CN1272445C - Method for purifying cephalosporin C broth using adsorbent - Google Patents

Method for purifying cephalosporin C broth using adsorbent Download PDF

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CN1272445C
CN1272445C CNB2004100015501A CN200410001550A CN1272445C CN 1272445 C CN1272445 C CN 1272445C CN B2004100015501 A CNB2004100015501 A CN B2004100015501A CN 200410001550 A CN200410001550 A CN 200410001550A CN 1272445 C CN1272445 C CN 1272445C
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cpc
aca
cephalosporin
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deacetylation
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CN1517442A (en
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金正优
申相圭
尹钟赞
尹仁均
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CKD Bio Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P35/00—Preparation of compounds having a 5-thia-1-azabicyclo [4.2.0] octane ring system, e.g. cephalosporin
    • C12P35/06—Cephalosporin C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01093—Glutaryl-7-aminocephalosporanic-acid acylase (3.5.1.93)

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Abstract

A method for purification of cephalosporin C broth using absorbent is provided, thereby effectively removing diacetyl CPC(cephalosporin C) from the cultured medium. The method for purification of cephalosporin C broth using absorbent comprises the steps of: filtering and absorbing cephalosporin C(CPC) cultured medium using absorbent such as polystyrene resin, and eluting it to remove diacetyl CPC from the CPC cultured medium; reacting cephalosporin C(CPC) cultured medium with D-amino acid oxidase(DAOD) to prepare glutaryl-7-aminocephalosporanic acid(Gl-7-ACA); and reacting glutaryl-7-aminocephalosporanic acid(Gl-7-ACA) with glutaryl-7-aminocephalosporanic acid acylase(Gl-7-ACA acylase) to prepare 7-aminocephalosporanic acid(7-ACA), wherein the CPC cultured medium has pH 5.0 to 6.0; and the eluting solution is phosphate buffer solution with pH 6 to 9, acetate buffer solution, carbonate buffer solution or weak alkali solution.

Description

Utilize the method for sorbent material purification of cephalosporin C meat soup
Technical field
The present invention relates to by enzyme method purification of cephalosporin C (CPC) meat soup with preparation 7-amino-cephalosporanic acid (7-aminocephalosporanic acid, method 7-ACA).More particularly, the present invention relates to method by using sorbent material from CPC meat soup, to separate and remove deacetylation cephalosporin (deacetylation CPC).
Background technology
Usually will be divided into chemical process and enzyme method from the method that CPC prepares 7-ACA.
According to chemical process, obtain the crystal of CPC salt from CPC meat soup, and it is hydrolyzed to synthesize 7-ACA (No. the 615955th, belgian patent).Chemical process has supervirulent pollution reactant (for example chlorine solvent, chlorosilane, xylidine etc.) because of using, and needs strict operational condition (extremely low temperature is as-50 ℃), thereby is defective.
According to enzyme method, after purifying CPC meat soup, make the reaction of the following route 1 of filtrate experience of purifying, and preparation 7-ACA.That is to say; CPC and D-amino-acid oxidase (DAOD) reaction; form glutaryl-7-amino-cephalosporanic acid (G1-7-ACA); and make formed G1-7-ACA and glutaryl-7-amino-cephalosporanic acid acyltransferase (G1-7-ACA acyltransferase) reaction, form 7-ACA.
Route 1
Figure C20041000155000041
Enzyme method is better than chemical synthesis process aspect yield, because it has omitted the crystallisation process that forms CPC salt.But because the deacetylation CPC that is included in the purifying filtrate produces by product by reaction identical with CPC shown in the following route 2, therefore deacetylation 7-ACA is difficult to prepare high-purity 7-ACA.
Route 2
Figure C20041000155000042
(deacetylation CPC) (deacetylation 7-ACA)
Although the deacetylation CPC in the CPC meat soup also is present in the CPC salt that obtains by crystallization; but under the situation of chemical process; during the response procedures shown in following route 3, be removed, so its quality to end product 7-ACA does not have much affect by lactonizing.Yet, under the situation of enzyme method, owing to deacetylation CPC directly is present among the 7-ACA with the form of deacetylation 7-ACA, so the process that must from CPC meat soup, remove deacetylation CPC.
Route 3
Figure C20041000155000043
Design the present invention solves aforesaid general issues, and the method for preparing 7-ACA by effectively removing the deacetylation CPC that comprises in the CPC meat soup with high yield is provided.
Summary of the invention
The present invention relates to purification of cephalosporin C (CPC) to prepare the method for 7-amino-cephalosporanic acid (7-ACA).More particularly, the present invention relates to method by using sorbent material from CPC meat soup, to separate and remove deacetylation cephalosporin (deacetylation CPC).
That is to say that the present invention relates to the preparation method of 7-amino-cephalosporanic acid, described method comprises:
Step 1: prepare glutaryl-7-amino-cephalosporanic acid (G1-7-ACA) by making the reaction of cephalosporin (CPC) meat soup and D-amino-acid oxidase (DAOD), and
Step 2: prepare 7-amino-cephalosporanic acid (7-ACA) by G1-7-ACA and glutaryl-7-amino-cephalosporanic acid acyltransferase (G1-7-ACA acyltransferase) reaction that makes such formation,
It is characterized in that, in described step 1, CPC meat soup is filtered, and use sorbent material, remove deacetylation CPC in the filtrate, CPC bouillon filtrate (BF) and DAOD are reacted by absorption and elution program.
CPC meat soup is the tunning of Acremonium chrysogenium by fermenting process, and preferably to use the pH value be 5.0～6.0 meat soup.
Described CPC meat soup is carried out general filtration procedure to remove impurity, for example substratum etc.In addition, if desired, can after in CPC meat soup, adding acid, carry out described filtration procedure.The solution that filters the back gained is called the CPC bouillon filtrate (BF).
Use sorbent material to carry out the purifying of described filtrate, and the absorption by filtrate and elution program and from filtrate, remove deacetylation CPC.Polystyrene resin is suitable sorbent material.Although the p-poly-phenyl ethenoid resin has no particular limits, can enumerate the highly porous multipolymer of highly porous poly styrene polymer, styrene monomer and alkene monomer, perhaps their big cross-linked network polymkeric substance.On the other hand, described cinnamic benzene ring hydrogen can replace with other substituting group.Although described substituting group is not had concrete restriction, the alkyl that can use alkyl or halogen to replace.As commercial resin, can enumerate Mitsubishi Chemical Corporation, the SEPABEADS of Japan SP series, particularly SEPABEADS SP-700, SEPABEADS SP-825 and SEPABEADS SP-850, and can use the Amberlite of Rohmand Hass XAD series, particularly Amberlite XAD 16.
Using sorbent material to separate from the filtrate of CPC meat soup under the situation of purifying deacetylation CPC also, the flow velocity of CPC bouillon filtrate (BF) is 0.1～10BVH (column volume/hour), is preferably 0.5～5BVH.Then, the phosphate buffered saline buffer, acetate buffer, carbonate buffer solution or the weak caustic solution that use pH 6～9 be as eluent, wash-out CPC solution from resin.The flow velocity of eluent is 0.1～10BVH, is preferably 0.5～6BVH.The temperature of absorption and wash-out is 2～25 ℃, is preferably 5～15 ℃.
Finish the CPC meat soup of fermentation in filtration after,, produce elution curve as shown in Figure 1 by the absorb-elute filtrate on polystyrene resin.As can be seen from Figure 1, deacetylation CPC and CPC can be separated from one another.At this moment, the transparence of formed 7-ACA improves by the colouring effect of removing that causes owing to absorption back wash-out.
On the other hand, use prepare 7-ACA through pretreated CPC meat soup can following further explanation.At first, make direct and DAOD reaction, preparation 7-ACA by the CPC solution of preprocessor.The concentration of substrate of CPC solution is 25～50g/L, is preferably 30～40g/L.Consider reaction conditions, with the scope of pH regulator to 6.5～8.0, be preferably 7～7.5, and temperature is controlled at 15～30 ℃, be preferably 20～25 ℃.At this moment, the pressure with reactor is adjusted to 0.5～1.5kgf/cm 2, and introduce oxygen with the speed of 0.1～0.5vvm (volume/liquor capacity/minute).
Then, described G1-7-ACA solution and G1-7-ACA acyltransferase are reacted under following reaction conditions: pH 7.0～9.0, preferred pH 7.5～8.5, and 15～30 ℃ of temperature, preferred 20～25 ℃, and normal atmosphere.
Hereinafter, more specifically describe the present invention, but scope of the present invention is not subjected to their restriction by embodiment.
Description of drawings
Fig. 1 represents cephalosporin (CPC) meat soup finish fermentation when filtering, and the typical elution curve (deacetylation CPC (), CPC (＜〉)) of gained during by the absorb-elute filtrate on polystyrene resin.
Fig. 2 as the result of embodiment 1, is to show to use SEPABEADS The absorption of the cephalosporin bouillon filtrate (BF) of SP-700 resin and wash-out result's figure (deacetylation CPC (), CPC (＜)).
Fig. 3 as the result of embodiment 2, is to show to use SEPABEADS The absorption of the cephalosporin bouillon filtrate (BF) of SP-825 resin and wash-out result's figure (deacetylation CPC (), CPC (＜)).
Fig. 4 as the result of embodiment 3, is to show to use SEPABEADS The absorption of the cephalosporin bouillon filtrate (BF) of SP-850 resin and wash-out result's figure (deacetylation CPC (), CPC (＜)).
Fig. 5 as the result of embodiment 4, is to show to use Amberlite The absorption of the cephalosporin bouillon filtrate (BF) of XAD 1600 resins and wash-out result's figure (deacetylation CPC (), CPC (＜)).
Embodiment
Describe the present invention in more detail with following embodiment, but they do not limit the scope of the invention.
Embodiment 1: use SEPABEADS The absorption and the wash-out of the cephalosporin bouillon filtrate (BF) of SP-700 resin
With the speed of 3BVH with CPC bouillon filtrate (BF) (deacetylation CPC content: 6%) be adsorbed on and be filled with SEPABEADS In the post of SP-700 resin (diameter: 6 centimetres, highly: 40 centimetres, column volume: 3000 milliliters) and wash-out.Behind absorption and wash-out, the distribution of CPC and deacetylation CPC as shown in Figure 2.
Under the situation of gathering elutriant more than the 6BV of Fig. 2 point, the deacetylation CPC content in the CPC solution is 0.9%, and is 3.5% at the deacetylation CPC content at 6BV point place.
Embodiment 2: use SEPABEADS The absorption and the wash-out of the cephalosporin bouillon filtrate (BF) of SP-825 resin
Except using SEPABEADS The SP-825 resin replaces SEPABEADS Beyond the SP-700 resin, according to method absorption and the wash-out CPC bouillon filtrate (BF) identical with embodiment 1.The result provides in Fig. 3.
Embodiment 3: use SEPABEADS The absorption and the wash-out of the cephalosporin bouillon filtrate (BF) of SP-850 resin
Except using SEPABEADS The SP-850 resin replaces SEPABEADS Beyond the SP-700 resin, according to method absorption and the wash-out CPC bouillon filtrate (BF) identical with embodiment 1.The result provides in Fig. 4.
Embodiment 4: use Amberiite The absorption and the wash-out of the cephalosporin bouillon filtrate (BF) of XAD 1600 resins
Except using Amberlite XAD 1600 resins replace SEPABEADS Beyond the SP-700 resin, according to method absorption and the wash-out CPC bouillon filtrate (BF) identical with embodiment 1.The result provides in Fig. 5.
The preparation of embodiment 5:G1-7-ACA
Press the CPC solution concentration of having removed deacetylation CPC that will obtain among the embodiment 1 concentration by inverse osmosis, and 1000 milliliters of these solution are put into reactor to 75mM.Add D-amino-acid oxidase (DAOD) 2kU, temperature is remained on 20 ℃ simultaneously, the ammonia of adding 5% is to remain on 7.2 with pH.At this moment, introduce oxygen, the interior pressure of reactor is maintained 1kgf/cm with the speed of 0.1vvm (volume/working volume/minute) 2In 45 minutes, transformation efficiency reaches 99%, and the composition of product provides in table 1 when finishing.
Table 1
Compound Ratio
CPC 0.0
G1-7-ACA 90.8
Deacetylation G1-7-ACA 1.2
The preparation of embodiment 6:7-ACA
To described wherein having finished to the solution of the conversion of G1-7-ACA adds G1-7-ACA acyltransferase 5kU as embodiment 5, and under atmospheric pressure in 20 ℃ of reactions down.At this moment, the ammonia of use 5% maintains 8.0 with pH.After 50 minutes, reach the maximum conversion of 7-ACA, yield is 95%.In the solution of finishing reaction, add 10%HCl, the pH value is adjusted to 3.5, and the filtered and recycled crystal.The crystal yield is 88%.The ratio of components of 7-ACA is as shown in table 2.
Table 2
Compound Ratio (%)
7-ACA 97.8
Deacetylation 7-ACA 1.00
The preparation of comparative example 1:G1-7-ACA
After filtering cephalosporin meat soup, do not carry out 1 described sepn process as embodiment, cephalosporin filtrate 1000ml (75mM) is put into reactor, and carry out 5 described identical reactions with embodiment.Ratio of components when reaction is finished provides in table 3.
Table 3
Compound Ratio (%)
CPC 0.0
G1-7-ACA 85.6
Deacetylation G1-7-ACA 4.7
The preparation of comparative example 2:7-ACA
Make the reaction soln that changes into G1-7-ACA in the comparative example 1 carry out 6 described identical reactions with embodiment.After finishing reaction, reclaim the 7-ACA crystal, 7-ACA crystalline ratio is as shown in table 4.
Table 4
Compound Ratio (%)
7-ACA 95.7
Deacetylation 7-ACA 2.5
Industrial applicibility
The deacetylation CPC that comprises in the adsorbent separation of C PC meat soup used according to the invention causes preparing the 7-ACA of the purity more much higher than routine techniques.

Claims (7)

1, the preparation method of 7-amino-cephalosporanic acid (7-ACA), described method comprises:
Step 1: prepare glutaryl-7-amino-cephalosporanic acid (G1-7-ACA) by making the reaction of cephalosporin (CPC) meat soup and D-amino-acid oxidase (DAOD), and
Step 2: prepare 7-amino-cephalosporanic acid (7-ACA) by G1-7-ACA and glutaryl-7-amino-cephalosporanic acid acyltransferase (G1-7-ACA acyltransferase) reaction that makes such formation,
It is characterized in that; in described step 1; CPC meat soup is filtered; and use polystyrene resin as sorbent material; the phosphate buffered saline buffer, acetate buffer, carbonate buffer solution or the weak caustic solution that use pH 6～9 are as eluent; remove deacetylation CPC in the described filtrate by absorption and wash-out, react with DAOD then.
2, the method for claim 1 is characterized in that, the pH of described CPC meat soup is 5.0～6.0.
3, the method for claim 1 is characterized in that, described resin is the porous copolymers of highly porous poly styrene polymer, styrene monomer and alkene monomer, perhaps their big cross-linked network polymkeric substance.
4, the method for claim 1 is characterized in that, during the adsorption process in described step 1, the flow velocity of filtrate is 0.1～10BVH.
5, the method for claim 1 is characterized in that, the flow velocity of described eluent is 0.1～10BVH.
6, the method for each of claim 1～5 is characterized in that, the pH value in the step 1 is 6.5～8.0.
7, the method for each of claim 1～5 is characterized in that, the pH value in the step 2 is 7.0～9.0.
CNB2004100015501A 2003-01-15 2004-01-13 Method for purifying cephalosporin C broth using adsorbent Expired - Fee Related CN1272445C (en)

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CN102584561B (en) * 2011-12-14 2014-12-24 伊犁川宁生物技术有限公司 Method for recovering glutaric acid by-products produced in enzymic method prepared 7-aminocephalosporin acid process
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