阅读说明：本技术 一种从伯克霍尔德菌发酵液提取fr901464的方法 (Method for extracting FR901464 from Burkholderia fermentation broth ) 是由 朱进伟 陈世敏 郑玲辉 彭湘屏 张敏 石磊 高祥 汪超 于 2021-08-17 设计创作，主要内容包括：本发明提供了一种从发酵液提取FR901464的方法,发酵液由伯克霍尔德菌发酵得到。发酵方法包括：将大孔树脂加入发酵液,混合吸附后过滤；将过滤得到的大孔树脂用有机溶剂浸泡分离,并将分离后的有机溶剂浓缩；浓缩后产物用正己烷与乙酸乙酯混合溶剂溶解,用硅胶进行色谱分离后,用乙酸乙酯洗脱得到洗脱液；浓缩洗脱液至膏状,随后配制成上柱液,上柱液用高压液相色谱仪进行分离纯化,填料为C18填料,用液相洗脱剂洗脱得到洗脱液；洗脱液经萃取后收集有机相,蒸干后形成固体粉末。(The invention provides a method for extracting FR901464 from fermentation liquor, wherein the fermentation liquor is obtained by fermenting Burkholderia. The fermentation method comprises the following steps: adding the macroporous resin into the fermentation liquor, mixing, adsorbing and filtering; soaking and separating the macroporous resin obtained by filtering with an organic solvent, and concentrating the separated organic solvent; dissolving the concentrated product with a mixed solvent of n-hexane and ethyl acetate, performing chromatographic separation by using silica gel, and eluting with ethyl acetate to obtain an eluent; concentrating the eluent to paste, preparing a column loading solution, separating and purifying the column loading solution by using a high-pressure liquid chromatograph, wherein the packing is C18 packing, and eluting by using a liquid-phase eluent to obtain an eluent; and extracting the eluent, collecting an organic phase, and evaporating to dryness to form solid powder.)

1. A method for extracting FR901464 from fermentation liquor is characterized by comprising the following steps:

the fermentation liquor is obtained by fermenting Burkholderia, and the extraction method comprises the following steps:

step 1: adding the macroporous resin into the fermentation liquor, mixing, adsorbing and filtering;

step 2: soaking the macroporous resin obtained by filtering in an organic solvent, separating, and concentrating the separated organic solvent;

and step 3: dissolving the product concentrated in the step 2 by using a mixed solvent, separating and filtering the product in a silica gel column, and leaching the product by using an eluent A to obtain an eluent;

and 4, step 4: concentrating the eluent to paste, preparing a column loading solution, and separating and purifying by using an eluent B through a high pressure liquid chromatograph.

2. The method of claim 1, wherein:

the mixed solvent is formed by mixing a polar solvent and a non-polar solvent.

3. The method of claim 2, wherein:

the polar solvent is selected from alcohols, acids, esters, nitriles, water, preferably from isopropanol, n-butanol, tetrahydrofuran, chloroform, ethanol, ethyl acetate, methanol, acetone, acetonitrile, acetic acid, more preferably ethyl acetate;

the non-polar solvent is selected from saturated hydrocarbons, benzene, preferably n-hexane.

4. The method of claim 3, wherein:

and mixing n-hexane and ethyl acetate to form the mixed solvent, wherein the volume fraction of the ethyl acetate is not more than 70%.

5. The method of claim 4, wherein:

in step 4, a column loading solution is prepared with 30% volume fraction acetonitrile.

6. The method of claim 5, wherein:

the eluent A is ethyl acetate, and the eluent A is ethyl acetate,

eluent B is selected from acetonitrile or methanol water solution.

7. The method of claim 6, wherein:

the organic solvent in step 2 is selected from ethyl acetate, dichloromethane and chloroform.

8. The method of claim 7, wherein:

the eluent B is acetonitrile solution with the volume concentration of 30-45 percent or 30-40 percent.

9. The method of claim 8, wherein:

the specification of the silica gel filler in the silica gel column is 100-200 meshes, and the pore diameter

10. The method of claim 8, wherein:

in step 4, the flow rate of the upper column liquid in the high pressure liquid chromatograph is 50-70ml/min, preferably 70 ml/min.

Technical Field

The invention relates to the technical field of fermentation liquor purification, and particularly relates to a method for extracting FR901464 from Burkholderia fermentation liquor.

Background

FR901464 is a natural compound with highly potent antitumor activity, originally isolated and purified by Nakajima et al in 1996 from Pseudomonas sp by fermentation (Nakajima, H.; Sato, B.; Fujita, T.; Takase, S.; Terano, H.; Okuhara, M.Antitumor substations, FR901463, FR901464and FR901465.I.taxonomy, promotion, isolation, physico-chemical properties and biological activities [ J.Antibiot.,1996,49: 1196-1203), wherein Pseudomonas sp.No. 360mg/L was fermented. The chemical structural formula is shown as formula 1.

As a splicing inhibitor of precursor mRNA in eukaryotic cells, FR901464 has strong inhibition effect on the growth of tumor cells, and activity tests show that FR901464 has very obvious inhibition effect on human solid tumor cells, and IC50 values obtained by experimental tests are 0.3-3.4 nM. Since FR901464 shows extremely high activity in resisting tumors and has unique physiological activity, it has been discovered that it has attracted the attention of scientists.

Nakajima H et al, although in New inhibitors substations, FR901463, FR901464and FR901465 I.Taxolomy, promotion, isolation, physico-chemical properties and biological activities, disclose a purification method which requires the consumption of a large amount of ethyl acetate for extraction and which by applicant's attempts could not achieve FR901464 of high purity.

Disclosure of Invention

In order to overcome the problems, the invention provides a method for extracting FR901464 from fermentation liquor, which is characterized by comprising the following steps:

the fermentation liquor is obtained by fermenting Burkholderia, and the extraction method comprises the following steps:

step 1: adding the macroporous resin into the fermentation liquor, mixing, adsorbing and filtering;

step 2: soaking the macroporous resin obtained by filtering in an organic solvent, separating, and concentrating the separated organic solvent;

and step 3: dissolving the product concentrated in the step 2 by using a mixed solvent, separating and filtering the product in a silica gel column, and leaching the product by using an eluent A to obtain an eluent;

and 4, step 4: concentrating the eluent to paste, preparing a column loading solution, and separating and purifying by using an eluent B through a high pressure liquid chromatograph.

Preferably, step 4 is followed by step 5: and 4, extracting the eluent in the step 4, collecting an organic phase, and evaporating to dryness to form solid powder.

Preferably, the filler of the high pressure liquid chromatograph is a C18 filler.

Preferably, in step 1, the macroporous resin is added to the fermentation broth and stirred.

Preferably, in step 1, the adsorption time is not less than 3h, or 4 h.

Preferably, in step 2, the soaking time is not less than 1 hour, or 2 hours.

Preferably, in step 2, the macroporous resin is soaked with the organic solvent 2 times or more, respectively.

Preferably, the mixed solvent is formed by mixing a polar solvent and a non-polar solvent.

Preferably, the polar solvent is selected from alcohols, acids, esters, nitriles, water, preferably from isopropanol, n-butanol, tetrahydrofuran, chloroform, ethanol, ethyl acetate, methanol, acetone, acetonitrile, acetic acid, more preferably ethyl acetate; the non-polar solvent is selected from saturated hydrocarbons, benzene, preferably n-hexane.

Preferably, n-hexane is mixed with ethyl acetate to form the mixed solvent, wherein the volume fraction of ethyl acetate is not more than 70%.

Preferably, in step 4, the upper column liquid is prepared with 30% volume fraction acetonitrile.

Preferably, the organic solvent in step 2 is selected from ethyl acetate, dichloromethane, chloroform.

Preferably, eluent a is ethyl acetate and eluent B is selected from acetonitrile or aqueous methanol.

Preferably, the eluent B is acetonitrile solution with a volume concentration of 30% to 45%, or 30% to 40%.

Preferably, step 3 and step 4 comprise between step 3.1: concentrating the eluent obtained in the step 3 into paste, and repeating the step 3 to obtain the eluent.

Preferably, the specification of the silica gel filler in the silica gel column is 100-200 meshes, and the pore diameter

Preferably, in step 4, the flow rate of the supernatant in the HPLC is 50-70ml/min, preferably 70 ml/min.

Preferably, in step 5, the collected organic phase is dehydrated by anhydrous magnesium sulfate, filtered and evaporated to dryness to form solid powder.

The invention is suitable for fermentation liquor obtained by fermenting any Burkholderia available in the market, preferably, the Burkholderia is Burkholderia (Burkholderia sp.) HDCC00024 which is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.22290, and the preservation date is 2021, 05 and 08 days.

Has the advantages that:

1. by adopting the purification method, the purity of FR901464 in the final product is high;

2. the purification method of the invention has simple operation steps. Is suitable for industrial production;

3. the FR901464 analogue which has a similar structure and is difficult to separate can be effectively removed by utilizing a high-pressure preparation liquid chromatograph and a mobile phase with a specific solvent and concentration;

4. most of the more polar and pigment impurities can be removed by the silica gel filler.

Drawings

FIG. 1 is a liquid chromatogram of a fermentation broth;

fig. 2 is a liquid phase map of FR901464 finished product powder in example 8.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

The materials, reagents and the like used in the following examples are all common commercially available products and are commercially available unless otherwise specified.

The invention will now be further described by way of the following examples, which are not intended to limit the scope of the invention in any way. It will be understood by those skilled in the art that the equivalent substitutions and modifications made in the present disclosure are within the scope of the present invention.

The purity of acetonitrile, ethyl acetate, dichloromethane, chloroform, n-hexane and methanol used in the purification is industrial grade.

The fermentation broth referred to in the following examples was obtained according to the patent application with application No. CN202110681279.4, and the strain used was Burkholderia sp HDCC00024(CGMCC NO. 22290). The specific fermentation method comprises the following steps:

(1) preparing and culturing slant strains:

slant culture medium formula (g/L): 4.0g/L of yeast extract powder, 10.0g/L of malt extract, 4.0g/L of glucose, 20.0g/L of agar, 7.2-7.4 of pH before digestion, 30 x 200mm of test tube and 15mL of package, sterilizing at 121 ℃ for 20min, cooling to 55-60 ℃, placing an inclined plane, inoculating to the inclined plane after cooling and solidification, and culturing at 28 +/-1 ℃ for 3 days to ensure that the strain is mature.

(2) Preparing and culturing a seed solution:

seed culture medium formula (g/L): 30g/L of glucose, 5g/L of sorbitol, 10g/L of cottonseed cake powder, 20g/L of yeast extract powder, 10g/L of calcium chloride, 10g/L of magnesium sulfate, 1g/L of monopotassium phosphate and 7.0 of pH before digestion; a250 mL triangular shake flask with the specification is filled with 50mL and sterilized at 121 ℃ for 20 min. Inoculation 10⁷～10⁸cfu/mL into seed culture medium, culturing at 28 + -1 deg.C and 250rpm under shaking for 24 hr, wherein the pH of culture solution is 6.8-7.2, and the thallus OD600 is 15-20.

(3) Preparing and culturing a fermentation medium:

20g/L of corn starch, 30g/L of glucose, 10g/L of sorbitol, 10g/L of mannitol, 6g/L of yeast extract powder, 8g/L of soybean cake powder, 7g/L of cottonseed cake powder, 3g/L of magnesium sulfate, 6g/L of monopotassium phosphate, 3g/L of potassium chloride and 3g/L of calcium chloride. pH 6.0 before digestion. A250 mL triangular shake flask with the specification is filled with 20mL and sterilized at 121 ℃ for 20 min. The seed liquid was inoculated at an inoculum size of 10% (by volume). The cells were cultured at 26. + -. 1 ℃ for 96 hours with shaking at 250 rpm.

EXAMPLE 1 macroporous resin adsorption

And taking the four groups of fermentation liquor, respectively adding macroporous resins with different specifications according to the following table, stirring and adsorbing, taking fermentation supernatant, and detecting the titer of FR901464 in the supernatant by an HPLC method.

Adsorption rate (%). 100% -supernatant titer/fermentation tank titer 100%

EXAMPLE 2 solvent extraction

And (3) obtaining a mixture of the fermentation liquor and the macroporous resin LX-30 according to the adsorption mode of the group 1 in the example 1, separating by using a vibrating screen to obtain the macroporous resin LX-30, adding the leaching solvent into the macroporous resin LX-30 according to the mode shown in the table below, stirring for 2 hours, and separating and collecting the leaching liquor. The potency of FR901464 in the leach solution was checked by HPLC.

Leach yield ═ leach liquor titer ═ leach liquor volume/(resin weight ×) resin titer · 100%

Example 3 silica gel separation

Referring to example 2, 2.5L of ethyl acetate was added to 1.27kg of the adsorbed macroporous resin LX-30 to leach, the leaching solution was separated after stirring for 2 hours, and the total leaching was performed twice to obtain 4.7L of mixed leaching solution, and the mixed leaching solution was separated by standing and then separated to obtain 4.5L of organic phase. The organic phase was concentrated to a paste by a rotary evaporator at 40 ℃.

Dissolving the above paste in the mixed solvent of n-hexane and ethyl acetate, filtering, preparing the paste into 3g/L with the mixed solvent, and filling the solution with silica gel (100 mesh and 200 mesh, pore diameter)) Carrying out chromatographic separation.

N-hexane: ethyl acetate (v: v)	Adsorption Rate (%)
		30:70	98.9
40:60	100
		50:50	100

The result shows that in the chromatographic process, when the proportion of ethyl acetate in the upper column liquid is increased, the adsorption rate of the silica gel is reduced, and when the proportion is 30: at 70, FR901464 leaks out from the upper column filtration.

Through the step, impurities with small polarity flow out with the liquid, and FR901464and substances with large polarity are adsorbed in the silica gel.

Example 4 silica gel separation

Referring to example 3, with n-hexane: ethyl acetate (v: v) 50:50, filtering, and separating with silica gel filler, eluting FR901464 adsorbed on silica gel with ethyl acetate, and adsorbing more polar impurities and pigment on silica gel. The purity of FR901464 in the eluate was measured by peak area distribution by HPLC (i.e., the percentage of the peak area of FR901464 to the total peak area, purity in the following examples was obtained by this method), and was 58.25%. The eluate was concentrated to a paste by a rotary evaporator at 40 ℃. Repeating the above steps to obtain eluent with purity of 75.68%, concentrating into paste, and preparing upper column solution with 30% volume fraction acetonitrile.

Example 5 separation and purification by high pressure liquid chromatography

And (3) separating and purifying the upper column liquid in the example 4 by using a high pressure liquid chromatograph, wherein the filler is a Huaban C18 filler, the liquid-phase eluent respectively uses acetonitrile and methanol, the volume concentration of the solvent in the eluent is 35%, the flow rate is 70ml/min, and the column temperature is room temperature. The experimental data are shown in the table below, in which the amount of eluent is a multiple of the volume of the filler.

Example 6 separation and purification by high pressure liquid chromatography

And (3) separating and purifying the upper column liquid in the example 4 by using a high pressure liquid chromatograph, wherein the packing is a Huaban C18 packing, 4 acetonitrile with different volume concentrations are respectively used as eluent, the flow rate is 70ml/min, and the column temperature is room temperature. The result shows that the concentration of acetonitrile is 30-40%, and the effect is better. The results of the experiment are as follows:

acetonitrile concentration (%)	Purity of qualified Components (%)	Yield (%)
			30	97.08	74.34
35	95.91	69.74
			40	95.44	62.73
45	92.01	40.36

Example 7 separation and purification by high pressure liquid chromatography

The upper column liquid in the example 4 is used for separation and purification by a high pressure liquid chromatograph, the eluent is 35% acetonitrile, the separation and purification experiments are respectively carried out by 3 types of C18 packing (nano-micro C18 packing, Huaban C18 packing and Kromasil C18 packing), the flow rate is 70ml/min, the column temperature is room temperature, and the experimental results are as follows:

type of filler	QualifiedPurity of the Components (%)	Yield (%)
			Nano-micro C18 filler	95.55	65.81
Chinese staff C18 filler	95.91	69.74
			Kromasil C18 filler	96.03	63.18

Example 8 separation and purification by high pressure liquid chromatography

And (3) separating and purifying the upper column liquid in the example 4 by using a high pressure liquid chromatograph, wherein the eluent is 35% acetonitrile, the filler is a Huaban C18 filler, the flow rate is 70ml/min, and the column temperature is room temperature to obtain the eluent with the purity of 96.14%. Extracting the eluent with ethyl acetate, and standing and separating to obtain an organic phase. The organic phase was dehydrated with anhydrous magnesium sulfate and filtered. The dehydrated organic phase was concentrated to a solid powder at 40 ℃ using a rotary evaporator, with a purity of 96.75% and a total yield of 26.6%.

Example 9

Step 1: taking 25L FR901464 fermentation liquor, adding 2.5kg macroporous resin HP20, stirring for 12h, and sampling to detect that the FR901464 content in the fermentation liquor is 11.34 ug/ml. The fermentation broth and macroporous resin were then separated by shaker to give 2.3kg of macroporous resin HP 20.

Step 2: adding 4.2L chloroform into 2.3kg macroporous resin HP20 obtained by separation, leaching for 4h, leaching twice to obtain 8.2L mixed leaching solution.

And step 3: standing the mixed leaching solution for liquid separation to obtain 8L of an organic phase. Concentrating the organic phase at 40 deg.C by rotary evaporator, and concentrating the mixed extractive solution to paste.

And 4, step 4: dissolving the paste with mixed solvent of n-hexane and ethyl acetate (volume ratio 50:50), filtering, and separating the filtrate by chromatography with silica gel filler to obtain eluent with purity of 57.22%. Concentrating the eluate at 40 deg.C by rotary evaporator, and concentrating the eluate to paste.

And 5: repeat step 4 to obtain an eluent with a purity of 68.68%. Concentrating the eluate at 40 deg.C by rotary evaporator, and concentrating the eluate to paste.

Step 6: the paste was prepared into a column-loading solution using 30% by volume acetonitrile. And (4) separating and purifying the upper column liquid by using a high-pressure liquid chromatograph, wherein the filler is Kromasil C18 filler. Elution with 40% by volume aqueous acetonitrile gave a purity of 95.68%.

And 7: and (4) extracting the eluent in the step 6 by using ethyl acetate, and standing and separating to obtain an organic phase. The organic phase was dehydrated with anhydrous magnesium sulfate and filtered. The dehydrated organic phase was concentrated to a solid powder at 40 ℃ using a rotary evaporator, with a purity of 95.66% and a total yield of 25.1%.

Example 10:

step 1: taking 25L FR901464 fermentation liquor, adding 2kg of macroporous resin HZ818, stirring for 8h, and sampling to detect that the FR901464 content in the fermentation liquor is 126 ug/ml. The fermentation broth and the macroporous resin were then separated by shaker to give 1.8kg of macroporous resin HZ 818.

Step 2: 3.6L of dichloromethane is added into 1.8kg of macroporous resin HZ818 for leaching, the leaching time is 2 hours, and the leaching is carried out twice, so that 7.1L of mixed leaching liquor is obtained.

And step 3: standing the mixed leaching solution for liquid separation to obtain 7L of an organic phase. Concentrating the organic phase at 40 deg.C by rotary evaporator, and concentrating the mixed extractive solution to paste.

And 4, step 4: dissolving the paste with a mixed solvent of n-hexane and ethyl acetate (volume ratio 40:60) and filtering. The filtrate was chromatographed on silica gel packing to give an eluent of 52.23% purity. Concentrating the eluate at 40 deg.C by rotary evaporator, and concentrating the eluate to paste.

And 5: repeat step 4 to obtain an eluent with a purity of 68.37%. Concentrating the eluate at 40 deg.C by rotary evaporator, and concentrating the eluate to paste.

Step 6: the paste is prepared into upper column liquid by 30 percent of acetonitrile by volume fraction. And (4) separating and purifying the upper column liquid by using a high-pressure liquid chromatograph, wherein the filler is nano-micro C18 filler. Elution was carried out with 35% by volume acetonitrile in water to give a purity of 96.30%.

And 7: and (4) extracting the eluent in the step 6 by using ethyl acetate, and standing and separating to obtain an organic phase. The organic phase was dehydrated with anhydrous magnesium sulfate and filtered. The dehydrated organic phase was concentrated to a solid powder at 40 ℃ using a rotary evaporator, with a purity of 96.02% and an overall yield of 27.2%.