CN107778357B - Extraction and purification method of pneumocandin B0 - Google Patents
Extraction and purification method of pneumocandin B0 Download PDFInfo
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Abstract
The invention discloses an extraction and purification method of pneumocandin B0, which comprises the following steps: 1) adjusting the pH value of fermentation liquor containing pneumocandin B0, adding filter aid, and filtering to obtain mushroom dregs; 2) leaching the mushroom dregs with high-concentration methanol water solution or ethanol water solution; 3) adsorbing the leaching solution with adsorption resin, eluting with 80-90% (V/V) -water solution of methanol (ethanol), and collecting eluate rich in pneumocandin B0; 4) adding activated carbon into the eluent of the pneumocandin B0 for decolorization; 5) adsorbing with adsorbent resin, eluting the resin with 80-100% (v/v) methanol (ethanol) -water solution, and collecting eluate rich in pneumocandin B0; 6) and concentrating in vacuum to obtain a crude product of the nemadectin B0. By improving the extraction and purification method, the decoloring and impurity removing effects are obvious, the product purity is obviously improved, the production cost is low, the process steps are simple, and the method is suitable for industrial production.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to an extraction and purification method of pneumocandin B0.
Background
Caspofungin is echinocandin which is the first marketed globally, has a unique action mechanism, takes a fungal cell wall as a target position, specifically inhibits the synthesis of beta (1, 3) -D-glucan of the cell wall, destroys the integrity of the fungal cell wall, makes the osmotic pressure in the fungal cell unstable, and finally leads to the dissolution of the fungal cell. Mammalian cells do not contain beta- (1, 3) -glucan and have no similar cell wall synthesis process, so that the caspofungin is antifungal and does not damage and influence human cells. In vitro pharmacological studies have shown that caspofungin is effective against a variety of candida species, endemic fungi, aspergillus species and pneumocystis carinii and has received increasing attention.
Pneumocandin B0(Pneumocandin B0) is a secondary metabolite produced by the fungus Zaleron arB0ricola and is an intermediate in the synthesis of the antifungal drug CasPofungin.
As an intermediate for synthesizing an antifungal drug CasPofungin, pneumocandin B0 is produced by fungal fermentation, and byproducts with a structure similar to that of pneumocandin B0 and a large amount of pigments are produced in the fermentation process, so that the extraction and purification method of pneumocandin B0 is complicated. The proline substituents are mainly classified into three main groups according to the difference of the proline substituents in the structure: a0(32 hydroxy 242 methyl proline), B0(32 hydroxy proline) and C0(42 hydroxy proline), and in addition, A0 can be divided into five subclasses of AO, Al, AZ, A3 and A4 according to different nemoconazole substituents on the cyclic polypeptide, and B0 can be divided into two subclasses of B0 and B2.
In The publication Of Pneumocandins From Yeast architectural I.discovery And Isolation (The Journal Of Antibiotics 45(12): 1853. sup. 1866), The extraction And purification method Of pneumocandin B0 was described. The method is briefly described as follows:
1) adjusting the pH value of the fermentation liquor, centrifuging at high speed, taking mycelium, soaking with methanol with twice volume, centrifuging, soaking with 80% methanol again to extract pneumocandin B0, and mixing methanol leaching liquor;
2) diluting the methanol extract with water, purifying with 40% -50% methanol on HP-207 resin column, washing the column with 65:35 methanol, and eluting with 100% methanol to obtain pneumocandin B0;
3) slowly adding isopropyl acetate into the pneumocandin B0 solution at 25 ℃, precipitating, filtering, centrifuging and evaporating to dryness to obtain pneumocandin B0 with purity of about 67% and yield of about 84%;
4) purifying again by silica gel chromatography and HP20 resin adsorption, washing the column with 85:10:5 EtOAC-MEOH-5% AcOH, collecting, dissolving in 50% methanol, washing with HP20, washing with 50% methanol and then with 100% methanol, and recovering 50% ethanol;
5) add capronitrile to precipitate pneumocandin B0, and filter to give 85% purity.
6) In n-propanol, 50mg/ml concentration, 60 ℃ heating, adding water and cooling to room temperature for crystallization. The main disadvantages of this process are the complicated separation procedure, the difficult thorough removal of the pigment from the upstream fermentation broth, and the most important disadvantages of the unstable silica gel in silica gel chromatography, which makes it difficult to carry out mass production.
The U.S. Pat. No. 6,416,327 also provides a Process for extracting and purifying pneumocandin B0, i.e. extraction and back extraction are repeatedly used, and extraction and washing are repeatedly carried out, the Process uses a large amount of organic solvents such as isobutanol, methanol and heptane, the operation is complicated, and the purity of the pneumocandin B0 solid obtained by final precipitation is only about 80%. Not only has low purity, but also has incomplete removal of pigment impurities. Is not suitable for large-scale production at all.
CN200910133118.0 "a method for preparing pneumocandin B0" describes a new pneumocandin B0 purification method, which mainly comprises the following steps:
1) centrifuging the fermentation broth, collecting mycelium, and leaching pneumocandin B0 with methanol;
2) evaporating the methanol leaching solution to dryness, and leaching the pneumocandin B0 with n-butanol;
3) evaporating n-butanol leaching solution to dryness, leaching pneumocandin B0 with 70-80% methanol, passing through acidic alumina column, and collecting eluate;
4) evaporating collected solution of pneumocandin B0 to dryness, dissolving with 60-70% methanol, adsorbing with HP20 adsorbent resin, eluting with 85-95% methanol, collecting eluate of pneumocandin B0 with purity of 50-65%;
5) evaporating collected solution of pneumocandin B0 to dryness, dissolving in 60-70% methanol, adding reversed phase resin YPR-II, eluting with 85-95% methanol, and collecting pneumocandin B0 with purity of more than 90%;
6) evaporating collected solution of pneumocandin B0 to dryness, dissolving with methanol, and dropping small amount of water to supersaturate and crystallize to obtain pneumocandin B0 with purity up to 96%.
Although the purification method can improve the quality of the pneumocandin B0 product, remove a large amount of pigment and improve the product purity, the whole process step is complex, almost every step needs to evaporate the pneumocandin B0 solution to dryness, the method can only be used for small-scale research and application in production, the energy consumption and loss are large, and the pneumocandin B0 is easy to damage under long-time heating. More importantly, the purity of the pneumocandin cannot be effectively improved by using the so-called reversed-phase resin YPR-II, particularly, analogues of the pneumocandin B0 are difficult to remove, the purity of 96 percent is required to be further verified, and therefore, the method is not suitable for industrial production of the pneumocandin B0.
CN201110266790.4 extraction and purification method of pneumocandin B0 describes a new extraction and purification method of pneumocandin B0, which mainly comprises the following steps:
1) adjusting pH of fermentation liquid containing pneumocandin B0 to 2.0-4.0, filtering, and leaching the mushroom residue with low molecular alcohol to obtain leaching solution;
2) adjusting the alcohol concentration and pH of the leaching solution;
3) adsorbing the leaching solution by using adsorption resin, sequentially washing the resin by using water and acetone-water solution, eluting the resin by using acetone-acid solution, and collecting eluent rich in pneumocandin B0;
4) diluting pneumocandin B0 eluate with water, adsorbing with adsorbent resin, washing the resin with water, eluting with low molecular alcohol, and collecting eluate rich in pneumocandin B0;
5) crystallizing by a conventional method to obtain pneumocandin B0, wherein the solid is light yellow or similar white, and the purity is more than 85%; the pneumocandin B0 solid is dissolved by low molecular alcohols, such as methanol, ethanol, propanol, butanol and the like, a small amount of water is added, and the solution is supersaturated and crystallized to be separated out, or other known crystallization methods are adopted, so that the pneumocandin B0 with the purity of more than 90 percent can be prepared, and through the improvement of the extraction and purification method, the extraction process steps of the pneumocandin B0 are like simple treatment, the product purity can be relatively improved, but the decolorization effect in the extraction and purification process is not obvious.
The main disadvantages of this process are:
1) the pH value of the fermentation liquor is adjusted to be too low, so that a large amount of impurities such as pigment and the like are precipitated, and the downstream purification difficulty is increased;
2) the purity of the product is still not ideal enough, and the difficulty of downstream synthesis, extraction and purification is increased.
Therefore, a more effective decoloring method is searched and a simple extraction and purification method is established, so that the industrial production of the pneumocandin B0 can be realized.
Disclosure of Invention
In order to solve the technical problems that pigments in fermentation liquor are difficult to remove in a process for preparing the pneumocandin B0 in the prior art and the whole process is complicated, the invention provides a simple method for preparing the pneumocandin B0, and the method not only can well remove the pigments in the pneumocandin B0, but also can improve the purity of the pneumocandin B0 to more than 93%. Accordingly, the present invention provides a process for preparing pneumocandin B0, the process comprising the steps of:
1) adjusting pH value of fermentation liquid containing pneumocandin B0 to 4.2-6 under stirring, adding filter aid, mixing, and filtering to obtain solid mushroom residue;
2) leaching the mushroom dregs with high-concentration low-molecular alcohol-water solution to obtain leaching liquor; adjusting alcohol concentration of the leaching solution to 20-30% (V/V);
3) adsorbing the leaching solution by using adsorption resin, sequentially washing the resin by using purified water and 30-50% (V/V) low molecular alcohol-water solution, eluting the resin by using 80-90% (V/V) low molecular alcohol-water solution, and collecting eluate rich in pneumocandin B0;
4) adding 0.5-5% active carbon into the eluate, stirring at 20-50 deg.C for 30-60 min, filtering, and decolorizing;
5) ultrafiltering the pneumocandin B0 decolorized solution, adding water to dilute alcohol to 20-30% (V/V), adsorbing with adsorption resin, washing the resin with 30-50% (V/V) solution containing low molecular alcohol, eluting with 80-100% (V/V) solution containing low molecular alcohol, and collecting the eluate rich in pneumocandin B0;
6) vacuum concentrating collected pneumocandin B0 eluate at 30-50 deg.C to obtain white crude product; the purity of the crude product is more than 96.5 percent.
In the step 1) of the method, due to the comprehensive application of pH regulation and the filter aid, excessive precipitated impurities caused by too low pH are avoided, the filtration efficiency of the filtrate is improved, the pneumocandin can be completely precipitated along with the thalli, the yield is improved, and the post-treatment difficulty is reduced. Wherein the pH of the fermentation broth of pneumocandin B0 is adjusted to 4.2-6, preferably pH 4.5, with phosphoric acid.
In the step 1) of the method, the amount of the added filter aid is 0.5-5% of the mass of the fermentation liquor, preferably 2%, the filter aid is one or more of diatomite, perlite, cellulose, magnesium oxide, gypsum and acid clay, and the filter aid is preferably perlite;
in step 2), the aqueous solution of high-concentration low-molecular alcohol is extracted, wherein the low-molecular alcohol is: one of methanol, ethanol and propanol; ethanol aqueous solution with concentration of 90-98% (V/V), preferably 95%; adding the leaching liquor in an amount of 2-5 times, preferably 3 times, of the mass of the mushroom dregs;
in the step 3) of the method, the types of the adsorption resin are skp-10-4300, D101, HPD300, HPD700 and BS-5, and the skp-10-4300 is preferred; adding pure water to make alcohol concentration in leaching solution be 20-30% (V/V), preferably 30%; 30-50% (V/V) methanol (ethanol) -aqueous solution is used for washing the resin, the alcohol concentration of the resin column is preferably 45% (V/V), and the alcohol concentration of the eluent is preferably 80% (V/V).
Because the adsorption resin has no adsorption effect on substances with larger polarity and has weak adsorption effect on impurities with stronger polarity, polar impurities in the solution can be basically removed through sample loading and purified water washing, and most of water-soluble pigments can be removed; elution of the resin with 80-90% (V/V) low molecular alcohol-water solution allowed relatively concentrated elution of pneumocandin B0 while leaving pigments and other impurities of very low polarity on the column. By these means, the purity of pneumocandin B0 was significantly improved.
In the method, in the step 4), activated carbon is used in a high-concentration alcohol solution, the decolorization effect is very remarkable, the color of the nemodadine B0 solution is changed from light brown to extremely light yellow, the loss of nemodadine B0 is controlled within 5%, the adding amount of the activated carbon is preferably 2%, and the decolorization temperature is preferably 40 ℃.
In the step 5) of the method, the selected adsorption resin is skp-40-4300, and the resin is a macroporous copolymer prepared by polymerizing two monomers of lipophilic divinylbenzene and hydrophilic N-vinyl pyrrolidone according to a certain proportion. The retention mechanism is reversed phase, and a special polar trapping group is used for increasing the retention of the polar substance and providing good water wettability. By utilizing the characteristics of the polymer, impurities with stronger polarity can be eluted when the pneumocandin B0 is adsorbed, so that the pneumocandin B0 is further purified, the purity is further improved, the pigment is basically removed completely, and meanwhile, the polymer has an obvious enrichment effect on a target product, and is beneficial to vacuum concentration in the next step. After concentration in vacuo, pneumocandin B0 was essentially colorless.
The resin is preferably skp-40-4300, the concentration of the alcohol in the column-loading solution is preferably adjusted to 30% (V/V), the alcohol concentration of the washing solution is preferably 50% (V/V), and the alcohol concentration of the eluent is preferably 90% (V/V).
In the step 6) of the invention, due to the enrichment effect in the step 5), the volume of the received eluent is small, the alcohol concentration is high, and the eluent is suitable for vacuum concentration, so that the target product is basically not lost by adopting a vacuum concentration method. The crystallization process of the macromolecular peptide substance is difficult to control (the cooling crystallization time is long), the yield is low, and the purification effect is poor. Through a number of comparative tests, the vacuum concentration method is much better than the traditional crystallization method. In the experiment, purity of Pneumocandin B096% can meet the demand of caspofungin synthesis, and if further improvement of purity is needed, effluent rich in Pneumocandin B0 can be collected by silica gel preparation method disclosed in article Pneumocandin from Zolerboriclo in journal of antibiotics at 6.15.1992, and purity can be further improved to 95% or more. Of course, a mature preparation method is developed, and the purity of the pneumocandin B0 can be further purified to reach more than 98%.
Compared with the prior art, the invention has obvious advantages and is easy to realize industrial production. Meanwhile, the process provided by the invention can be used for basically removing pigments and impurities in the fermentation liquor, so that the purity of the pneumocandin B0 reaches a level of more than 96.5%.
Detailed Description
The advantageous effects of the present invention will now be further described by the following examples, which are for illustrative purposes only and do not limit the scope of the present invention, and variations and modifications apparent to those of ordinary skill in the art according to the present invention are also included in the scope of the present invention.
Detailed Description
The detection conditions of the pneumocandin B0HPLC are as follows: a chromatographic column: ODS-C18 (5 um);
detection wavelength: 210 nm;
mobile phase: water: acetonitrile (55: 45); flow rate: 1.0ml/min
Column temperature: at 40 ℃.
The fermentation conditions of the pneumocandin B0 are as follows:
strain: ATCC 20957
Slant culture
Culture medium: PDA (personal digital Assistant)
Conditions are as follows: 25 ℃, 12 days
Seed culture
Culture medium: glucose 1.0%, mannitol 0.5%, glycerol 1.0%, peptone 0.5%, cotton seed powder 2.5%, KH2P040.5%, and pH of 6.8-7.0 before digestion
Conditions are as follows: 25 ℃,4 days, shaker speed: 250rpm
Fermentation culture
Culture medium: mannitol 8.0%, glucose 2.0%, cottonseed meal 3.0%, yeast powder 0.5%, KHZPO 40.5%, and adjusting pH to 7.0 before digestion.
Sterilizing at 121 deg.C for 30min, inoculating 10%, shaking in 200ml/1000ml bottle, placing in 25 deg.C thermostatic chamber, rotating shaking table at 250rpm, shaking, and fermenting for 12 days to obtain fermentation broth of pneumocandin B0.
Example 1
1) 1000ml of fermentation liquor containing pneumocandin B0, the purity of which is 2.6 percent and the fermentation titer is 675mg/L, is stirred, the pH value is adjusted by phosphoric acid to be 4.2, 2 percent of filter aid diatomite is added, the mixture is uniformly mixed, the mixture is kept stand for 0.5 hour and filtered, and solid mushroom dregs are obtained;
2) leaching the mushroom dregs with 2 times of 95% ethanol, and filtering to remove hypha to obtain leaching liquor with deep red color; adjusting the alcohol concentration of the leaching solution to 20(V/V), wherein the yield of the step is 81%;
3) loading a D101 column with the height of 25cm and the diameter of about 4cm, enabling a sample solution to flow through the D101 column at the speed of 5ml/min, sequentially washing the resin column with purified water and 30% (V/V) ethanol-water solution, then eluting the resin column with 80% (V/V) ethanol-water solution, collecting eluent rich in pneumocandin B0, detecting the purity of the eluent to be 46% through a high-pressure liquid phase, and obviously lightening the color, wherein the yield of the step is 90%;
4) adding 0.5% of activated carbon into the eluent, stirring for 30 minutes at 20 ℃, filtering and decoloring, wherein the decoloring effect is better, the color is light yellow, and the yield of the step is 96%;
5) the pneumocandin B0 decolorized solution is ultrafiltered and diluted to 30% (V/V) with water. Loading a KB-SPE-HLB column with the height of 25cm and the diameter of about 4cm, enabling the sample solution to flow through the KB-SPE-HLB column at the speed of 5ml/min, washing the resin column by using a solution containing 30% (V/V) of ethanol, then eluting by using an ethanol solution containing 80% (V/V), collecting an eluent rich in pneumocandin B0, wherein the liquid phase detection purity is 96.5%, the color is obviously lightened to be light yellow, and the yield of the step is 91%;
6) vacuum concentrating collected pneumocandin B0 eluate at 30 deg.C to obtain pneumocandin B0 crude product; the purity of the crude product is 96.5%.
The total yield of the steps is 64 percent, and the purity of the pneumocandin B0 reaches 96.5 percent.
Example 2
1) 1000ml of fermentation liquor containing pneumocandin B0, the purity of the fermentation liquor is 2.7 percent, the fermentation titer is 701mg/L, the pH value is adjusted to 6 by phosphoric acid under the stirring state, 5 percent of filter aid perlite is added, the mixture is uniformly mixed, the mixture is kept stand for 0.5 hour and filtered to obtain solid mushroom dregs, and the color of the mushroom dregs is lighter;
2) leaching the mushroom dregs with 5 times of 95% ethanol, and filtering to remove hypha to obtain leaching liquor with deep red color; adjusting the alcohol concentration of the leaching solution to 30% (V/V), wherein the yield of the step is 87%;
3) loading an HPD300 column with the height of 25cm and the diameter of about 4cm, enabling a sample solution to flow through the HPD300 column at the speed of 5ml/min, sequentially washing the resin column with purified water and 40% (V/V) ethanol-water solution, then eluting the resin column with 90% (V/V) ethanol-water solution, collecting eluate rich in pneumocandin B0, detecting the purity of the eluate to be 41% by a high-pressure liquid phase, and enabling the eluate to be red in color and obviously lighter, wherein the yield of the step is 93%;
4) adding 5% of activated carbon into the eluent, stirring for 60 minutes at 50 ℃, filtering and decoloring, wherein the decoloring effect is better, the color is light yellow, and the yield of the step is 88%;
5) the pneumocandin B0 decolorized solution is ultrafiltered and diluted to 20% (V/V) with water. Loading a skp-40-4300 column with the height of 25cm and the diameter of about 4cm, enabling the sample liquid to flow through the skp-40-4300 column at the speed of 5ml/min, washing the resin column by using a 50% (V/V) solution containing ethanol, then eluting by using a 90% (V/V) solution containing ethanol, collecting an eluent rich in pneumocandin B0, wherein the liquid phase detection purity is 97.9%, the color is obviously lightened, and the yield of the step is 94%;
6) vacuum concentrating collected pneumocandin B0 eluate at 50 deg.C to obtain pneumocandin B0 crude product; the purity of the crude product is 97.9%.
The total yield of the steps is 67 percent, and the purity of the pneumocandin B0 reaches 97.9 percent.
Example 3
1) 1000ml of fermentation liquor containing pneumocandin B0, the purity of which is 2.2 percent and the fermentation titer of which is 694mg/L, is stirred, phosphoric acid is used for adjusting the pH value to 5, 2 percent of filter aid acid clay is added, the mixture is uniformly mixed, the mixture is kept stand for 0.5 hour and filtered to obtain solid mushroom dregs, and the color of the mushroom dregs is lighter;
2) leaching the mushroom dregs with 3 times of 95% ethanol, and filtering to remove hypha to obtain leaching liquor with deep red color; adjusting the alcohol concentration of the leaching solution to 30% (V/V), wherein the yield of the step is 85%;
3) loading a skp-10-4300 column with the height of 25cm and the diameter of about 4cm, enabling a sample solution to flow through the skp-10-4300 column at the speed of 5ml/min, sequentially washing the resin column by using purified water and 50% (V/V) ethanol-water solution, then eluting the resin column by using 80% (V/V) ethanol-containing solution, collecting an eluent rich in pneumocandin B0, and detecting the purity of the eluent to be 39.8% by using a high-pressure liquid phase, wherein the color of the eluent is red and obviously lightened, and the yield of the step is 95%;
4) adding 3% of activated carbon into the eluent, stirring for 40 minutes at 40 ℃, filtering and decoloring, wherein the decoloring effect is better, the color is light yellow, and the yield of the step is 92%;
5) the pneumocandin B0 decolorized solution is ultrafiltered and diluted to 30% (V/V) with water. Loading a Waters Oasis HLB column with the height of 25cm and the diameter of about 4cm, enabling a sample solution to flow through the Waters Oasis HLB column at the speed of 5ml/min, washing the resin column by using a solution containing 45 percent (V/V) of ethanol, then eluting by using an absolute ethanol solution, collecting an eluent rich in pneumocandin B0, wherein the liquid phase detection purity is 97.1 percent, the color is obviously lightened, and the yield of the step is 95 percent;
6) vacuum concentrating collected pneumocandin B0 eluate at 40 deg.C to obtain pneumocandin B0 crude product; the purity of the crude product is 97.1%.
The total yield of the steps is 70 percent, and the purity of the pneumocandin B0 reaches 97.1 percent.
Example 4
1) 1000ml of fermentation liquor containing pneumocandin B0, the purity of the fermentation liquor is 2.8 percent, the fermentation titer is 639mg/L, the pH value is adjusted by phosphoric acid under the stirring state to be 4.2, 2 percent of filter aid perlite is added, the mixture is uniformly mixed, the mixture is kept stand for 0.5 hour and filtered to obtain solid mushroom dregs, and the color of the mushroom dregs is lighter;
2) leaching the mushroom dregs with 3 times of anhydrous methanol, and filtering to remove hyphae to obtain leaching liquor with deep red color; adjusting the alcohol concentration of the leaching solution to 30% (V/V), wherein the yield of the step is about 80%;
3) loading a skp-10-4300 column with the height of 25cm and the diameter of about 4cm, enabling a sample solution to flow through the skp-10-4300 column at the speed of 5ml/min, sequentially washing the resin column by using purified water and 50% (V/V) methanol-water solution, then eluting the resin column by using 80% (V/V) methanol-containing solution, collecting an eluent rich in pneumocandin B0, and detecting the purity of the eluent by using a high-pressure liquid phase to be 44.9%, wherein the color of the eluent is red and obviously lightened, and the yield of the step is 91%;
4) adding 3% of activated carbon into the eluent, stirring for 30 minutes at 30 ℃, filtering and decoloring, wherein the decoloring effect is better, the color is light yellow, and the yield of the step is 89%;
5) the pneumocandin B0 decolorized solution is ultrafiltered and diluted to 30% (V/V) with water. Loading a skp-40-4300 column with the height of 25cm and the diameter of about 4cm, enabling the sample solution to flow through the skp-40-4300 column at the speed of 5ml/min, washing the resin column with a solution containing 45% (V/V) of methanol, eluting with an anhydrous methanol solution, collecting an eluent rich in pneumocandin B0, wherein the liquid phase detection purity is 97.8%, the color is obviously lightened, and the yield of the step is 96%;
6) vacuum concentrating collected pneumocandin B0 eluate at 30 deg.C to obtain pneumocandin B0 crude product; the purity of the crude product is 97.8%.
The total yield of the steps is 62 percent, and the purity of the pneumocandin B0 reaches 97.8 percent.
Example 5
1) 1000ml of fermentation liquor containing pneumocandin B0, the purity of the fermentation liquor is 2.9 percent, the fermentation titer is 679mg/L, the pH value is adjusted to 4.5 by phosphoric acid under the stirring state, 2 percent of filter aid perlite is added, the mixture is uniformly mixed, the mixture is kept stand for 0.5 hour and filtered to obtain solid mushroom dregs, and the color of the mushroom dregs is lighter;
2) leaching the mushroom dregs with 3 times of 95% ethanol, and filtering to remove hypha to obtain leaching liquor with deep red color; adjusting the alcohol concentration of the leaching solution to 30% (V/V), wherein the yield of the step is 88%;
3) loading a skp-10-4300 column with the height of 25cm and the diameter of about 4cm, enabling a sample solution to flow through the skp-10-4300 column at the speed of 5ml/min, sequentially washing the resin column by using purified water and 45% (V/V) ethanol-water solution, then eluting the resin column by using 80% (V/V) ethanol-containing solution, collecting eluate rich in pneumocandin B0, and detecting the purity of the eluate to be 48.7% by using a high-pressure liquid phase, wherein the color of the eluate is red and obviously lightened, and the yield of the step is 94%;
4) adding 2% of activated carbon into the eluent, stirring for 40 minutes at 40 ℃, filtering and decoloring, wherein the decoloring effect is better, the color is light yellow, and the yield of the step is 93%;
5) the pneumocandin B0 decolorized solution is ultrafiltered and diluted to 30% (V/V) with water. Loading a skp-40-4300 column with the height of 25cm and the diameter of about 4cm, enabling the sample liquid to flow through the skp-40-4300 column at the speed of 5ml/min, washing the resin column by using a 50% (V/V) solution containing ethanol, then eluting by using an 80% (V/V) solution containing ethanol, collecting an eluent rich in pneumocandin B0, wherein the liquid phase detection purity is 99.2%, the color is obviously lightened, and the yield of the step is 96%;
6) vacuum concentrating collected pneumocandin B0 eluate at 40 deg.C to obtain pneumocandin B0 crude product; the purity of the crude product is 99.2%.
The total yield of the steps is 74 percent, and the purity of the pneumocandin B0 reaches 99.2 percent.
Claims (3)
1. An extraction and purification method of pneumocandin B0 comprises the following steps:
1) adjusting pH value of fermentation liquid containing pneumocandin B0 to 4.2-6 under stirring, adding filter aid, mixing, and filtering to obtain solid mushroom residue;
2) leaching the mushroom dregs with high-concentration low-molecular alcohol-water solution, and filtering to obtain a leaching solution; adding pure water to make the alcohol concentration in the leaching solution be 20-30% (V/V);
3) adsorbing the leaching solution by using adsorption resin, sequentially washing the resin by using purified water and 30-50% (V/V) low molecular alcohol-water solution, eluting the resin by using 80-90% (V/V) low molecular alcohol-water solution, and collecting eluate rich in pneumocandin B0;
4) adding active carbon with the mass of 0.5-5% of the eluent into the eluent, stirring for 30-60 minutes at the temperature of 20-50 ℃, filtering and decoloring;
5) filtering a pneumocandin B0 decolorized solution by ultrafiltration, adding water to dilute the solution until the concentration of alcohol is 20-30% (V/V), adsorbing the solution by using an adsorption resin, washing the resin by using 30-50% (V/V) low molecular alcohol-water solution, eluting by using 80-100% (V/V) low molecular alcohol-water solution, and collecting an eluent rich in pneumocandin B0;
6) vacuum concentrating collected pneumocandin B0 eluate at 30-50 deg.C to obtain white crude product;
in the step 1), the pH value is adjusted and the filter aid is added for use at the same time;
the filter aid added in the step 1) is one or more of diatomite, perlite, cellulose, magnesium oxide, gypsum and acid clay;
the low molecular alcohol is: one of methanol, ethanol and propanol; the high-concentration low-molecular alcohol-water solution is 90-98% (V/V);
in the step 3), the types of the used adsorption resins are skp-10-4300, D101, HPD300, HPD700 and BS-5;
in the step 5), the types of the used adsorption resins are skp-40-4300, KB-SPE-HLB and Waters oasisHLB.
2. The method of claim 1, wherein the amount of the filter aid added in step 1) is 0.5% to 5% by mass of the fermentation broth.
3. The method according to claim 1, wherein in the step 2), the adding amount of the leaching solution is 2-5 times of the mass of the mushroom dregs.
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| US20020028916A1 (en) * | 2000-09-01 | 2002-03-07 | Chandler Martin A. | Purification process |
| CN102295686A (en) * | 2011-09-09 | 2011-12-28 | 杭州华东医药集团生物工程研究所有限公司 | Method for extracting and purifying pneumocandin B0 |
| US20130030149A1 (en) * | 2010-03-29 | 2013-01-31 | Kushal Rastogi | Process for purification of pneumocandin |
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| CN103936837A (en) * | 2014-02-14 | 2014-07-23 | 博瑞生物医药泰兴市有限公司 | Method used for high-efficient purification of pneumocandins B0 |
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