CN105131053A - Separation and purification method of flavomycoin A component - Google Patents
Separation and purification method of flavomycoin A component Download PDFInfo
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Abstract
The invention discloses a separation and purification method of a flavomycoin A component and belongs to the technical field of bio-chemical engineering. In the technical scheme, the method includes following steps: 1) pre-purifying a flavomycoin fermentation liquid through polymethacrylate macroporous adsorption resin; 2) removing the solvent through anion exchange resin; and 3) finally performing purification through reversion-phase chromatography to obtain the flavomycoin A component. In the invention, by means of the anion exchange resin for removing the solvent, the process is compact. Through the polymethacrylate macroporous adsorption resin for pre-purification, the purity and yield of the flavomycoin A component are both better than those through a styrene-divinylbenzene macroporous adsorption resin. In addition, the method is much less in consumption of solvent and is low in cost since a preparation-grade reversion-phase chromatography filling material is employed. The method is more easy to popularize industrially.
Description
Technical field
The invention belongs to technical field of biochemical industry, relate to a kind of separation purification method of xanthomycin A prescription.
Background technology
Bambermycin, has another name called moenomycin (moenomycin), is a kind of phospholipid microbiotic, by spot Bai Shi streptomycete fermentation gained.Similar by the natural antibiotics of fermentative Production with great majority, as a kind of microbial secondary meta-bolites, bambermycin (hereinafter referred to as Mon) is the general designation of some analogs.
Chinese patent CN1194984A discloses the infection that xanthomycin A (hereinafter referred to as MonA) and bismuth salt composition thereof are applicable to control helicobacter pylori, and treats various stomach trouble as stomach ulcer etc. with this.This new therepic use of bambermycin just proposes requirement to the separation and purification of xanthomycin A component.
English Patent GB1068639 adopts the methods such as methyl alcohol lixiviate → n-butanol extraction → precipitation → dialysis → n-butanol extraction → precipitation → Magnesium Silicate q-agent column chromatography decolouring → gel filtration chromatography → anion-exchange chromatography, obtains the sterling of bambermycin mixture.Chinese patent ZL200510051382.1 also adopts methyl alcohol lixiviate → n-butanol extraction → silica gel normal-phase chromatography → Magnesium Silicate q-agent column chromatography decolouring → C8 or C18 reverse-phase chromatography to obtain the sterling of bambermycin mixture, but aforesaid method all fails the sterling of obtained xanthomycin A component.
At present, about the document of the separation and purification of xanthomycin A component is rare, following report is only had:
Based on the feed liquid of US Patent No. 3660569 after Magnesium Silicate q-agent decolouring, be separated by normal-phase chromatography (filler is silica gel) and obtain xanthomycin A component, eluent is the mixture of n-propyl alcohol and ammoniacal liquor.But due to the not reproducible use of purification on normal-phase silica gel filler, the reasons such as the large and solvent consumption of labour intensity is large, are not suitable for promoting the use of.
The people such as the Sun Chenghang (separation andpreconcentration of PGL class microbiotic moenomycinA, China's microbiotic magazine, 2003,28 (6): 325-327,360) on the basis of US Patent No. 3660569 normal-phase chromatography, adopt the AG C18 chromatographic column of packing material size 5 μm, obtain xanthomycin A component sterling, also point out " the sterling preparation method based on new high performance liquid phase separation condition; cost is high, only for the preparation of the sample size of milligram level " the ending author of article.Chinese patent ZL201210029457.6 adopts the little column extracting of 3mLC18 solid phase, packing material size 5 μm of C18 analytical columns are separated, C18 solid phase extraction column is refined further, and all reagent is chromatographically pure and ultrapure water, finally obtains flaxen MonA.Obviously, this invention is only applicable to laboratory and prepares on a small quantity, is difficult to industrialization promotion.
US Patent No. 5986089 also discloses a kind of preparation technology (see Fig. 4) of xanthomycin A component.Whole technical process can be divided into 3 steps substantially, and the first step adopts styrene-divinylbenzene class macroporous adsorbent resin (MCIGELCHP20P, DIAIONEHP20SS, MIT; AmberliteXAD16, AmberliteXAD1180s, Rhom and Hass of the U.S.) pre-separation or ultrafiltration-solvent extraction pre-separation, the former impurity-eliminating effect is better; Second step is the key of whole separation purifying technique, clastotype used is ion-exchange chromatography, filler is polymethacrylate skeleton, anion chromatographic filling material with DEAE weak base group, why thinks second step to be the core procedure of whole technique be that the purity based on xanthomycin A component obtains maximum lifting through this step; 3rd step is reverse-phase chromatography pattern, its object is to desalination or is further purified the sterling obtaining xanthomycin A component.
Three one step process disclosed in US Patent No. 5986089 its essence is the combination of reverse-phase chromatography-anion-exchange chromatography-reverse-phase chromatography.According to chromatographic theory, there is following weak point:
First, as a complete unit, whole technique is connected unreasonable.It is to be noted that this is not only the problem of US Patent No. 5986089, is also the common problem of aforementioned bambermycin or the existing technology of preparing of xanthomycin A component.As mentioned above, DEAE anion-exchange chromatography is the core of US Patent No. 5986089, tentatively do not consider the problem of cost, directly with ion-exchange chromatogram purification fermented liquid theoretically and infeasible, its reason is that a large amount of negatively charged ion existed in fermented liquid can compete the site of DEAE with electronegative xanthomycin A component, therefore xanthomycin A component cannot be adsorbed on DEAE filler, naturally also cannot reach the object of separation and purification.In order to address this problem, applicant arranged reverse-phase chromatography process before anion-exchange chromatography, reached the object of preliminary purification by reverse-phase chromatography desalination.But reverse-phase chromatography can introduce organic solvent, and ion-exchange chromatography is generally aqueous phase loading, therefore have to again by ultrafiltration and dry desolvation, then desciccate is dissolved in water, the condition of anion-exchange chromatography could be met like this.Xanthomycin A component color spectral purity contained in the elutriant of anion-exchange chromatography reaches requirement, but the salinity in elutriant must remove again, and therefore applicant's rearmounted reverse-phase chromatography of having to again carrys out desalination.And in the end before a step reverse-phase chromatography, carry out ultrafiltration, drying to the elutriant of ion-exchange chromatography again, desciccate is dissolved in water and carries out final step reverse-phase chromatography operation (see Fig. 5).This just makes whole complex process, and effective constituent yield reduces and cost raises.
Secondly, this patent adopts reverse-phase chromatography pretreated fermentation liquid also to have weak point.Mainly contain: 1) main purpose of this step is to adsorb the bambermycin in fermented liquid, therefore with the maximum carrying capacity of bambermycin for main starting point, and the pH of fermented liquid is most important to absorption carrying capacity, we find, best pH is between 3.0 ~ 4.5 but not the pH7.5 of this patent; 2) the styrene-divinylbenzene macroporous adsorbent resin selected by is nonpolar very strong, very easily adsorb the impurity of a large amount of existence in fermented liquid and regenerate difficulty, this is also the of paramount importance factor affecting styrene-divinylbenzene class macroporous adsorbent resin work-ing life in the middle of microbiotic industrial practice.
Summary of the invention
In order to overcome the defect that above-mentioned prior art exists, the object of the present invention is to provide a kind of separation purification method of xanthomycin A component, the method operating procedure is connected compact, and solvent consumption is few, environmental friendliness, is suitable for industrial amplification production.
The present invention is achieved through the following technical solutions:
The separation purification method of xanthomycin A component disclosed by the invention, first polymethacrylate macroporous adsorbent resin is adopted to carry out prepurification to bambermycin fermented liquid, then by anionite-exchange resin desolventizing, refine finally by reverse-phase chromatography, obtain xanthomycin A component.
Preferably, the separation purification method of xanthomycin A prescription disclosed by the invention, comprises the following steps:
1) bambermycin fermented liquid pH value is adjusted to 3.0 ~ 4.5, in 4 DEG C of refrigerated overnight, centrifugal, get supernatant liquor;
2) supernatant liquor is continued to flow through polymethacrylate macroporous adsorption resin chromatography post with the flow velocity of 1 ~ 3BV/hr, the carrying capacity controlling xanthomycin A component is 15 ~ 30g/L, then carry out gradient elution, collect cut, merge the elutriant of enrichment xanthomycin A component;
3) pH value of the elutriant of enrichment xanthomycin A component is adjusted to 5.0 ~ 8.0, continues to flow through the chromatography column being filled with anionite-exchange resin with 1 ~ 20BV/hr flow velocity, then carry out wash-out, collect the xanthomycin A component elutriant after desolventizing;
4) pH value of the xanthomycin A component elutriant after desolventizing is adjusted to 3.0 ~ 4.5, the chromatography column being filled with reverse-phase chromatography filler is pumped into the flow velocity of 50 ~ 250cm/hr, then with the advanced line linearity gradient elution of identical flow velocity, carry out Gradient elution again, until the complete desorb of xanthomycin A prescription, merge high purity xanthomycin A component, remove solvent postlyophilization, obtain xanthomycin A component.
Step 1) described in centrifugal be centrifugal 10min under the condition of 8000r/min.
Step 2) described in gradient elution be: carry out wash-out with 2BV deionized water, 1BV20% methanol aqueous solution, 1BV40% methanol aqueous solution, 3BV60% methanol aqueous solution and 2BV80% methanol aqueous solution successively, elution flow rate is 1 ~ 3BV/hr.
Step 3) in the elutriant of enrichment xanthomycin A component and the volume ratio of anionite-exchange resin be 4 ~ 10:1, described anionite-exchange resin is the ion exchange resin with quaternary amine base or tertiary amine groups.
Step 3) described in wash-out be: carry out wash-out with 2BV deionized water and 5BV buffered soln successively, the pH value of damping fluid is 3.5, containing the KH of 67mM in damping fluid
2pO
4and the NaCl of 1M; Elution flow rate is 1 ~ 20BV/hr.
Step 4) in reverse-phase chromatography filler aperture used be
particle diameter is 20 ~ 75 μm; Reverse-phase chromatography filler is C8, C18 filler of silica gel modification, or is the chromatograph packing material of polymeric matrix.
Described polymeric matrix is styrene-divinylbenzene polymkeric substance or polymethacrylate polymkeric substance.
Step 4) in advanced line linearity gradient elution, then the concrete operations carrying out Gradient elution are:
Take pH value as 20% methanol solution of 8.30 be A moving phase, with pure methyl alcohol for B moving phase, according to 0 ~ 40%B moving phase linear gradient elution 40min, then use 40%B moving phase Gradient elution to the complete desorb of xanthomycin A component.
Step 4) in be filled with reverse-phase chromatography filler chromatography column adopt the chromatography column of 20 × 250mm.
Compared with prior art, the present invention has following useful technique effect:
The separation purification method of xanthomycin A component disclosed by the invention, first by polymethacrylate macroporous adsorbent resin prepurification bambermycin, the purity of xanthomycin A component and yield are all better than styrene-divinylbenzene class macroporous adsorbent resin, and solvent consumption is less, in addition, polymethacrylate macroporous adsorbent resin more anti-pollution, be easier to regeneration, thus extend the work-ing life of resin, reduce cost; Secondly, macroporous adsorbent resin elutriant is without ultrafiltration-drying or revolve the method removal solvents such as steaming, but directly by anionite-exchange resin desolvation, thus whole separation and purification process is connected closely; Finally, larger, that price is the more cheap preparation scale reverse-phase chromatography filler of particle diameter is adopted to refine xanthomycin A component, easier industrial application.
Accompanying drawing explanation
Fig. 1 is that the HPLC before and after XAD7HP purifying detects collection of illustrative plates; Wherein, (a) is (fermented liquid) before XAD7HP process; B () is for after XAD7HP wash-out;
Fig. 2 is through the purity of FPA98Cl plastic resin treatment xanthomycin A and yield under different pH condition;
Fig. 3 is the HPLC detected result of bambermycin fermented liquid and final purified product;
Fig. 4 is US Patent No. 5986089 technical process figure;
Fig. 5 is US Patent No. 5986089 complete process flow figure;
Fig. 6 is complete process flow figure of the present invention.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail, and the explanation of the invention is not limited.
Process flow sheet of the present invention is see Fig. 6, and the separation purification method of disclosed xanthomycin A component, comprises the following steps:
(1) polymethacrylate macroporous adsorbent resin extracts
By bambermycin ferment filtrate adjustment pH to 3.0 ~ 4.5, be put in cold compartment of refrigerator (4 DEG C) and spend the night (making some protein depositions), centrifugal 10min under 8000r/min condition, remove throw out.Centrifuged supernatant is continued to flow through polymethacrylate macroporous adsorption resin chromatography post with 1-3BV/hr, do not spill with column outlet effluent liquid xanthomycin A component and be advisable, according to the difference of different batches fermented liquid quality, control carrying capacity 15 ~ 30g/L resin of xanthomycin A component.Use 2BV deionized water, 1BV20% methanol aqueous solution, 1BV40% methanol aqueous solution, 3BV60% methanol aqueous solution and 2BV80% methanol aqueous solution wash-out subsequently successively, elution flow rate is 1 ~ 3BV/hr, collects cut and merges the elutriant of enrichment MonA.
(2) anionite-exchange resin desolventizing
Macroporous adsorbent resin elutriant pH=6.0 ~ 8.0 of adjustment containing solvent, this elutriant is continued to flow through the chromatography column being filled with anionite-exchange resin with 1-20BV/hr flow velocity, the ratio of elutriant and resin is 4:1 ~ 10:1 (v/v).2BV deionized water, 5BV67mMKH is used successively under 1 ~ 20BV/hr flow velocity
2pO
4+ 1MNaCl (pH=3.50) buffered soln wash-out, collects the MonA elutriant after purifying.
(3) reverse-phase chromatography is refined
Get the MonA elutriant that 160 ~ 400ml anion exchange method obtains, regulate pH=3.0 ~ 4.5, pump into the chromatography column (20 × 250mm) being filled with reverse-phase chromatography filler with 50 ~ 250cm/hr flow velocity.Then with same flow velocity, with 20% methyl alcohol of pH=8.30 as A moving phase, pure methyl alcohol is B moving phase, according to 0 ~ 40%B linear gradient mode wash-out 40min, then is eluted to the complete desorb of MonA component by 40%B constant gradient mode, merges high purity MonA cut.
Through ultrafiltration or revolve and boil off solvent, lyophilize obtains product.
Described macroporous adsorbent resin is polymethacrylate macroporous adsorbent resin;
Described anionite-exchange resin is the ion exchange resin with quaternary amine base or tertiary amine groups;
Described reverse-phase chromatography filler aperture is
particle diameter 20 ~ 75 μm, reverse-phase chromatography condiment comprises C8 or the C18 filler of silica gel modification and the reverse phase filler of polymeric matrix, and described polymeric matrix comprises styrene-divinylbenzene polymkeric substance or polymethacrylate polymkeric substance.
1, acrylic acid or the like macroporous adsorbent resin pre-separation
Bambermycin fermented liquid (solid content is 60g/L) is used rare H
2sO
4regulate pH=3.50, be put in cold compartment of refrigerator (4 DEG C) and spend the night (making some protein depositions), centrifugal 10min under 8000r/min condition, remove throw out, supernatant liquor is for subsequent use.
Respectively get 300mLAmberliteXAD7HP and AmberliteXAD1180 and fill post (Ф=25mm, H=600mm), 600mL bambermycin fermented liquid is respectively flowed through the chromatography column being filled with AmberliteXAD7HP and AmberliteXAD1180 macroporous adsorbent resin with the flow velocity of 5mL/min.Use 600mL deionized water, 300mL20% methyl alcohol, 300mL40% methyl alcohol, 900mL60% methyl alcohol, 600mL80% methyl alcohol, 600mL100% methyl alcohol with the flow velocity wash-out of 5mL/min successively.HPLC detects elutriant, and merge the elutriant containing high purity MonA, the purification effect of AmberliteXAD7HP and AmberliteXAD1180 is in table 1.
The table main physical and chemical of 1XAD7HP, XAD1180 and the purification effect to MonA thereof
Obviously, the purification effect of XAD7HP macroporous adsorbent resin to MonA is better than XAD1180 macroporous adsorbent resin, and target compound MonA is mainly present in 80% meoh eluate, comparatively uses XAD1180 resin to save organic solvent.And follow-up resin regeneration test show XAD7HP comparatively XAD1180 more easily regenerate.
The composition of each cut of XAD7HP loading, elution process is in table 2.
The table distribution of 2MonA in each elutriant (XAD7HP)
Effect before and after XAD7HP purifying is in table 3 and Fig. 1.
Show the purity of Mon and MonA in fermented liquid before and after 3XAD7HP purifying
2, anionite-exchange resin desolventizing
Scheme 1 (not desolventizing)
Get the MonA elutriant (containing 80% methyl alcohol) that four parts of 400mL embodiments 1 obtain, pH value is adjusted to 4.0,6.0,8.0,10.0 respectively, under 5mL/min flow velocity, loading to AmberliteFPA98Cl anion exchange resin layer analyses post (Ф=16mm, H=500mm).Under identical flow velocity, use deionized water, 67mMKH successively
2pO
4+ 1MNaCl (pH=3.50) buffered soln wash-out, HPLC detects, and collects the MonA elutriant after purifying.
Scheme 2 (desolventizing)
After MonA elutriant 400mLMonA embodiment 1 obtained revolves and boils off solvent methanol, must the about 80mL aqueous solution, add deionized water and be diluted to 400mL, dilute NaOH solution regulates pH=8.00, carry out purifying according to the loading-elution requirement in scheme 1, collect the MonA elutriant after purifying.
In the elutriant collected under scheme 1 condition of different pH, MonA purity and yield the results are shown in Figure 2.As pH=6-8, FPA98Cl is better to the purification effect of MonA, and wherein during pH=8, MonA purity is 49.4%, and yield is 90%.
Contain organic solvent methyl alcohol in Mon sample liquid in scheme 1, the Mon sample liquid in scheme 2 boils off except methyl alcohol by revolving, and compares its purification effect, obtain table 4 under optimizing pH condition.As can be seen from Table 4, the purification effect of scheme 1 is obviously better than scheme 2, and what this means methyl alcohol in Mon sample liquid has the purifying being beneficial to MonA.
The comparison of table 4 two schemes purification effect
In addition, in scheme 2, the extra increase of palpus is revolved and is boiled off this technique of methyl alcohol, just can obtain aqueous phase Mon sample liquid.Clearly this can cause and extend operation, reduces yield, improve the problems such as production cost.Based on the linking of technique and the simplicity of operation, we select not remove the scheme 1 that methyl alcohol is directly splined on anion-exchange column.Utilize FPA98Cl anionresin column purification MonA, the purity of MonA is increased to 49.8% by 43.8%, and yield is up to 90%.Remove while methyl alcohol reaching, again due to high salt concentration (1MNaCl) strong wash-out power thus concentrate Mon sample liquid (becoming 200mL by 400mL), be conducive to next step treating process.
The composition of scheme 1 loading, each cut of elution process is in table 5.
The content of MonA in each elutriant of table 5FPA98Cl purifying MonA
3, reverse-phase chromatography refines MonA
Get the MonA elutriant that 320mL anion exchange method obtains, regulate pH=4.5.Be splined on 100cm/hr flow velocity and be filled with FujiChromatorexC18 reverse-phase chromatographic column (Ф=20mm, H=250mm).Then use 20% methyl alcohol of pH=8.30 as A moving phase, 100% methyl alcohol is B moving phase, with 100cm/hr flow velocity, according to 0-40%B linear gradient mode wash-out 40min, be eluted to the complete desorb of MonA component by 40%B constant gradient mode again, merge high purity MonA cut, through ultrafiltration or revolve and boil off solvent, lyophilize obtains colourless xanthomycin A product, and purity is between 95%-99%.
Embodiment 1
A separation purification method for xanthomycin A prescription, comprises the following steps:
1) bambermycin fermented liquid pH value is adjusted to 3.0, in 4 DEG C of refrigerated overnight, under the condition of 8000r/min, centrifugal 10min, gets supernatant liquor;
2) supernatant liquor is continued to flow through polymethacrylate macroporous adsorbent resin ((DiaionHP2MG with the flow velocity of 1BV/hr, Mitsubishi chemistry) chromatography column, the carrying capacity controlling xanthomycin A component is 15g/L, then gradient elution is carried out, collect cut, merge the elutriant of enrichment xanthomycin A component;
Described gradient elution is: carry out wash-out with 2BV deionized water, 1BV20% methanol aqueous solution, 1BV40% methanol aqueous solution, 3BV60% methanol aqueous solution and 2BV80% methanol aqueous solution successively, elution flow rate is 1BV/hr;
3) pH value of the elutriant of enrichment xanthomycin A component is adjusted to 6.0, continues to flow through the chromatography column being filled with anionite-exchange resin with 1BV/hr flow velocity, then carry out wash-out, collect the xanthomycin A component elutriant after purifying;
Wherein, the elutriant of enrichment xanthomycin A component and the volume ratio of anionite-exchange resin are 4:1; Described anionite-exchange resin is the ion exchange resin (AmberliteFPA53, Rhom and Hass of the U.S.) with tertiary amine groups;
Described elution requirement is: carry out wash-out with 2BV deionized water and 5BV buffered soln successively, the pH value of damping fluid is 3.5, the KH containing 67mM in damping fluid
2pO
4and the NaCl of 1M; Elution flow rate is 1BV/hr;
4) pH value of the xanthomycin A component elutriant after purifying is adjusted to 3.0, the chromatography column of the 20 × 250mm being filled with reverse-phase chromatography filler is pumped into the flow velocity of 50cm/hr, then with identical flow velocity, take pH value as 20% methanol solution of 8.30 be A moving phase, with pure methyl alcohol for B moving phase, according to 0 ~ 40%B linear gradient mode wash-out 40min, the complete desorb of xanthomycin A component is eluted to again by 40%B constant gradient mode, until the complete desorb of xanthomycin A prescription, merge high purity xanthomycin A component, remove solvent postlyophilization, obtain xanthomycin A component;
Wherein, reverse-phase chromatography filler used is C8 filler (SP-300-40/60-C8-P, the aperture of silica gel modification
particle diameter 50 μm, Japanese Daiso company).
Embodiment 2
A separation purification method for xanthomycin A prescription, comprises the following steps:
1) bambermycin fermented liquid pH value is adjusted to 3.8, in 4 DEG C of refrigerated overnight, under the condition of 8000r/min, centrifugal 10min, gets supernatant liquor;
2) supernatant liquor is continued to flow through polymethacrylate macroporous adsorbent resin (PurosorbPAD610 with the flow velocity of 2BV/hr, Piao Laite company of Britain) chromatography column, the carrying capacity controlling xanthomycin A component is 25g/L, then gradient elution is carried out, collect cut, merge the elutriant of enrichment xanthomycin A component;
Described gradient elution is: carry out wash-out with 2BV deionized water, 1BV20% methanol aqueous solution, 1BV40% methanol aqueous solution, 3BV60% methanol aqueous solution and 2BV80% methanol aqueous solution successively, elution flow rate is 1BV/hr;
3) pH value of the elutriant of enrichment xanthomycin A component is adjusted to 7.0, continues to flow through the chromatography column being filled with anionite-exchange resin with 12BV/hr flow velocity, then carry out wash-out, collect the xanthomycin A component elutriant after purifying;
Wherein, the elutriant of enrichment xanthomycin A component and the volume ratio of anionite-exchange resin are 7:1; Described anionite-exchange resin is the ion exchange resin (AmberliteFPA98, Rhom and Hass of the U.S.) with quaternary amine base;
Described wash-out is: carry out wash-out with 2BV deionized water and 5BV buffered soln successively, the pH value of damping fluid is 3.5, the KH containing 67mM in damping fluid
2pO
4and the NaCl of 1M; Elution flow rate is 15BV/hr;
4) pH value of the xanthomycin A component elutriant after purifying is adjusted to 3.8, the chromatography column of the 20 × 250mm being filled with reverse-phase chromatography filler is pumped into the flow velocity of 150cm/hr, then with identical flow velocity, take pH value as 20% methanol solution of 8.30 be A moving phase, with pure methyl alcohol for B moving phase, according to 0 ~ 40%B linear gradient mode wash-out 40min, the complete desorb of xanthomycin A component is eluted to again by 40%B constant gradient mode, until the complete desorb of xanthomycin A prescription, merge high purity xanthomycin A component, remove solvent postlyophilization, obtain xanthomycin A component;
Wherein, reverse-phase chromatography filler used is C18 filler (ChromatorexC18MB100-40/75, the aperture of silica gel modification
particle diameter 40 ~ 75 μm, Japanese Fuji company).
Embodiment 3
A separation purification method for xanthomycin A prescription, comprises the following steps:
1) bambermycin fermented liquid pH value is adjusted to 4.5, in 4 DEG C of refrigerated overnight, under the condition of 8000r/min, centrifugal 10min, gets supernatant liquor;
2) supernatant liquor is continued to flow through polymethacrylate macroporous adsorbent resin (AmberliteXAD7HP with the flow velocity of 3BV/hr, Rhom and Hass of the U.S.) chromatography column, the carrying capacity controlling xanthomycin A component is 30g/L, then gradient elution is carried out, collect cut, merge the elutriant of enrichment xanthomycin A component;
Described gradient elution is: carry out wash-out with 2BV deionized water, 1BV20% methanol aqueous solution, 1BV40% methanol aqueous solution, 3BV60% methanol aqueous solution and 2BV80% methanol aqueous solution successively, elution flow rate is 1BV/hr;
3) pH value of the elutriant of enrichment xanthomycin A component is adjusted to 8.0, continues to flow through the chromatography column being filled with anionite-exchange resin with 20BV/hr flow velocity, then carry out wash-out, collect the xanthomycin A component elutriant after purifying;
Wherein, the elutriant of enrichment xanthomycin A component and the volume ratio of anionite-exchange resin are 10:1; Described anionite-exchange resin is the ion exchange resin (AmberliteIRA958, Rhom and Hass of the U.S.) with quaternary amine base;
Described wash-out is: carry out wash-out with 2BV deionized water and 5BV buffered soln successively, the pH value of damping fluid is 3.5, the KH containing 67mM in damping fluid
2pO
4and the NaCl of 1M; Elution flow rate is 20BV/hr;
4) pH value of the xanthomycin A component elutriant after purifying is adjusted to 4.5, the chromatography column of the 20 × 250mm being filled with reverse-phase chromatography filler is pumped into the flow velocity of 250cm/hr, then with identical flow velocity, take pH value as 20% methanol solution of 8.30 be A moving phase, with pure methyl alcohol for B moving phase, according to 0 ~ 40%B linear gradient mode wash-out 40min, the complete desorb of xanthomycin A component is eluted to again by 40%B constant gradient mode, until the complete desorb of xanthomycin A prescription, merge high purity xanthomycin A component, remove solvent postlyophilization, obtain xanthomycin A component;
Wherein, reverse-phase chromatography filler used is C18 filler (ChromatorexC18MB100-20/45, the aperture of silica gel modification
particle diameter 20 ~ 45 μm, Japanese Fuji company).
In sum, the inventive method had both obtained xanthomycin A component, overcame again the deficiency of prior art processes, by anionite-exchange resin desolventizing, thus whole technique was connected closely; By adopting polymethacrylate macroporous adsorbent resin at pH3.0 ~ 4.5 prepurification bambermycin, the purity of xanthomycin A component and yield are all better than vinylbenzene-divinylbenzene class macroporous adsorbent resin, and solvent consumption is less.Due to polymethacrylate macroporous adsorbent resin in essence more anti-pollution, more easily regenerate and the life-span of resin extended greatly; The industrial preparation scale reverse-phase chromatography filler of particle diameter 20-75 μm is adopted to refine xanthomycin A component, the low price of filler and easier industrial application.
Claims (10)
1. the separation purification method of an xanthomycin A component, it is characterized in that, first adopt polymethacrylate macroporous adsorbent resin to carry out prepurification to bambermycin fermented liquid, then by anionite-exchange resin desolventizing, refine finally by reverse-phase chromatography, obtain xanthomycin A component.
2. the separation purification method of xanthomycin A prescription according to claim 1, is characterized in that, comprise the following steps:
1) bambermycin fermented liquid pH value is adjusted to 3.0 ~ 4.5, in 4 DEG C of refrigerated overnight, centrifugal, get supernatant liquor;
2) supernatant liquor is continued to flow through polymethacrylate macroporous adsorption resin chromatography post with the flow velocity of 1 ~ 3BV/hr, the carrying capacity controlling xanthomycin A component is 15 ~ 30g/L, then carries out gradient elution, collects cut, merges the elutriant of enrichment xanthomycin A component;
3) pH value of the elutriant of enrichment xanthomycin A component is adjusted to 5.0 ~ 8.0, continues to flow through the chromatography column being filled with anionite-exchange resin with 1 ~ 20BV/hr flow velocity, then carry out wash-out, collect the xanthomycin A component elutriant after desolventizing;
4) pH value of the xanthomycin A component elutriant after desolventizing is adjusted to 3.0 ~ 4.5, the chromatography column being filled with reverse-phase chromatography filler is pumped into the flow velocity of 50 ~ 250cm/hr, then with the advanced line linearity gradient elution of identical flow velocity, carry out Gradient elution again, until the complete desorb of xanthomycin A prescription, merge high purity xanthomycin A component, remove solvent postlyophilization, obtain xanthomycin A component.
3. the separation purification method of xanthomycin A prescription according to claim 2, is characterized in that, centrifugal described in step 1) is centrifugal 10min under the condition of 8000r/min.
4. the separation purification method of xanthomycin A prescription according to claim 2, it is characterized in that, step 2) described in gradient elution be: carry out wash-out with 2BV deionized water, 1BV20% methanol aqueous solution, 1BV40% methanol aqueous solution, 3BV60% methanol aqueous solution and 2BV80% methanol aqueous solution successively, elution flow rate is 1 ~ 3BV/hr.
5. the separation purification method of xanthomycin A prescription according to claim 2, it is characterized in that, in step 3), the elutriant of enrichment xanthomycin A component and the volume ratio of anionite-exchange resin are 4 ~ 10:1, and described anionite-exchange resin is the ion exchange resin with quaternary amine base or tertiary amine groups.
6. the separation purification method of xanthomycin A prescription according to claim 2, is characterized in that, the wash-out described in step 3) is: carry out wash-out with 2BV deionized water and 5BV buffered soln successively, the pH value of damping fluid is 3.5, the KH containing 67mM in damping fluid
2pO
4and the NaCl of 1M; Elution flow rate is 1 ~ 20BV/hr.
7. the separation purification method of xanthomycin A prescription according to claim 2, is characterized in that, reverse-phase chromatography filler aperture used in step 4) is
particle diameter is 20 ~ 75 μm; Reverse-phase chromatography filler is C8, C18 filler of silica gel modification, or is the chromatograph packing material of polymeric matrix.
8. the separation purification method of xanthomycin A prescription according to claim 7, is characterized in that, described polymeric matrix is styrene-divinylbenzene polymkeric substance or polymethacrylate polymkeric substance.
9. the separation purification method of xanthomycin A prescription according to claim 2, is characterized in that, advanced line linearity gradient elution in step 4), then the concrete operations carrying out Gradient elution are:
Take pH value as 20% methanol solution of 8.30 be A moving phase, with pure methyl alcohol for B moving phase, according to 0 ~ 40%B moving phase linear gradient elution 40min, then use 40%B moving phase Gradient elution to the complete desorb of xanthomycin A component.
10. the separation purification method of xanthomycin A prescription according to claim 2, is characterized in that, the chromatography column being filled with reverse-phase chromatography filler in step 4) adopts the chromatography column of 20 × 250mm.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1068639A (en) * | 1964-09-21 | 1967-05-10 | Hoechst Ag | A purified form of the antibiotic moenomycin and a process for its manufacture |
| US5986089A (en) * | 1997-04-17 | 1999-11-16 | Hoechst Aktiengesellschaft | Process for the preparation of moenomycin A |
| CN1683549A (en) * | 2005-03-09 | 2005-10-19 | 中牧实业股份有限公司 | Purifying method for moenomycin |
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2015
- 2015-07-21 CN CN201510430606.3A patent/CN105131053B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB1068639A (en) * | 1964-09-21 | 1967-05-10 | Hoechst Ag | A purified form of the antibiotic moenomycin and a process for its manufacture |
| US5986089A (en) * | 1997-04-17 | 1999-11-16 | Hoechst Aktiengesellschaft | Process for the preparation of moenomycin A |
| CN1683549A (en) * | 2005-03-09 | 2005-10-19 | 中牧实业股份有限公司 | Purifying method for moenomycin |
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