CN105131053B - A kind of isolation and purification method of xanthomycin A component - Google Patents
A kind of isolation and purification method of xanthomycin A component Download PDFInfo
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Abstract
The invention discloses a kind of isolation and purification method of xanthomycin A component, belong to technical field of biochemical industry, technical scheme is：Prepurification is carried out to flavomycoin zymotic fluid using polymethacrylate macroporous absorbent resin first, then by anion exchange resin desolvation, last inverted chromatographic refining obtains xanthomycin A component.The inventive method causes process intensification using anion exchange resin desolvation, SDEB styrene diethylenebenzene class macroporous absorbent resin is superior to using the purity and yield of polymethacrylate macroporous adsorbent prepurification xanthomycin A component, and solvent consumption is less, industrial application is more easy to using reverse-phase chromatography filler cheap of preparation scale.
Description
Technical field
The invention belongs to technical field of biochemical industry, it is related to a kind of isolation and purification method of xanthomycin A prescription.
Background technology
Flavomycoin, also known as moenomycin (bambermycin) (moenomycin), are a kind of phospholipid antibiotic, by spot Bai Shi streptomycete fermentations
Gained.It is similar with most of natural antibiotics produced by fermentation method, it is used as a kind of microbial secondary metabolite, flavomycoin
(hereinafter referred to as Mon) is the general designation of some analogues.
Chinese patent CN1194984A discloses xanthomycin A (hereinafter referred to as Mon A) and its bismuth salt composition is applied to
The infection of helicobacter pylori is controlled, and various stomach trouble such as gastric ulcer etc. is treated with this.This new therapeutical uses of flavomycoin are just right
Isolating and purifying for xanthomycin A component proposes requirement.
BP GB1068639 is using methanol extraction → extracting n-butyl alcohol → precipitation → dialysis → extracting n-butyl alcohol → heavy
The methods such as shallow lake → magnesium silicate column chromatography decolouring → gel filtration chromatography → anion-exchange chromatography, have obtained flavomycoin mixture
Sterling.Chinese patent ZL 200510051382.1 also uses methanol extraction → extracting n-butyl alcohol → silica gel normal-phase chromatography → silicon
Sour magnesium column chromatography decolouring → C8 or C18 reverse-phase chromatographies have obtained the sterling of flavomycoin mixture, but the above method all fails system
Obtain the sterling of xanthomycin A component.
At present, the document isolated and purified of relevant xanthomycin A component is rare, only following to report：
United States Patent (USP) US 3660569 by magnesium silicate decolourize after feed liquid based on, pass through normal-phase chromatography (filler is silica gel)
Isolated xanthomycin A component, eluant, eluent is the mixture of normal propyl alcohol and ammoniacal liquor.But, because purification on normal-phase silica gel filler can not be weighed
Use again, the reason such as labor intensity is big and solvent consumption is big is not suitable for promoting the use of.
Sun Cheng boats et al. (phosphoric acid sugar lipid antibiotic moenomycin A separation and identification, Chinese antibiotic magazine,
2003,28(6):325-327,360) on the basis of the normal-phase chromatographies of United States Patent (USP) US 3660569, using 5 μm of packing material size
Analysis level C18 chromatographic columns, have obtained xanthomycin A component sterling, are also indicated that in the ending author of article " with new efficient liquid phase point
From the sterling preparation method based on condition, cost is high, is only used for the preparation of the sample size of milligram level ".Chinese patent ZL
201210029457.6 is small using the small column extracting of 3mL C18 solid phases, 5 μm of C18 analyses post separations, C18 SPEs of packing material size
Post is further refined, and all reagents are chromatographically pure and ultra-pure water, finally obtains flaxen Mon A.Obviously, the invention is only fitted
Prepared on a small quantity for laboratory, it is difficult to industrialization promotion.
United States Patent (USP) US 5986089 also discloses that a kind of preparation technology of xanthomycin A component (referring to Fig. 4).Whole technique
Flow can substantially be divided into 3 steps, the first step using styrene-divinylbenzene class macroporous absorbent resin (MCI GEL CHP20P,
DIAIONE HP20SS, MIT；Amberlite XAD16, Amberlite XAD1180s, U.S.'s ROHM AND HAAS are public
Department) pre-separation or ultrafiltration-solvent extraction pre-separation, the former impurity-eliminating effect is more preferably；Second step is whole separation purifying technique
Key, clastotype used is ion-exchange chromatography, and filler is polymethacrylates skeleton, with DEAE weak base groups
Anion chromatographic filling material, why think second step be whole technique core procedure be based on xanthomycin A component purity warp
The step obtains the lifting of maximum；3rd step is reverse-phase chromatography pattern, its object is to desalination or is further purified and obtains xanthomycin A
The sterling of component.
Three one step process disclosed in United States Patent (USP) US 5986089 its essence is reverse-phase chromatography-anion-exchange chromatography-
The combination of reverse-phase chromatography.According to chromatographic theory, there is following weak point：
First, as a complete unit, whole technique linking is unreasonable.It is to be noted that this is not only United States Patent (USP) US
The problem of 5986089 the problem of is also foregoing flavomycoin or the common existing technology of preparing of xanthomycin A component.As described above, DEAE
Anion-exchange chromatography is United States Patent (USP) US 5986089 core, tentatively the problem of not considering cost, directly uses ion exchange
Chromatogram purification zymotic fluid is theoretically and infeasible, its reason be substantial amounts of anion present in zymotic fluid can with it is negatively charged
The xanthomycin A component of lotus competes DEAE site, therefore xanthomycin A component can not be adsorbed on DEAE fillers, naturally also can not
Reach the purpose isolated and purified.In order to solve this problem, applicant sets reverse-phase chromatography mistake before anion-exchange chromatography
Journey, by reverse-phase chromatography desalination and reaches the purpose of preliminary purification.But reverse-phase chromatography can introduce organic solvent, and ion exchange
Chromatogram is generally aqueous phase loading, therefore has to by ultrafiltration and dry desolvation, then desciccate is dissolved in water,
The condition of anion-exchange chromatography could so be met.Contained xanthomycin A component color in the eluent of anion-exchange chromatography
Spectral purity has reached that the salinity in requirement, but eluent must be driven off again, therefore applicant has to rearmounted reverse-phase chromatography
Desalination.And carry out ultrafiltration, drying, desciccate before final step reverse-phase chromatography, and to the eluent of ion-exchange chromatography
It is dissolved in water progress final step reverse-phase chromatography operation (referring to Fig. 5).This allows for whole complex process, active ingredient yield
Reduce and cost rise.
Secondly, the patent also has weak point using reverse-phase chromatography pretreated fermentation liquid.Mainly have：1) the main mesh of the step
Be adsorb the flavomycoin in zymotic fluid, therefore using the maximum carrying capacity of flavomycoin as main starting point, and pH pairs of zymotic fluid
Adsorb carrying capacity most important, it has been found that optimal pH is between 3.0~4.5 rather than pH7.5 of the patent；2) the benzene second selected by
Alkene-divinylbenzene macroporous absorbent resin is nonpolar very strong, easily adsorbs the impurity largely existed in zymotic fluid and regenerates difficulty, this
Be also influence styrene-divinylbenzene class macroporous absorbent resin service life among antibiotic industrial practice it is mostly important because
Element.
The content of the invention
In order to overcome the defect that above-mentioned prior art is present, it is an object of the invention to provide a kind of xanthomycin A component
Isolation and purification method, this method operating procedure linking is compact, and solvent consumption is few, environment-friendly, suitable for industrial amplification production.
The present invention is to be achieved through the following technical solutions：
The isolation and purification method of xanthomycin A component disclosed by the invention, is inhaled using polymethacrylate macropore first
Attached resin carries out prepurification to flavomycoin zymotic fluid, then by anion exchange resin desolventizing, last inverted chromatogram essence
System, obtains xanthomycin A component.
It is preferred that, the isolation and purification method of xanthomycin A prescription disclosed by the invention comprises the following steps：
1) flavomycoin zymotic fluid pH value is adjusted to 3.0~4.5, in 4 DEG C of refrigerated overnights, centrifugation takes supernatant；
2) supernatant is continued to flow through into polymethacrylate macroporous adsorption resin chromatography with 1~3BV/hr flow velocity
Post, the carrying capacity for controlling xanthomycin A component is 15~30g/L, then carries out gradient elution, collects cut, merges enrichment xanthomycin A
The eluent of component；
3) pH value that will be enriched with the eluent of xanthomycin A component is adjusted to 5.0~8.0, continuous with 1~20BV/hr flow velocitys
The chromatographic column for being filled with anion exchange resin is flowed through, is then eluted, the xanthomycin A component elution after desolventizing is collected
Liquid；
4) pH value of the xanthomycin A component eluent after desolventizing is adjusted to 3.0~4.5, with 50~250cm/hr's
Flow velocity is pumped into the chromatographic column for being filled with reverse-phase chromatography filler, then with the advanced row linear gradient elution of identical flow velocity, then carries out
Gradient elution, until xanthomycin A prescription is desorbed completely, merges high-purity xanthomycin A component, removes and is freeze-dried after solvent,
Obtain xanthomycin A component.
Step 1) described in centrifugation be that 10min is centrifuged under conditions of 8000r/min.
Step 2) described in gradient elution be：2BV deionized waters, the methanol aqueous solutions of 1BV 20%, 1BV 40% are used successively
Methanol aqueous solution, the methanol aqueous solutions of 3BV 60% and the methanol aqueous solutions of 2BV 80% are eluted, and elution flow rate is 1~3BV/
hr。
Step 3) in enrichment xanthomycin A component eluent and anion exchange resin volume ratio be 4~10：1, it is described
Anion exchange resin be the ion exchange resin with quaternary amine base or tertiary amine groups.
Step 3) described in elution be：Eluted successively with 2BV deionized waters and 5BV cushioning liquid, the pH of buffer solution
It is worth for 3.5, the KH containing 67mM in buffer solution2PO4And 1M NaCl；Elution flow rate is 1~20BV/hr.
Step 4) in used reverse-phase chromatography filler aperture beParticle diameter is 20~75 μm；Reverse-phase chromatography is filled out
Expect C8, C18 filler being modified for silica gel, or be the chromatograph packing material of polymer substrate.
Described polymer substrate is styrene-divinylbenzene polymer or polymethacrylate polymer.
Step 4) in advanced row linear gradient elution, then carry out the concrete operations of Gradient elution and be：
It is A mobile phases by 8.30 20% methanol solution of pH value, using pure methanol as B mobile phases, is flowed according to 0~40%B
Dynamic phase linear gradient elution 40min, then desorbed completely with 40%B mobile phases Gradient elution to xanthomycin A component.
Step 4) in be filled with reverse-phase chromatography filler chromatographic column use 20 × 250mm chromatographic column.
Compared with prior art, the present invention has following beneficial technique effect：
The isolation and purification method of xanthomycin A component disclosed by the invention, is inhaled by polymethacrylate macropore first
Attached resin prepurification flavomycoin, purity and the yield of xanthomycin A component are superior to styrene-divinylbenzene class macroporous absorption tree
Fat, and solvent consumption is less, in addition, polymethacrylate macroporous absorbent resin is more resistant to polluting, being more easily regenerated into, so as to prolong
The service life of resin has been grown, cost is reduced；Secondly, macroporous absorbent resin eluent is without the side such as ultrafiltration-drying or revolving
Method removes solvent, but directly by anion exchange resin desolvation, so that the whole process that isolates and purifies is connected non-
It is often compact；Finally, using particle diameter is bigger, price more cheap preparation scale reverse-phase chromatography filler refine xanthomycin A component, be more easy to
Industrial application.
Brief description of the drawings
Fig. 1 be XAD7HP before purification after HPLC detection collection of illustrative plates；Wherein, (a) is XAD7HP before processings (zymotic fluid)；(b)
After XAD7HP elutions；
Fig. 2 is purity and yield through FPA98Cl resin treatment xanthomycin As under different pH condition；
Fig. 3 is the HPLC testing results of flavomycoin zymotic fluid and final purified product；
Fig. 4 is United States Patent (USP) US5986089 technical process figures；
Fig. 5 is United States Patent (USP) US5986089 complete process flow figures；
Fig. 6 is complete process flow figure of the present invention.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
The process chart of the present invention is referring to Fig. 6, the isolation and purification method of disclosed xanthomycin A component, including following step
Suddenly：
(1) polymethacrylate macroporous absorbent resin is extracted
Flavomycoin ferment filtrate is adjusted into pH to 3.0~4.5, cold compartment of refrigerator (4 DEG C) is put in and stays overnight (so that some albumen
Sedimentation), 10min is centrifuged under the conditions of 8000r/min, sediment is removed.Centrifuged supernatant is continued to flow through into poly- first with 1-3BV/hr
Base esters of acrylic acid macroporous adsorption resin chromatography post, is not spilt with column outlet efflux xanthomycin A component and is advisable, according to difference batch
The difference of secondary zymotic fluid quality, controls carrying capacity 15~30g/L resins of xanthomycin A component.Then successively with 2BV deionized waters,
The methanol aqueous solutions of 1BV 20%, the methanol aqueous solutions of 1BV 40%, the methanol aqueous solutions of 3BV 60% and the methanol aqueous solutions of 2BV 80%
Elution, elution flow rate is 1~3BV/hr, collects cut and merges enrichment Mon A eluent.
(2) anion exchange resin desolventizing
Macroporous absorbent resin eluent pH=6.0~8.0 containing solvent are adjusted, the eluent is flowed with 1-20BV/hr
Speed continues to flow through the chromatographic column for being filled with anion exchange resin, and the ratio of eluent and resin is 4:1~10:1(v/v).According to
It is secondary that 2BV deionized waters, 5BV 67mMKH are used under 1~20BV/hr flow velocitys2PO4+ 1M NaCl (pH=3.50) cushioning liquid is washed
It is de-, collect Mon A eluents after purification.
(3) reverse-phase chromatography is refined
Take the Mon A eluents that 160~400ml anion exchange methods are obtained, adjust pH=3.0~4.5, with 50~
250cm/hr flow velocitys are pumped into the chromatographic column (20 × 250mm) for being filled with reverse-phase chromatography filler.Then with same flow velocity, pH=is used
8.30 20% methanol is B mobile phases as A mobile phases, pure methanol, and 40min is eluted according to 0~40%B linear gradient modes,
Mon component As are eluted to 40%B constant gradient modes to desorb completely, merge high-purity Mon A cuts again.
Solvent is removed through ultrafiltration or revolving, freeze-drying obtains product.
Described macroporous absorbent resin is polymethacrylate macroporous absorbent resin；
Described anion exchange resin is the ion exchange resin with quaternary amine base or tertiary amine groups；
Described reverse-phase chromatography filler aperture be 100~20~75 μm of particle diameter, reverse-phase chromatography condiment includes silica gel
Modified C8 or C18 fillers and the reverse phase filler of polymer substrate, described polymer substrate include styrene-divinylbenzene
Polymer or polymethacrylate polymer.
1st, acrylic compounds macroporous absorbent resin pre-separation
Flavomycoin zymotic fluid (solid content is 60g/L) is used into dilute H2SO4PH=3.50 is adjusted, cold compartment of refrigerator (4 DEG C) is put in
Overnight (so that some protein depositions), 10min is centrifuged under the conditions of 8000r/min, sediment is removed, supernatant is standby.
300mL Amberlite XAD 7HP and Amberlite XAD 1180 are respectively taken to fill post (Ф=25mm, H=
600mm), 600mL flavomycoins zymotic fluid is respectively flowed through with 5mL/min flow velocity be filled with Amberlite XAD7HP and
The chromatographic column of Amberlite XAD1180 macroporous absorbent resins.Successively with 600mL deionized waters, the methanol of 300mL 20%,
The methanol of 300mL 40%, the methanol of 900mL 60%, 600mL80% methanol, the methanol of 600mL 100% are washed with 5mL/min flow velocity
It is de-.HPLC detects eluent, merges the eluent containing high-purity Mon A, Amberlite XAD7HP and Amberlite
XAD1180 purification effect is shown in Table 1.
Table 1 XAD7HP, XAD1180 main physical and chemical and its purification effect to Mon A
Obviously, XAD7HP macroporous absorbent resins are better than XAD1180 macroporous absorbent resins, mesh to Mon A purification effect
Mark thing Mon A are primarily present in 80% meoh eluate, relatively save organic solvent using XAD1180 resins.And follow-up
Resin regeneration experiment shows that XAD7HP is more easy to regeneration compared with XAD1180.
The composition of each cut of XAD7HP loadings, elution process is shown in Table 2.
Distributions (XAD7HP) of the Mon A of table 2 in each eluent
XAD7HP before purification after effect be shown in Table 3 and Fig. 1.
The XAD7HP of table 3 before purification in after fermentation liquid Mon and Mon A purity
2nd, anion exchange resin desolventizing
Scheme 1 (not desolventizing)
Take the Mon A eluents (containing 80% methanol) that four parts of 400mL embodiments 1 obtain, pH value is adjusted to 4.0 respectively, 6.0,
8.0th, 10.0, under 5mL/min flow velocitys, loading to Amberlite FPA98Cl anion exchange resin chromatographic column (Ф=
16mm, H=500mm).Under identical flow velocity, successively with deionized water, 67mM KH2PO4+ 1M NaCl (pH=3.50) cushioning liquid
Elution, HPLC detections, collects Mon A eluents after purification.
Scheme 2 (desolventizing)
The Mon A eluents revolving that 400mL Mon A embodiments 1 are obtained is gone after solvent methanol, obtains the about 80mL aqueous solution,
Add deionized water and be diluted to 400mL, dilute NaOH solution regulation pH=8.00, carried out according to the loading in scheme 1-elution requirement
Purifying, collects Mon A eluents after purification.
Mon A purity is shown in Fig. 2 with yield result in the eluent being collected under the condition of different pH of scheme 1.Work as pH=6-8
When, FPA98Cl is preferable to Mon A purification effect, and during wherein pH=8, Mon A purity is 49.4%, and yield is 90%.
The Mon sample liquids contained in scheme 1 in Mon sample liquids in organic solvent methanol, scheme 2 eliminate methanol by revolving,
Compare its purification effect under the conditions of optimization pH, obtain table 4.From table 4, it can be seen that the purification effect of scheme 1 is substantially better than scheme
2, it means that the presence of methanol has the purifying beneficial to Mon A in Mon sample liquids.
The comparison of the two schemes purification effect of table 4
In addition, in scheme 2, must additionally increase revolving and go this technique of methanol, can just obtain aqueous phase Mon sample liquids.It is very bright
Show the problems such as this can cause extension process, reduction yield, improve production cost.The simplicity of linking and operation based on technique,
We select not removing the scheme 1 that methanol is directly splined on anion-exchange column.Purified using FPA98Cl anion-exchange columns
Mon A, Mon A purity are improved to 49.8% by 43.8%, and high income is up to 90%.While removal methanol is reached, and by
In the strong elution power of high salt concentration (1M NaCl) so as to concentrate Mon sample liquids (becoming 200mL by 400mL), be conducive to next step
Subtractive process.
The loading of scheme 1, the composition of each cut of elution process are shown in Table 5.
Mon A content in the FPA98Cl of table 5 purifying each eluents of Mon A
3rd, reverse-phase chromatography refines Mon A
The Mon A eluents that 320mL anion exchange methods are obtained are taken, pH=4.5 is adjusted.It is splined on 100cm/hr flow velocitys
It is filled with Fuji Chromatorex C18 reverse-phase chromatographic columns (Ф=20mm, H=250mm).Then the 20% of pH=8.30 is used
Methanol is B mobile phases as A mobile phases, 100% methanol, with 100cm/hr flow velocitys, is eluted according to 0-40%B linear gradient modes
40min, then be eluted to Mon component As with 40%B constant gradient modes and desorb completely, merges high-purity Mon A cuts, through ultrafiltration or
Revolving removes solvent, and freeze-drying obtains colourless xanthomycin A product, and purity is between 95%-99%.
Embodiment 1
A kind of isolation and purification method of xanthomycin A prescription, comprises the following steps：
1) flavomycoin zymotic fluid pH value is adjusted to 3.0, in 4 DEG C of refrigerated overnights, centrifuged under conditions of 8000r/min
10min, takes supernatant；
2) supernatant is continued to flow through into polymethacrylate macroporous absorbent resin ((Diaion with 1BV/hr flow velocity
HP2MG, Mitsubishi chemistry) chromatographic column, the carrying capacity for controlling xanthomycin A component is 15g/L, then carries out gradient elution, is collected
Cut, merges the eluent of enrichment xanthomycin A component；
Described gradient elution is：2BV deionized waters, the methanol aqueous solutions of 1BV 20%, the methanol-waters of 1BV 40% are used successively
Solution, the methanol aqueous solutions of 3BV 60% and the methanol aqueous solutions of 2BV 80% are eluted, and elution flow rate is 1BV/hr；
3) pH value that will be enriched with the eluent of xanthomycin A component is adjusted to 6.0, is continued to flow through and is filled with 1BV/hr flow velocitys
The chromatographic column of anion exchange resin, is then eluted, and collects xanthomycin A component eluent after purification；
Wherein, the volume ratio of the eluent and anion exchange resin that are enriched with xanthomycin A component is 4：1；It is described it is cloudy from
Sub-exchange resin is the ion exchange resin (Amberlite FPA53, Rhom and Hass of the U.S.) with tertiary amine groups；
Described elution requirement is：Eluted successively with 2BV deionized waters and 5BV cushioning liquid, the pH value of buffer solution
For 3.5, the KH containing 67mM in buffer solution2PO4And 1M NaCl；Elution flow rate is 1BV/hr；
4) pH value of xanthomycin A component eluent after purification is adjusted to 3.0, filling is pumped into 50cm/hr flow velocity
There is 20 × 250mm of reverse-phase chromatography filler chromatographic column, then with identical flow velocity, 20% methanol solution using pH value as 8.30
For A mobile phases, using pure methanol as B mobile phases, 40min is eluted according to 0~40%B linear gradient modes, then use 40%B constant gradients
Mode is eluted to xanthomycin A component and desorbed completely, until xanthomycin A prescription is desorbed completely, merges high-purity xanthomycin A component,
Remove and be freeze-dried after solvent, obtain xanthomycin A component；
Wherein, reverse-phase chromatography filler used is C8 fillers (SP-300-40/60-C8-P, aperture that silica gel is modified50 μm of particle diameter, Japanese Daiso companies).
Embodiment 2
A kind of isolation and purification method of xanthomycin A prescription, comprises the following steps：
1) flavomycoin zymotic fluid pH value is adjusted to 3.8, in 4 DEG C of refrigerated overnights, centrifuged under conditions of 8000r/min
10min, takes supernatant；
2) supernatant is continued to flow through into polymethacrylate macroporous absorbent resin (Purosorb with 2BV/hr flow velocity
PAD610, Piao Laite companies of Britain) chromatographic column, the carrying capacity for controlling xanthomycin A component is 25g/L, then carries out gradient elution, is received
Collect cut, merge the eluent of enrichment xanthomycin A component；
Described gradient elution is：2BV deionized waters, the methanol aqueous solutions of 1BV 20%, the methanol-waters of 1BV 40% are used successively
Solution, the methanol aqueous solutions of 3BV 60% and the methanol aqueous solutions of 2BV 80% are eluted, and elution flow rate is 1BV/hr；
3) pH value that will be enriched with the eluent of xanthomycin A component is adjusted to 7.0, and filling is continued to flow through with 12BV/hr flow velocitys
There is the chromatographic column of anion exchange resin, then eluted, collect xanthomycin A component eluent after purification；
Wherein, the volume ratio of the eluent and anion exchange resin that are enriched with xanthomycin A component is 7：1；It is described it is cloudy from
Sub-exchange resin is the ion exchange resin (Amberlite FPA98, Rhom and Hass of the U.S.) with quaternary amine base；
Described elution is：Eluted successively with 2BV deionized waters and 5BV cushioning liquid, the pH value of buffer solution is
3.5, the KH containing 67mM in buffer solution2PO4And 1M NaCl；Elution flow rate is 15BV/hr；
4) pH value of xanthomycin A component eluent after purification is adjusted to 3.8, filling is pumped into 150cm/hr flow velocity
There is 20 × 250mm of reverse-phase chromatography filler chromatographic column, then with identical flow velocity, 20% methanol solution using pH value as 8.30
For A mobile phases, using pure methanol as B mobile phases, 40min is eluted according to 0~40%B linear gradient modes, then use 40%B constant gradients
Mode is eluted to xanthomycin A component and desorbed completely, until xanthomycin A prescription is desorbed completely, merges high-purity xanthomycin A component,
Remove and be freeze-dried after solvent, obtain xanthomycin A component；
Wherein, reverse-phase chromatography filler used is C18 fillers (the Chromatorex C18MB 100-40/ that silica gel is modified
75, aperture40~75 μm of particle diameter, Japanese Fuji companies).
Embodiment 3
A kind of isolation and purification method of xanthomycin A prescription, comprises the following steps：
1) flavomycoin zymotic fluid pH value is adjusted to 4.5, in 4 DEG C of refrigerated overnights, centrifuged under conditions of 8000r/min
10min, takes supernatant；
2) supernatant is continued to flow through into polymethacrylate macroporous absorbent resin with 3BV/hr flow velocity
(Amberlite XAD7HP, Rhom and Hass of the U.S.) chromatographic column, the carrying capacity for controlling xanthomycin A component is 30g/L, Ran Houjin
Row gradient elution, collects cut, merges the eluent of enrichment xanthomycin A component；
Described gradient elution is：2BV deionized waters, the methanol aqueous solutions of 1BV 20%, the methanol-waters of 1BV 40% are used successively
Solution, the methanol aqueous solutions of 3BV 60% and the methanol aqueous solutions of 2BV 80% are eluted, and elution flow rate is 1BV/hr；
3) pH value that will be enriched with the eluent of xanthomycin A component is adjusted to 8.0, and filling is continued to flow through with 20BV/hr flow velocitys
There is the chromatographic column of anion exchange resin, then eluted, collect xanthomycin A component eluent after purification；
Wherein, the volume ratio of the eluent and anion exchange resin that are enriched with xanthomycin A component is 10：1；It is described it is cloudy from
Sub-exchange resin is the ion exchange resin (Amberlite IRA958, Rhom and Hass of the U.S.) with quaternary amine base；
Described elution is：Eluted successively with 2BV deionized waters and 5BV cushioning liquid, the pH value of buffer solution is
3.5, the KH containing 67mM in buffer solution2PO4And 1M NaCl；Elution flow rate is 20BV/hr；
4) pH value of xanthomycin A component eluent after purification is adjusted to 4.5, filling is pumped into 250cm/hr flow velocity
There is 20 × 250mm of reverse-phase chromatography filler chromatographic column, then with identical flow velocity, 20% methanol solution using pH value as 8.30
For A mobile phases, using pure methanol as B mobile phases, 40min is eluted according to 0~40%B linear gradient modes, then use 40%B constant gradients
Mode is eluted to xanthomycin A component and desorbed completely, until xanthomycin A prescription is desorbed completely, merges high-purity xanthomycin A component,
Remove and be freeze-dried after solvent, obtain xanthomycin A component；
Wherein, reverse-phase chromatography filler used is C18 fillers (the Chromatorex C18MB100-20/ that silica gel is modified
45, aperture20~45 μm of particle diameter, Japanese Fuji companies).
In summary, the inventive method had both obtained xanthomycin A component, and the deficiency of prior art processes is overcome again, led to
Anion exchange resin desolventizing is crossed, so that whole technique linking is closely；By using polymethacrylate
Macroporous absorbent resin is superior to styrene-bis- in the prepurification flavomycoin of pH3.0~4.5, purity and the yield of xanthomycin A component
Ethenylbenzene class macroporous absorbent resin, and solvent consumption is less.Due to substantially polymethacrylate macroporous absorbent resin more
Anti-pollution, be more easy to regeneration and so that the life-span of resin greatly prolong；Filled out using 20-75 μm of industrially prepared level reverse-phase chromatography of particle diameter
The refined xanthomycin A component of material, filler cheap and be more easy to industrial application.
Claims (6)
1. a kind of isolation and purification method of xanthomycin A component, it is characterised in that comprise the following steps：
1) flavomycoin zymotic fluid pH value is adjusted to 3.0~4.5, in 4 DEG C of refrigerated overnights, centrifugation takes supernatant；
2) supernatant is continued to flow through into polymethacrylate macroporous adsorption resin chromatography post with 1~3BV/hr flow velocity, controlled
The carrying capacity of manufacture-yellow mycin component A is 15~30g/L, then carries out gradient elution, collects cut, merges enrichment xanthomycin A component
Eluent；
Described gradient elution is：Successively with 2BV deionized waters, the methanol aqueous solutions of 1BV 20%, the methanol aqueous solutions of 1BV 40%,
The methanol aqueous solutions of 3BV 60% and the methanol aqueous solutions of 2BV 80% are eluted, and elution flow rate is 1~3BV/hr；
3) pH value that will be enriched with the eluent of xanthomycin A component is adjusted to 5.0~8.0, is continued to flow through with 1~20BV/hr flow velocitys
The chromatographic column of anion exchange resin is filled with, is then eluted, the xanthomycin A component eluent after desolventizing is collected；
The volume ratio of the eluent and anion exchange resin that are enriched with xanthomycin A component is 4~10：1, described anion exchange
Resin is the ion exchange resin with quaternary amine base or tertiary amine groups；
Described elution is：Eluted successively with 2BV deionized waters and 5BV cushioning liquid, the pH value of buffer solution is 3.5, is delayed
KH containing 67mM in fliud flushing2PO4And 1M NaCl；Elution flow rate is 1~20BV/hr；
4) pH value of the xanthomycin A component eluent after desolventizing is adjusted to 3.0~4.5, with 50~250cm/hr flow velocity
The chromatographic column for being filled with reverse-phase chromatography filler is pumped into, then with the advanced row linear gradient elution of identical flow velocity, then the ladder such as carries out
Degree elution, until xanthomycin A component is desorbed completely, merges high-purity xanthomycin A component, removes and is freeze-dried after solvent, is obtained
Xanthomycin A component.
2. the isolation and purification method of xanthomycin A component according to claim 1, it is characterised in that step 1) described in from
The heart is that 10min is centrifuged under conditions of 8000r/min.
3. the isolation and purification method of xanthomycin A component according to claim 1, it is characterised in that step 4) in it is used
Reverse-phase chromatography filler aperture isParticle diameter is 20~75 μm；Reverse-phase chromatography filler is C8, C18 that silica gel is modified
Filler, or be the chromatograph packing material of polymer substrate.
4. the isolation and purification method of xanthomycin A component according to claim 3, it is characterised in that described polymer matrix
Matter is styrene-divinylbenzene polymer or polymethacrylate polymer.
5. the isolation and purification method of xanthomycin A component according to claim 1, it is characterised in that step 4) in first carry out
Linear gradient elution, then carry out the concrete operations of Gradient elution and be：
It is A mobile phases by 8.30 20% methanol solution of pH value, using pure methanol as B mobile phases, according to 0~40%B mobile phases
Linear gradient elution 40min, then desorbed completely with 40%B mobile phases Gradient elution to xanthomycin A component.
6. the isolation and purification method of xanthomycin A component according to claim 1, it is characterised in that step 4) in be filled with
The chromatographic column of reverse-phase chromatography filler uses 20 × 250mm chromatographic column.
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| 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 |
| EP0872556A3 (en) * | 1997-04-17 | 2000-06-14 | Hoechst Aktiengesellschaft | Process for the preparation of moenomycin A |
| CN1290853C (en) * | 2005-03-09 | 2006-12-20 | 中牧实业股份有限公司 | Purifying method for moenomycin |
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