Prepared by reacting with alpha-hydroxypropionyloxy) succinimide to bind an alpha-hydroxy-beta-aminopropionyl group with an amine group at the carbon position of gentamicin B. can do. Therefore, in order to increase the productivity of isepamycin, it is required in the art to increase the purity of the tablet together with the improvement of the productivity of the gentamicin B, which is an essential precursor for the production of isepamycin.

It is known that the fermentation titer of the strain specialized in the production of gentamicin B is about 800 mg / l, and it is expected to reach 1,000 ~ 1,500 mg / l in a short time by improving the strain and optimizing the fermentation condition. However, in the fermentation process of the gentamicin B producing strain, various other gentamicin flexible components having a similar structure to the gentamicin B are generated together, and thus there is a problem in that purification is difficult even if the fermentation titer is high. Therefore, it is necessary to study a purification method capable of securing high purity gentamicin B. In connection with the method for separating or purifying antibiotics such as gentamicin, Korean Patent No. 10-1366216 discloses a method of preparing a pharmaceutical composition comprising: (a) dissolving a hydrochloride salt of cefcapene pivoxil with a primary solvent of an alcohol of C1 to C3; (b) adding and diluting a secondary solvent comprising at least one selected from the group consisting of dichloromethane, ethyl acetate, benzene, hexane, acetone, methyl isobutyl ketone and methyl ethyl ketone; And (c) further adding a hydrochloric acid aqueous solution. In addition, Korean Patent No. 10-0430202 discloses isoelectric focusing in a multi-compartment electrolyzer having a fixed zwitter ion membrane, wherein the support solution is an aqueous mixture of detergent and urea, which is a sulfobeta zwitterion derivative of cholic acid. A method for purifying a dalbaheptide antibiotic compound for use is disclosed. In addition, Korean Patent Publication No. 10-2001-0024027 discloses a solid β-lactam antibiotic obtained at a temperature between -5 and 20 ° C. such that the pH of the mixture is between 3 and 8 at a concentration of FG remaining in the solution. Β-lactam antibiotics and D-phenyl in solution consisting of recovering the temperature of the residual liquid, raising the temperature of the residual liquid to between 10 and 60 ° C., forming a solid FG, separating the FG as a solid and precipitating the mother liquor at least partially. A method for recovering β-lactam antibiotics from a mixture containing glycine (FG) is disclosed. In addition, Korean Patent Publication No. 10-2001-0024028 discloses that the pH of a mixture containing β-lactam antibiotics and D-phenyl glycine (FG) at a concentration of FG remaining in the solution is pH 3 to 8, and is obtained. Recover the solid β-lactam antibiotics, and form a slurry with solid β-lactam antibiotics and solid FG, bring the pH of the slurry to the pH at which β-lactam antibiotics dissolve, separate the FG as a solid, A method of recovering β-lactam antibiotics from a mixture containing β-lactam antibiotics and D-phenyl glycine is sent, wherein the residual liquid is sent to a concentration step using at least a portion of the β-lactam antibiotics present in the mother liquor. However, no method of purifying gentamicin B with high purity from microbial fermentation broth containing gentamicin B and other gentamicins having a structure similar to gentamicin B is disclosed in the prior art.

The present invention has been derived under the conventional technical background, and an object of the present invention is to provide a method for recovering gentamicin in high yield or purifying it with high purity in a microbial fermentation broth containing gentamicin B and a flexible substance thereof.

One embodiment of the present invention to solve the above object is a step of obtaining a gentamicin B-containing filtrate by filtration of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B; Contacting the gentamicin B-containing filtrate with a weakly acidic cation exchange resin to adsorb gentamycin B; Eluting the gentamicin B adsorbed on the cation exchange resin with an eluent to obtain a gentamicin B-containing eluent; And it provides a method for recovering or purifying gentamicin B comprising the step of decolorizing the gentamicin B-containing eluent or concentrate thereof in contact with a strong base anion exchange resin.

In the method for recovering or purifying gentamicin B according to an embodiment of the present invention, the flexible substance of gentamicin B may be micronomicin, gentamicin A, gentamycin A1, gentamicin X2, gentamicin B1, gentamicin C1. , May be composed of one or more selected from gentamicin C1a or gentamicin C2, preferably may be composed of a combination of micronomicin and gentamicin C1a.

In the method for recovering or purifying gentamicin B according to an embodiment of the present invention, the microorganism is not limited to a kind thereof if the microorganism produces gentamicin B and a flexible substance thereof, for example, micromonospora purpuria ( Micromonospora). purpurea ), Micromonospora echinospora or variant strains thereof. Specifically, the present invention is a micromonospora purpuria ( Micromonospora ) as a microorganism to produce gentamicin B and its flexible material. purpurea ) ATCC 15835 strain, Micromonospora purpurea ) may comprise a KCTC 10506BP strain. The micromonospora purpuria ( Micromonospora) purpurea ) KCTC 10506BP strain is disclosed in Korean Patent No. 10-0519142.

Filtration of the microbial fermentation broth in the method for recovering or purifying gentamicin B according to an embodiment of the present invention may be performed by centrifugation, filter paper, filter cloth, diatomaceous earth, paracell, or a combination of two or more thereof. And is preferably performed by a combination of centrifugation and PARACELL. In this regard, the step of obtaining the gentamicin B-containing filtrate in the method of recovery or purification of gentamicin B according to an embodiment of the present invention is a1) of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B adjusting the pH to 1.5-4.5 and centrifuging to obtain a gentamicin B-containing supernatant; And a2) adjusting the pH of the gentamicin B-containing supernatant to 5.5 to 7.0 and filtration with any one selected from filter paper, filter cloth, diatomaceous earth, or paracells (PARACELL). In addition, in step a1), the pH of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B is adjusted to 2 to 3 and left for 10 hours or more (for example, 12 to 24 hours) at 2 to 8 ° C. More preferably, by centrifugation followed by centrifugation to obtain the gentamicin B-containing supernatant. In addition, the step a2) is more preferably configured to adjust the pH of the gentamicin B-containing supernatant to 6.0 ~ 6.8 and left for 8 to 20 hours and then filtered with a paracell (PARACELL).

In the method for recovering or purifying gentamicin B according to an embodiment of the present invention, the weakly acidic cation exchange resin is preferably porous and has a particle size of 10 to 60 mesh. In addition, the weakly acidic cation exchange resin is more preferably a particle size of 15 ~ 50 mesh (mesh). In addition, it is more preferable that the weakly acidic cation exchange resin uses carboxylic acid as an exchanger. The weakly acidic cation exchange resin is commercially available, and commercial products include Amberite IRC-50, Amberite IRC-50S, Amberite IRP-64, and Dowex CCR-3.

In the method for recovering or purifying gentamicin B according to an embodiment of the present invention, the eluent is preferably 0.1-0.5 N concentration of ammonia water, and preferably 0.1-0.3 N concentration of ammonia water. In this regard, the step of obtaining the gentamicin B-containing eluent in the method for recovering or purifying the gentamicin B according to an embodiment of the present invention may be performed by washing the cation exchange resin in which the gentamicin B is adsorbed with water and using an eluent of 0.1-0.3N. Preferably, the ammonia water is contacted with a cation exchange resin to elute the flexible material of gentamicin B and gentamicin b adsorbed on the cation exchange resin. In addition, the content of gentamicin B in the gentamicin B-containing eluent is preferably 90% or more and more preferably 95% or more based on the total weight of the gentamicin flexible material.

In the method for recovering or purifying gentamicin B according to one embodiment of the present invention, the strongly basic anion exchange resin is preferably a gel type and has a particle size of 50 to 400 mesh. In addition, the strongly basic anion exchange resin is more preferably a particle size of 50 ~ 200 mesh (mesh). In addition, the strongly basic anion exchange resin is more preferably used a quaternary ammonium group as the exchanger. The strong basic anion exchange resin is commercially available, and commercial products include Dowex 1X2-200, Dowex 1X2-100, Dowex 1X2-400, Amberite IRA-743, DIAION SA10A, DIAION SA11A, DIAION SA12A, DIAION SA20A, etc. There is this. In addition, the gentamicin B-containing eluent is preferably concentrated before contact with the strong basic anion exchange resin until the gentamicin B concentration reaches a predetermined value. For example, the concentration of gentamicin B in the concentrate of the gentamicin B-containing eluate is preferably 8,000 to 30,000 µg / ml, and more preferably 8,000 to 15,000 µg / ml. In addition, the gentamicin B-containing eluate or concentrate thereof is preferably adjusted to a pH of 5.5 to 7.5, and more preferably to 5.5 to 6.5 before contact with the strong basic anion exchange resin.

The method for recovering or purifying gentamicin B according to one embodiment of the present invention may preferably further include powdering the gentamicin B-containing eluent or its concentrated solution decolorized by a strong basic anion exchange resin. As the powdering method, spray drying, lyophilization, or the like may be used. In addition, the gentamicin B-containing eluent decolored by the strong basic anion exchange resin or concentrate thereof may be further concentrated to facilitate powdering.

Another example of the present invention to solve the above object is a step of obtaining a gentamicin B-containing filtrate by filtration of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B; The gentamicin B-containing filtrate was contacted with the weakly acidic cation exchange resin to adsorb the gentamycin B, the cation exchange resin was washed with water, and the gentamicin B adsorbed on the cation exchange resin was eluted with the first eluent to contain the gentamicin B. Obtaining a first eluent; The gentamicin B-containing first eluate was contacted with the weakly acidic cation exchange resin to resorb the gentamicin B, the cation exchange resin was washed with water, and the gentamicin B resorbed to the cation exchange resin was eluted with a second eluent. Obtaining a mycin B-containing second eluent; And it provides a method for recovering or purifying gentamicin B comprising the step of decolorizing the gentamicin B-containing second eluent or concentrate thereof in contact with a strong base anion exchange resin. The recovery or purification method of gentamicin B according to another embodiment of the present invention uses a resorption method in which gentamicin B is repeatedly adsorbed on a weakly acidic cation exchange resin and eluted with an eluent.

In the method for recovering or purifying gentamicin B according to another embodiment of the present invention, it is preferable that the weakly acidic cation exchange resin has a porous type and has a particle size of 10 to 60 mesh and uses carboxylic acid as an exchanger.

In the method for recovering or purifying gentamicin B according to another embodiment of the present invention, it is preferable that the first eluent is ammonia water at a concentration of 0.1 to 0.3 N and the second eluent is ammonia water at a concentration of 0.4 to 0.6 N. In this regard, the step of obtaining the gentamicin B-containing first eluent is contacting the gentamicin B-containing filtrate with the weakly acidic cation exchange resin to adsorb the gentamicin B, wash the cation exchange resin with water, The adsorbed gentamicin B may be eluted with ammonia water at a concentration of 0.15 to 0.25 N, which is the first eluent. In addition, the step of obtaining the gentamicin B-containing second eluent is contacting the gentamicin B-containing first eluent with the weakly acidic cation exchange resin to resorb the gentamicin B, wash the cation exchange resin with water, 0.1 ~ 0.2N concentration After preliminarily eluting with ammonia water of, may be composed of eluting the gentamicin B reabsorbed in the cation exchange resin with an eluent of 0.4 ~ 0.6N concentration of the second eluent. In addition, the gentamicin B-containing second eluent is preferably concentrated to have a concentration of gentamicin B of 8,000 to 30,000 μg / ml (preferably 8,000 to 15,000 μg / ml) before contacting the strong acid anion exchange resin. In addition, the gentamicin B-containing second eluate or concentrate thereof is preferably adjusted to a pH of 5.5 to 7.5 (preferably 6.0 to 6.8) before contacting the strong acid anion exchange resin.

In the method for recovering or purifying gentamicin B according to another embodiment of the present invention, the strongly basic anion exchange resin is a gel type, has a particle size of 50 to 200 mesh, and uses a quaternary ammonium group as an exchanger. Do.

In the method of recovering or purifying gentamicin B according to another embodiment of the present invention, the step of obtaining the gentamicin B-containing filtrate may include a1) pH of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B of 1.5 to Adjusting to 4.5 and then centrifuging to obtain a gentamicin B-containing supernatant; And a2) adjusting the pH of the gentamicin B-containing supernatant to 5.5 to 7.0 and filtration with any one selected from filter paper, filter cloth, diatomaceous earth, or paracells (PARACELL).

In the method for recovering or purifying gentamicin B according to another embodiment of the present invention, the obtaining of the eluent of the method for recovering or purifying the gentamicin B according to one embodiment of the present invention includes obtaining the first eluent and obtaining the second eluent. For the features of the rest of the configuration is different in that it refers to the contents described in the method for recovering or purifying gentamicin B according to an embodiment of the present invention.

The recovery or purification method of gentamicin B according to the present invention can overcome the economic burden, complicated process and low purification yield of the purification process generally performed because the process is simple and the recovery yield or purification purity of gentamicin B is high. It is cost-competitive and can be applied to mass production. Therefore, by using the method for recovering or purifying gentamicin B according to the present invention, it is possible to secure high-quality gentamicin B used as a precursor of an antibiotic such as isepamycin.

1 is a result of HPLC analysis of purified gentamicin B powder obtained by the method of Example 12, Figure 2 is a result of HPLC-ELSD analysis of the purified gentamicin B powder obtained by the method of Example 12.

As used herein, the term "gentamicin analogue" refers to gentamicin A, A1, X2, B, B1, C1, C1a, C2 as well as micromycin (G-418), which constitutes gentamicin. , JI-20A, X2, JI-20B and the like, including various derivatives.

The term "flexible material of gentamicin B" used in the present invention is a component constituting gentamicin except for gentamicin B, and micronomicin, G-418, JI-20A, X2, The concept includes various derivatives such as JI-20B.

As used herein, the term "RV" is an abbreviation for Reactor Volume and means the volume of charged ion exchange resin in an ion exchange resin packed column system.

As used herein, the term "SV" is an abbreviation for Space Velocity and indicates the flow rate of the fluid through the packed bed.

Hereinafter, the present invention will be described in detail through examples. However, the following examples are only intended to clearly illustrate the technical features of the present invention, and do not limit the protection scope of the present invention.

Example  1: Preparation of microbial fermentation broth containing gentamicin B

45 g / l of dextrin, 15 g / l of corn powder, 35 g / l of degreasing, 35 g / l of peptone, 3 g of calcium carbonate, strain of gentamicin-producing micromonospora echinospora ATCC 15835 / l, ammonium sulfate 0.3 g / l, calcium nitrate 0.1 g / l, cobalt chloride 0.007 g / l inoculated in a medium and incubated for about 8 days at about 30 ℃ gentamicin B and gentamicin B analog A microbial fermentation broth containing was prepared.

Example  2: Selection of Gentamicin B Adsorption Resin

The pH of the fermentation broth prepared in Example 1 was adjusted to 2.5 using a 50 wt% sulfuric acid aqueous solution, and left at 4 ° C. for at least 12 hours, followed by centrifugation to obtain a fermentation supernatant. Then, 50 ml of the fermentation supernatant was mixed with 0.5 ml of the activated cation exchange resin (732, Amberlite IR-120) beads and stirred at 25 ° C. at a speed of 100 rpm for 2 hours and 30 minutes to add gentamicin B to the cation exchange resin beads. Adsorbed. In addition, 100 ml of the fermented supernatant was mixed with 0.5 ml of other activated cation exchange resin (Amberlite IRC-50) beads and stirred at 25 ° C. at 100 rpm for 2 hours 30 minutes to adsorb gentamycin B to the cation exchange resin beads. I was. In addition, 100 ml of the fermented supernatant was mixed with 0.5 ml of Diaion HP-20, which is a synthetic adsorption resin, and stirred at 25 ° C. at 100 rpm for 2 hours and 30 minutes to adsorb gentamicin B to the synthetic adsorption resin. The adsorption amount of gentamicin B etc. according to the kind of adsorption resin used in following Table 2 was shown.

Adsorption Resin Type Gentamicin B adsorption amount (mg / ml) Micromycin Adsorption Amount (mg / ml) Gentamicin C1a adsorption amount (mg / ml) 732 8.70 3.50 2.12 Amberlite IR-120 12.13 5.27 3.13 Amberlite IRC-50 73.65 42.85 21.06 Diaion HP-20 27.35 11.44 5.04

* 732 is a strongly acidic cation exchange resin consisting of a gel type polystyrene sulfonate, and the polymer backbone is a crosslinked styrene-divinylbenzene copolymer

Amberlite IR-120 is a strong acid cation exchange resin composed of gel-type polystyrene sulfonate

Amberlite IRC-50 is a porous acid weak acid cation exchange resin, has particle size of 16 ~ 50 mesh and uses carboxylic acid as exchanger

* Diaion HP-20: Synthetic adsorption resin composed of a copolymer of styrene and divinylbenzene, and has many large pores

As shown in Table 2, Amberlite IRC-50, a porous weakly acidic cation exchange resin, adsorbed a relatively large amount of the flexible material of gentamicin B and gentamicin B.

Example  3: adsorption amount of gentamicin B according to adsorption temperature

The pH of the fermentation broth prepared in Example 1 was adjusted to 2.5 using a 50 wt% sulfuric acid aqueous solution, and left at 4 ° C. for at least 12 hours, followed by centrifugation to obtain a fermentation supernatant. The pH of the fermentation supernatant was then adjusted to 6.5 using 30% by weight aqueous sodium hydroxide solution, and 70 ml of the fermentation supernatant was mixed with 0.5 ml of activated Amberlite IRC-50 (NH4 ⁺ ) beads and subjected to various temperatures (14, Gentamicin B was adsorbed onto the cation exchange resin beads by stirring at 100 rpm (24, 34 ° C.) for 2 hours 30 minutes. Table 3 below shows the adsorption amount of gentamicin B and the like to Amberlite IRC-50 beads.

Adsorption temperature Gentamicin B adsorption amount (mg / ml) Micromycin Adsorption Amount (mg / ml) Gentamicin C1a adsorption amount (mg / ml) 14 ℃ 14.12 15.22 9.10 24 ℃ 16.37 21.95 10.21 34 ℃ 18.60 19.94 10.75

As shown in Table 3 above, Amberlite IRC-50 adsorbed more of the gentamicin B and the gentamicin B related substances as the adsorption temperature was higher, but the difference was not large. Therefore, the adsorption of gentamicin B and the like to Amberlite IRC-50 is estimated to proceed at room temperature.

Example  4: Adsorption amount of gentamicin B according to pH of fermentation supernatant

The pH of the fermentation broth prepared in Example 1 was adjusted to 2.5 using a 50 wt% sulfuric acid aqueous solution, and left at 4 ° C. for at least 12 hours, followed by centrifugation to obtain a fermentation supernatant. Thereafter, the pH of the fermentation supernatant was adjusted to various values (5.0, 5.5, 6.0, 6.5, 7.0) using an aqueous 30% sodium hydroxide solution, and 70 ml of the fermentation supernatant was activated Amberlite IRC-50 (NH4 ^+). ) Gentamicin B was adsorbed onto cation exchange resin beads by mixing with 0.5 ml beads and stirring at 25 ° C. at 100 rpm for 2 hours 30 minutes. Table 4 shows the adsorption amount of the fermentation supernatant to Amberlite IRC-50 beads, such as gentamicin B by pH.

PH of fermentation supernatant Gentamicin B adsorption amount (mg / ml) Micromycin Adsorption Amount (mg / ml) Gentamicin C1a adsorption amount (mg / ml) 5.0 20.53 19.67 5.47 5.5 30.13 27.11 8.65 6.0 32.80 29.65 9.47 6.5 39.86 36.23 11.76 7.0 36.57 31.94 11.91

As shown in Table 4, when the pH of the fermentation supernatant was 6, the adsorption amount of gentamicin B and the like to Amberlite IRC-50 was the highest.

Example  5: in the cation exchange resin column system Eluent  By concentration Adsorbate Elution  characteristic

The pH of the fermentation broth prepared in Example 1 was adjusted to 2.5 using a 50 wt% sulfuric acid aqueous solution, and left at 4 ° C. for at least 12 hours, followed by centrifugation to obtain a fermentation supernatant. Then, the pH of the fermentation supernatant was adjusted to 6.5 using an aqueous 30% by weight sodium hydroxide solution and left for 12 hours, followed by filtration with PARACELL, a filter aid (Filter-Aid), to obtain a fermentation filtrate. It was. The fermentation filtrate was then passed through a column filled with activated Amberlite IRC-50 (NH4 ⁺ ) beads at a rate of 3 RV / hr to adsorb gentamicin B and the like. Thereafter, the column was washed with distilled water corresponding to a capacity of 6 RV, and then the adsorbate was eluted by passing ammonia water of different concentrations (0.15N, 0.2N) into the column at a flow rate of 0.5 SV, respectively, as an eluent. Table 5 shows the gentamicin B recovery rate when the ratio of gentamicin B was about 95% based on the total weight of the gentamicin analogue contained in the eluent.

Eluent Gentamicin B recovery of 95% or more purity (%) 0.15N ammonia water 21.8 0.2N ammonia water 68.5

Example  6: containing gentamicin B Eluent  Selection of Activated Carbon for Decolorization

Based on the total weight of the gentamicin analogue obtained in Example 5, the eluent having a concentration of about 95% of the gentamicin B was concentrated until the concentration of the gentamicin B became about 100,000-120,000 μg / ml. Thereafter, the pH of the gentamicin B-containing concentrate was adjusted to 6.0 using an aqueous 6 M sulfuric acid solution. Thereafter, 0.1 g of activated carbon was mixed with 0.5 ml of gentamicin B-containing concentrate and decolorized for 1 hour at room temperature. Table 6 below shows the recovery of gentamicin B and the degree of decolorization of gentamicin B-containing concentrate by activated carbon.

Measurement item Activated Carbon Type CA1 CN1 CGSP SX-PLUS Gentamicin B recovery (%) 98.0 90.0 75.5 86.0 Concentration Decolorization (%) 13.6 30.8 46.2 1.0

As shown in Table 6, when the Norit CN1 activated carbon was used, the decolorizing effect was relatively excellent, and the recovery rate of gentamicin B was 90%. Subsequent experiments on activated carbon decolorization were performed using Norit's CN1 activated carbon.

Example  7: Gentamicin B Recovery and Decolorization Effect by Decolorization Reaction Temperature

Based on the total weight of the gentamicin analogue obtained in Example 5, the eluent having a concentration of about 95% of the gentamicin B was concentrated until the concentration of the gentamicin B became about 100,000-120,000 μg / ml. Thereafter, the pH of the gentamicin B-containing concentrate was adjusted to 6.0 using an aqueous 6 M sulfuric acid solution. Thereafter, 0.1 g of CN1 activated carbon of Norit Inc. was mixed with 0.5 ml of a gentamicin B-containing concentrate and decolorized for 1 hour at various temperature conditions (20, 40, 60 ° C). Table 7 below shows the recovery of gentamicin B and the decolorization degree of gentamicin B-containing concentrate by decolorization reaction temperature in the decolorization test using CN1 activated carbon of Norit.

Measurement item Decolorization reaction temperature 20 ℃ 40 ℃ 60 ℃ Gentamicin B recovery (%) 97.1 92.2 91.0 Concentration Decolorization (%) 9.2 30.5 36.8

The decolorization effect using No1's CN1 activated carbon was excellent at higher decolorization reaction temperature, but the recovery of gentamishi B tended to decrease as the decolorization reaction temperature was higher. However, even when the decolorization reaction temperature is 60 ℃, the recovery of gentamicin B was found to be 90% or more.

Example  8: of gentamicin B-containing concentrate by pH  Decolorization Effect of Activated Carbon

Based on the total weight of the gentamicin analogue obtained in Example 5, the eluent having a concentration of about 95% of the gentamicin B was concentrated until the concentration of the gentamicin B became about 100,000-120,000 μg / ml. Thereafter, the pH of the gentamicin B-containing concentrate was adjusted to various values (5.0, 5.5, 6.0, 6.5, 7.0, 8.0) using 6M aqueous sulfuric acid solution. Thereafter, 0.1 g of CN1 activated carbon of Norit Inc. was mixed with 0.5 ml of a concentrated concentration of gentamicin B containing pH and subjected to a decolorization reaction at 60 ° C for 1 hour. Table 8 below shows the recovery of gentamicin B by pH of the gentamicin B-containing concentrate and the decolorization degree of the gentamicin B-containing concentrate in the decolorization test using CN1 activated carbon of Norit.

Measurement item PH of gentamicin B-containing concentrate 5.0 5.5 6.0 6.5 7.0 8.0 Gentamicin B recovery (%) 77.7 72.3 67.4 63.4 71.9 68.2 Concentration Decolorization (%) 90.4 90.1 90.0 84.5 84.3 72.6

Example  9: decolorization experiment using anion exchange resin

Based on the total weight of the gentamicin analogue obtained in Example 5, the eluent having a ratio of about 95% of the gentamicin B was concentrated until the concentration of the gentamicin B became about 10,000 to 13,000 μg / ml. Thereafter, the pH of the gentamicin B-containing concentrate was adjusted to various values (6.0, 7.0, 8.0, 9.0) using an aqueous 6 M sulfuric acid solution. Thereafter, 0.5 ml of an activated anion exchange resin (Dowex 1X2-200, SAR10MB of Samyang Co.) was mixed with 0.5 ml of a concentrated gentamicin B-containing concentrate and decolorized for 25 minutes at room temperature. Table 9 shows the recovery rate of gentamicin B by pH of the anion exchange resin type and gentamicin B-containing concentrate used in the decolorization experiment, Table 10 below by the pH of the anion exchange resin type and gentamicin B-containing concentrate used in the decolorization experiment The decolorization degree of the gentamicin B containing concentrate is shown.

PH of gentamicin B-containing concentrate Gentamicin B recovery (%) Dowex 1X2-200 SAR10MB 6 59.8 61.8 7 55.2 64.3 8 40.6 42.9 9 52.1 54.8

PH of gentamicin B-containing concentrate Concentration Decolorization (%) Dowex 1X2-200 SAR10MB 6 96.8 89.4 7 95.9 90.5 8 98.8 93.2 9 98.0 92.9

* Dowex 1X2-200: Porous strong base anion exchange resin with 100 ~ 200 mesh particle size, porous Styrene-Divinylbenzene copolymer as base matrix and quaternary ammonium group as exchanger

* SAR10MB: Gel-type strong basic anion exchange resin, with Styrene-Divinylbenzene copolymer as base matrix and quaternary ammonium group as exchanger

As shown in Table 9 and Table 10, Dowex 1X2-200 anion exchange resin showed a better decolorizing effect than Samyang's SAR10MB anion exchange resin, and the difference in pH decolorization of gentamicin concentrate was not large. Also, the difference in the recovery rate of gentamicin between Dowex 1X2-200 anion exchange resin and Samyang's SAR10MB anion exchange resin was not significant. When Dowex 1X2-200 was used as the anion exchange resin and the pH of the gentamicin concentrate was 6, both the recovery rate of the gentamicin B and the decolorization of the gentamicin B-containing concentrate were excellent.

Example  10: Decolorization Effect of Gentamicin B Concentrations by Gentamicin B Concentration in Bleaching Process Using Anion Exchange Resin

200,000 μg / ml of the concentration of gentamicin B in the eluate obtained in Example 5 with respect to 95% of the gentamicin B based on the total weight of the gentamicin analogue; 100,000 μg / ml; 50,000 μg / ml; Concentrate until 10,000 μg / ml. Thereafter, the pH of the gentamicin B-containing concentrate having a different concentration of gentamicin B was adjusted to 6.0 using an aqueous sulfuric acid solution having a 6 M concentration. Thereafter, 0.5 ml of the activated Dowex 1X2-200 anion exchange resin was mixed with 0.5 ml of the gentamicin B-containing concentrate and decolorized for 25 minutes at room temperature. Table 11 below shows the decolorization degree of gentamicin B and gentamicin B-containing concentrate according to the concentration of gentamicin B in the gentamicin B-containing concentrate when decolorizing the gentamicin B-containing concentrate using Dowex 1X2-200 anion exchange resin. will be.

Measurement item Gentamicin Concentration of Gentamicin-Containing Concentrates 200,000 μg / ml 100,000 μg / ml 50,000 μg / ml 10,000 μg / ml Gentamicin B recovery (%) 80.5 98.0 93.4 92.1 Concentration Decolorization (%) 28.9 20.2 41.4 97.1

As shown in Table 11, when the gentamicin B-containing concentrate having a relatively low concentration of gentamicin B was decolorized using the Dowex 1X2-200 anion exchange resin, the gentamicin recovery rate and the gentamicin B-containing concentrate were higher in decolorization. appear.

Example  11: Decolorization of Gentamicin B Concentrate in Anion Exchange Resin Column System

Dowex 1X2-200 anion exchange resin was charged and activated in an open column.

Based on the total weight of the gentamicin analogue obtained in Example 5, the eluate having a concentration of about 95% of the gentamicin B based on the total weight of the gentamicin analogue (genmamic B concentration is about 6,000 µg / ml) was immediately activated without concentration. Dewet by passing through a column packed with Dowex 1X2-200 anion exchange resin. In addition, the concentration of gentamicin B in the eluate of about 95% based on the total weight of the gentamicin analogue obtained in Example 7 was about 10,000 μg / ml. The gentamicin B-containing concentrate was then decolorized by passing it through a column filled with activated Dowex 1X2-200 anion exchange resin. The flow rate of the liquid passing through the column was 4 SV and the liquid flow amount was 1 RV. Table 12 shows the results of the decolorization experiment of the gentamicin B-containing concentrate in the anion exchange resin column system.

Measurement item Gentamicin B concentration in the gentamicin B-containing eluate or gentamicin B-containing concentrate supplied to the anion exchange resin column 6,000 μg / ml 10,000 μg / ml Gentamicin B recovery (%) 78.2 95.0 Decolorization (%) 99.9 99.6

As shown in Table 12, when the gentamicin B-containing eluate having a gentamicin B concentration lower than 10,000 ㎍ / ml is passed through a column filled with a Dowex 1X2-200 anion exchange resin, the decolorization is excellent, but the recovery rate of gentamicin B is greatly increased. Appeared to fall.

Example  12: Optimal Design of Gentamicin B Recovery / Separation Process Using Re-adsorption

Experiments were conducted by optimally designing the recovery / separation process of gentamicin B from the results of Examples 1 to 11, and the specific unit process combinations are as follows.

1) The pH of the microbial fermentation broth containing gentamicin B and its flexible substance is adjusted to 2.5 using a 50% by weight aqueous sulfuric acid solution and left at 4 ° C. for at least 12 hours, followed by centrifugation to obtain a fermentation supernatant.

2) The pH of the fermentation supernatant was adjusted to 6.5 using a 30% by weight aqueous sodium hydroxide solution and left for 12 hours, followed by filtration with a para-cell (PARACELL), a filter aid (Filter-Aid). Get

3) The fermentation filtrate is passed through a column filled with activated Amberlite IRC-50 (NH4 ⁺ ) cation exchange resin at a rate of 3 RV / hr to adsorb gentamicin B and the like.

4) Wash the cation exchange resin packed column adsorbed with gentamicin B with distilled water corresponding to the capacity of 6 RV, and pass ammonia water of 0.2N concentration as eluent at a rate of 0.5 SV through the cation exchange resin packed column. Recover the primary eluent containing B and its flexible material.

5) Re-adsorb gentamicin B and the like by adjusting the pH of the primary eluent back to 6.5 and passing the activated Amberlite IRC-50 (NH4 ⁺ ) cation exchange resin at a rate of 3 RV / hr.

6) Wash the cation exchange resin packed column re-adsorbed gentamicin B, etc. with distilled water corresponding to the capacity of 6 RV, and add ammonia water of 0.15N concentration as eluent at a rate of about 7 RV to the cation exchange resin packed column. Elution was carried out by the amount corresponding to the preliminary elution, and then the eluent 0.5N concentration of ammonia water was passed through the cation exchange resin packed column at a rate of 0.5 SV to recover a secondary eluate containing gentamycin B. The recovery rate of gentamicin B is about 80.5%.

7) Concentrate the secondary eluate containing gentamicin B until the concentration of gentamicin B is 10,000 ㎍ / ml to prepare a gentamicin B-containing concentrate, and adjust the pH of the gentamicin B-containing concentrate to 6.0. Pass through a column packed with activated Dowex 1X2-200 anion exchange resin to obtain a first bleached gentamicin B containing concentrate.

8) Adjust the pH of the first decolorized gentamicin B-containing concentrate to 5.5, and add 30% CN1 activated carbon based on the weight of gentamicin B and stir to obtain the second degenerated gentamicin B-containing concentrate.

9) Freeze-drying the secondary decolorized gentamicin B-containing concentrate immediately to obtain purified gentamicin B powder or further concentrateing the secondary decolorized gentamicin B-containing concentrate and lyophilized to obtain purified gentamicin B powder .

1 is a result of HPLC analysis of purified gentamicin B powder obtained by the method of Example 12, Figure 2 is a result of HPLC-ELSD analysis of the purified gentamicin B powder obtained by the method of Example 12. As shown in FIGS. 1 and 2, the purified gentamicin B powder obtained by the method of Example 12 showed very high purity (80% or more).

Although the present invention has been described through the above embodiments as described above, the present invention is not necessarily limited thereto, and various modifications can be made without departing from the scope and spirit of the present invention. Therefore, the protection scope of the present invention should be construed as including all embodiments falling within the scope of the claims appended to the present invention.

Claims (20)

Adjusting the pH of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B to 2 to 3, leaving for 10 to 24 hr at 2 to 8 ° C., and then centrifuging to obtain the gentamicin B containing supernatant;
After adjusting the pH of the gentamicin B-containing supernatant to 6.5 to 7.0, the gentamicin B was contacted with a weakly acidic cation exchange resin having a porous type and a particle size of 10 to 60 mesh and using a carboxylic acid as an exchanger. Adsorbing;
Eluting the gentamicin B adsorbed on the cation exchange resin with ammonia water at a concentration of 0.2-0.3 N to obtain a gentamicin B-containing eluent; And
The gentamicin B-containing eluate was concentrated to a concentration of gentamicin B of 8,000-15,000 ㎍ / ml, and the pH was adjusted to 5.5-6.5, and then gel-type and particle size of 50-400 mesh. A method for recovering or purifying gentamicin B, comprising contacting a strong basic anion exchange resin using a quaternary ammonium group as an exchanger to obtain a decolorized gentamicin B-containing concentrate.
The method of claim 1, wherein the flexible substance of gentamicin B is selected from micronomicin, gentamicin A, gentamycin A1, gentamicin X2, gentamycin B1, gentamycin C1, gentamycin C1a or gentamicin C2. Method for recovering or purifying gentamicin B, which is composed of one or more species.
The method of claim 1, wherein the microorganism is recovered or purification of gentamicin B, characterized in that selected micro mono spokes la greener Apulia (Micromonospora purpurea), micro mono spokes la Station North Fora (micromonospora echinospora) or a mutant strain Way.
delete delete delete delete delete delete delete The method for recovering or purifying gentamicin B according to any one of claims 1 to 3, wherein the cation exchange resin is washed with water before eluting the gentamicin B adsorbed on the cation exchange resin with ammonia water.
The recovery or purification of gentamicin B according to any one of claims 1 to 3, wherein the content of gentamicin B in the gentamicin B-containing eluent is 90% or more based on the total weight of the gentamicin analogue. Way.
delete delete The method for recovering or purifying gentamicin B according to any one of claims 1 to 3, further comprising pulverizing the decolorized gentamicin B-containing concentrate.
Adjusting the pH of the microbial fermentation broth containing the flexible material of gentamicin B and gentamicin B to 2 to 3, leaving for 10 to 24 hr at 2 to 8 ° C., and then centrifuging to obtain the gentamicin B containing supernatant;
Adjusting the pH of the gentamicin B-containing supernatant to 6.5 ~ 7.0 and left for 8 to 20 hr and filtered with any one selected from filter paper, filter cloth, diatomaceous earth or paracell (PARACELL) to obtain a gentamicin B-containing filtrate ;
The gentamicin B-containing filtrate was contacted with a weakly acidic cation exchange resin having a porous type and a particle size of 10 to 60 mesh and using carboxylic acid as an exchanger to adsorb gentamicin B, and wash the cation exchange resin with water. And eluting the gentamicin B adsorbed on the cation exchange resin with ammonia water at a concentration of 0.2-0.3 N to obtain a gentamicin B-containing first eluent;
After adjusting the pH of the gentamicin B-containing first eluent to 6.5 to 7.0, the gentamicin is brought into contact with a weakly acidic cation exchange resin having a porous type and a particle size of 10 to 60 mesh and using a carboxylic acid as an exchanger. Reabsorb B, wash the cation exchange resin with water, pre-elute gentamicin B resorbed on the cation exchange resin with 0.1-0.2 N concentration of ammonia water, then elute with 0.4-0.6 N concentration of ammonia water To obtain a gentamicin B-containing second eluent; And
After concentrating the gentamicin B-containing second eluent so that the gentamicin B concentration is 8,000-15,000 ㎍ / ml and adjusting the pH to 5.5-6.5, it is a gel type and has a particle size of 50-400 mesh. And contacting a strong basic anion exchange resin using a quaternary ammonium group as an exchanger to obtain a degenerated gentamicin B-containing concentrate.
17. The method according to claim 16, wherein the flexible substance of gentamicin B is selected from micronomicin, gentamycin A, gentamycin A1, gentamycin X2, gentamycin B1, gentamycin C1, gentamycin C1a or gentamycin C2. Method for recovering or purifying gentamicin B, which is composed of one or more species.
The method of claim 16, wherein the microorganism is recovered or purified gentamicin B, characterized in that selected from Micromonospora purpurea, micromonospora echinospora or a variant strain thereof. Way.
19. The method according to any one of claims 16 to 18, wherein the pH of the bleached gentamicin B-containing concentrate is adjusted to 5-6, followed by contact with activated charcoal to obtain a further bleached gentamicin B-containing concentrate. Method for recovering or purifying gentamicin B further comprising.
19. The method for recovering or purifying gentamicin B according to any one of claims 16 to 18, further comprising powdering the decolorized gentamicin B-containing concentrate.

Images