CN110755884A - Method for extracting biological product by reverse adsorption - Google Patents
Method for extracting biological product by reverse adsorption Download PDFInfo
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- CN110755884A CN110755884A CN201911111377.3A CN201911111377A CN110755884A CN 110755884 A CN110755884 A CN 110755884A CN 201911111377 A CN201911111377 A CN 201911111377A CN 110755884 A CN110755884 A CN 110755884A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1807—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using counter-currents, e.g. fluidised beds
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/16—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the fluid carrier
- B01D15/166—Fluid composition conditioning, e.g. gradient
- B01D15/168—Fluid composition conditioning, e.g. gradient pH gradient, chromatofocusing, i.e. separation according to the isoelectric point pI
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/18—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns
- B01D15/1864—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns
- B01D15/1871—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to flow patterns using two or more columns placed in series
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/42—Selective adsorption, e.g. chromatography characterised by the development mode, e.g. by displacement or by elution
- B01D15/424—Elution mode
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- Treatment Of Liquids With Adsorbents In General (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting biological products by reverse adsorption. The invention utilizes macroporous adsorption resin to directly extract weak polar or non-polar products from biological product liquid containing cells and mycelium fragments in a reverse column adsorption mode, and the extracted products have high purity. The method not only overcomes a series of defects caused by using organic solvent for extraction in the traditional biological product extraction, but also solves the contradiction that the column feeding liquid is strictly required in the traditional resin separation. The invention has simple technical process, strong selective adsorption to products, good product quality and less production waste; meanwhile, the invention can help realize the on-line extraction and continuous production of partial biological products.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a method for reversely adsorbing and extracting a biological product.
Background
Biotransformation and enzyme catalysis are two important methods for producing drugs and drug intermediates, and because the methods have the characteristics of low pollution and high stereoselectivity, chemical synthesis methods of many products are gradually replaced at present. Although the immobilized cell and immobilized enzyme technology in the prior art is applied to the production of certain biological products, and certain liquid enzymes are also used in the form of clear liquid, most of the biocatalysts participate in biological reactions in the form of whole cells or whole cell disruption liquid due to the instability of the biocatalysts, so that the product is difficult to extract from the reaction system after the reaction is finished. Most products produced by the biotransformation and enzyme catalysis methods have large molecular weight, low polarity and small water solubility, so the extraction by using an organic solvent becomes a commonly used and most classical method in the extraction process of biological products. However, the organic solvent extraction generates emulsification, water-soluble impurities generated in the emulsification affect the product quality, and a part of organic solvent remaining in the extraction mother liquor can cause heavy pressure on a sewage treatment system.
Resin separation methods are also used for bioproduct extraction, but traditionally the column fluid must be a clear fluid or the column of resin can become clogged. In production, solid substances such as mycelium and the like in biological product liquid are generally removed by adopting a filtering method, but a flocculating agent is added for pretreatment; since the optimum PH range for use of flocculants is generally from neutral to slightly acidic, this method is not suitable when the biological product liquid contains solid products, and the large amount of filter cake produced by filtration inevitably entrains a portion of the product, which also has a certain effect on the product yield.
At present, the resin separation method generally adopted in the experimental process is that the resin column feeding mode is generally positive column feeding, namely, the column feeding liquid enters from the upper part of the resin column and flows out from the bottom. The forward column loading has the advantages of clear chromatographic distribution of the product on the resin column, high exchange saturation, good product quality, long exchange period and high requirement on column loading liquid quality. The upper column liquid enters from the bottom of the resin column and is discharged from the upper part of the resin column, which is called as a reverse upper column, and the reverse upper column is generally considered to have the defects of unobvious chromatographic distribution and low adsorption quantity, so that no report of the current practical application is found in the reverse upper column method in production or scientific research.
Disclosure of Invention
In order to make up for the deficiencies of the prior art, the present invention provides a method for reverse adsorption extraction of a biological product.
The technical scheme adopted by the invention is as follows:
a method for reverse adsorption extraction of a biological product comprising the steps of:
step 1): filtering the biological product liquid through a 60-120 mesh filter screen, and adjusting the pH value;
step 2), arranging 40-60 mesh filter screens at the upper end and the lower end in the resin column to prevent the resin filler from losing;
step 3): adsorbing on the column in a reverse column loading mode, wherein the flow rate of the column loading is 3-5 BV/h; collecting column passing liquid, and treating the column passing liquid in a sewage treatment plant;
step 4): when the resin adsorption amount reaches 60-70% of the saturated adsorption amount, switching, and performing back flushing by using deionized water, wherein the flushing rate is 3-6BV/h, and the flushing time is 0.5-2 h;
step 5): desorbing at a desorption rate of 0.5-2 BV/h; collecting the desorption solution, and extracting the product.
Preferably, the biological product in step 1) is a medium polar substance, a low polar substance or a non-polar substance.
Preferably, the liquid source of the biological product in the step 1) is a biological conversion liquid, an enzyme catalysis reaction liquid or a biological fermentation liquid.
Preferably, the biological product liquid in step 1) is an aqueous phase and does not contain an organic solvent.
Preferably, the filler used in step 2) is macroporous adsorption resin, and the macroporous adsorption resin is medium-polarity macroporous adsorption resin, weak-polarity macroporous adsorption resin or non-polarity macroporous adsorption resin.
Preferably, the stem height ratio of the resin column in step 2) is 1: 8-14.
Preferably, the resin column in step 2) is used in the form of a single column or multiple columns in series.
Preferably, the indication of the end of rinsing in step 4): the rinsing effluent was clear and transparent.
Preferably, the desorption manner in step 5) comprises forward desorption or reverse desorption.
Compared with the prior art, the invention has the beneficial effects that:
the particle size range of the resin is generally 300-1000 microns, the size of cells or broken mycelia in the biological product liquid is micron and submicron, and the higher column feeding rate and washing rate in the application enable the resin particles to be in a semi-suspension state, so that the problem of resin column blockage in the adsorption process of the application can not be caused. The internal pore diameter of the macroporous adsorption resin is generally from several nanometers to dozens of nanometers, and only substances with molecular level sizes can enter the resin, so that suspended solid substances in the liquid of the biological product cannot influence the adsorption of the resin on the product. The larger stem height ratio of the resin column or the serial connection of the resin columns are used, so that the liquid of the biological product to be extracted is fully absorbed, and the absorption effect and the product recovery rate of the product are improved.
The invention successfully completes the separation of two mechanism directions by using a reverse adsorption method: substances with the particle size of more than 100 nanometers, including almost all cells and mycelium fragments, can not enter the resin particles to be adsorbed, flow through gaps of the resin particles and are discharged along with column passing liquid; the medium molecular weight and small molecular weight substances entering the resin particles almost have higher polarity and hydrophilicity except the product and are not adsorbed, and finally flow out along with column passing liquid and washing water, so that the high-efficiency adsorption separation of the product to be extracted is completed.
Many biological products are weakly acidic or weakly basic substances, and their solubility and polarity change with the change of pH. The invention controls the polarity of the extracted product to be at an ideal level by adjusting the PH of the biological product liquid, and then completes the accurate adsorption and separation of the product by selecting the adsorption resin with the proper polarity level for adsorption. It has been proved by sufficient experimental data that almost all biological products extracted by organic solvents can be separated and extracted by adsorption resins.
The invention carries out reverse adsorption extraction on the non-high-polarity biological product through the macroporous adsorption resin, and the regeneration of the resin is finished by flushing with deionized water after desorption, so that the production waste is reduced to the minimum degree.
The invention directly absorbs and extracts the biological product liquid containing the cell or mycelium fragments by a reverse column loading mode, thereby not only simplifying the production process, but also obtaining a high-purity product; in addition, the reverse adsorption technology provided by the invention is expected to realize on-line extraction, continuous extraction and continuous production of certain products.
Detailed Description
The first embodiment is as follows:
⑴ bioconversion liquid is prepared by culturing colletotrichum in 30L fermentation medium for 18h, adding sterile solid dehydroepiandrosterone (prasterone) 80g, ventilating at 29 deg.C and 1.8 Nm/h, bioconverting for about 30h, detecting by TLC, finishing the culture after the conversion to obtain bioconversion liquid, detecting by HPLC to obtain bioconversion liquid containing 2.21mg/ml dehydroepiandrosterone, 7-hydroxydehydroepiandrosterone and dihydroxyandrosterone (7 α,15 α -dihydroxyandrosterone), diluting the bioconversion liquid with 60L deionized water, performing primary crushing by colloid mill, and performing secondary crushing by high pressure homogenizer at 60MP to release the dihydroxyprasterone product from mycelia completely to obtain the liquid to be detected biological product.
⑵ A method for reverse adsorption extraction of bioproducts, comprising the steps of:
step 1): carrying out HPLC detection on 4800ml of the biological product liquid to be detected, wherein the detection result is that the content of the dihydroxyprasterone is 0.73 mg/ml; filtering the biological product liquid to be detected by a 120-mesh filter screen, detecting the PH of the filtrate to be 6.64, and adjusting the PH to be 7.2 by using liquid alkali;
step 2): adopting a resin column with the specification of theta 22 multiplied by 400, fixing a 60-mesh stainless steel filter screen at the bottom in the resin column, filling 80ml of nonpolar macroporous adsorption resin above the stainless steel filter screen, reserving a space with the height of 50mm at the upper part of the nonpolar macroporous adsorption resin, and then fixing another 60-mesh stainless steel filter screen; in the step, the upper opening of the resin column is sealed by a rubber plug with a liquid discharge rubber tube, and a latex tube with the length of about 1m is arranged at the lower opening of the resin column to be used as an upper column liquid inlet;
step 3): performing reverse column-loading adsorption on the biological product liquid subjected to filtration treatment in the step 1) by using the resin column treated in the step 2), wherein the column-loading flow rate is 300 ml/h; collecting the column-passing liquid, and pumping the column-passing liquid into a sewage treatment plant for treatment;
step 4): the adsorption capacity of the resin reaches 60-70% of the saturated adsorption capacity, and the column loading is finished; reversely washing with deionized water at a washing rate of 400ml/h, clarifying the washing effluent after washing for 0.5h, and finishing washing;
step 5): carrying out forward desorption by using acetone at the flow rate of 80ml/h, discarding 40ml of the initial desorption solution, collecting 340ml of the desorption solution, and forward washing and regenerating the resin column by using 200ml of deionized water; recovering acetone from the desorption solution, stopping distillation when the concentration volume is reduced to about 20ml, cooling to 4-8 ℃ for crystallization, filtering and drying to obtain a product 3.15g, wherein the content of the dihydroxyprasterone is 98.7%.
Example two:
⑴ preparation of the biotransformation liquid the same way as in the first example was carried out.
⑵ A method for reverse adsorption extraction of bioproducts, comprising the steps of:
step 1): 4000ml of the biological product liquid to be detected prepared in the second embodiment is taken, filtered by a 100-mesh filter screen, and the PH value is adjusted to 7.15 by liquid alkali;
step 2), adopting a resin column with the specification of theta 22 multiplied by 400, fixing a 40-mesh stainless steel filter screen at the bottom in the resin column, then filling 70ml of nonpolar macroporous adsorption resin above the stainless steel filter screen, reserving a space with the height of 60mm at the upper part of the nonpolar macroporous adsorption resin, and then fixing another 40-mesh stainless steel filter screen; in the step, the two same resin columns are connected in series by a rubber plug and a rubber tube, the upper opening of the upper resin column is sealed by the rubber plug with a liquid discharge rubber tube, and the lower opening of the lower resin column is provided with an emulsion tube with the length of about 1.5m as an upper column liquid inlet;
step 3): performing reverse column-loading adsorption on the biological product liquid subjected to filtration treatment in the step 1) by using the resin column treated in the step 2), wherein the column-loading flow rate is 350 ml/h; collecting the column-passing liquid, and treating the filtrate in a sewage treatment plant;
step 4): the adsorption capacity of the resin reaches 60-70% of the saturated adsorption capacity, and the column loading is finished; separating two resin columns connected in series, wherein the upper resin column is directly used for the next batch of first-stage loading columns, the lower resin column is reversely flushed with deionized water at the flow rate of 400ml/h, the effluent is clarified after flushing for 0.5h, and the flushing is finished;
step 5): carrying out forward desorption by using acetone at the flow rate of 70ml/h, discarding 35ml of desorption solution, collecting 300ml of desorption solution, and forward washing and regenerating the resin column by using 200ml of deionized water; recovering acetone from the desorption solution, evaporating to obtain a solid, collecting the solid, stirring and washing with 60ml of 4 ℃ n-heptane, filtering, and drying to obtain 2.78g of the product with the content of the dihydroxyl prasterone of 99.3%.
Example three:
⑴ preparing biotransformation liquid, which is prepared by recombining 7- β dehydrogenase complex glucose dehydrogenase gene into engineering escherichia coli, culturing, centrifuging and collecting thallus, taking 100g of thallus to suspend in 400g of deionized water, crushing by ultrasonic wave to obtain whole thallus enzyme liquid, adding 25g of glucose, adding 10g of 7-ketolithocholic acid, and carrying out catalytic reaction for 8h to obtain 627ml of ursodeoxycholic acid reaction liquid with the ursodeoxycholic acid content of 15.15 mg/ml;
⑵ A method for reverse adsorption extraction of bioproducts, comprising the steps of:
step 1): taking 300ml of ursodeoxycholic acid reaction solution, adding 300ml of deionized water, adjusting the pH value to 9.0 by using liquid alkali, and filtering by using a 80-mesh filter screen;
step 2), adopting a resin column with the specification of theta 22 multiplied by 400, fixing a 40-mesh stainless steel filter screen at the bottom in the resin column, then filling 80ml of medium-polarity macroporous adsorption resin above the stainless steel filter screen, reserving a space with the height of 60mm at the upper part of the medium-polarity macroporous adsorption resin, and then fixing another 40-mesh stainless steel filter screen; in the step, the upper opening of the resin column is sealed by a rubber plug with a liquid discharge rubber tube, and the lower opening of the resin column is provided with a latex tube with the length of about 1m as an upper column liquid inlet;
step 3): carrying out reverse column-loading adsorption on the ursodeoxycholic acid reaction solution subjected to filtering treatment in the step 1) by using the resin column treated in the step 2), wherein the column-loading flow rate is 300 ml/h; collecting the column-passing liquid, and treating the filtrate in a sewage treatment plant;
step 4): the adsorption capacity of the resin reaches 60-70% of the saturated adsorption capacity, and the column loading is finished; reversely washing with deionized water at a washing rate of 400ml/h, clarifying the washing effluent after washing for 0.5h, and finishing washing;
step 5): reversely desorbing with 50% ethanol solution at flow rate of 60ml/h, collecting 300ml of all desorption solution, and positively washing and regenerating the resin column with 200ml of deionized water; recovering ethanol from the desorption solution, concentrating to 180ml, adjusting pH to 2.5 with sulfuric acid, crystallizing for 4h, filtering, and drying to obtain ursodeoxycholic acid product 4.43g with ursodeoxycholic acid content of 98.9%.
The above-described embodiment is only one of the preferred embodiments of the present invention, and general changes and substitutions by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention.
Claims (9)
1. A method for reverse adsorption extraction of biological products, comprising: the method comprises the following steps:
step 1): filtering the biological product liquid by a filter screen, and adjusting the pH value;
step 2): the upper end and the lower end in the resin column are both provided with filter screens;
step 3): adsorbing on the column in a reverse column-loading mode, wherein the flow rate of column loading is 3-5 BV/h;
step 4): when the adsorption capacity of the resin reaches 60-70% of the saturated adsorption capacity, switching, and performing back flushing by using deionized water at a flushing rate of 3-6 BV/h;
step 5): desorbing at a desorption rate of 0.5-2 BV/h; collecting the desorption solution, and extracting the product.
2. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 1), the biological product is a medium-polarity substance, a weak-polarity substance or a non-polar substance.
3. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 1), the liquid source of the biological product is biological conversion liquid, enzyme catalysis reaction liquid or biological fermentation liquid.
4. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 1), the biological product liquid is a water phase and does not contain an organic solvent.
5. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 2), the used filler is macroporous adsorption resin which is medium-polarity macroporous adsorption resin, weak-polarity macroporous adsorption resin or nonpolar macroporous adsorption resin.
6. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 2), the stem height ratio of the resin column is 1: 8-14.
7. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 2), the resin column is used in a single-column mode or a multi-column mode in series.
8. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 4), the index of the washing end is that the washing effluent is clear and transparent.
9. The method of reverse sorption extraction of a bioproduct of claim 1, wherein: in the step 5), the desorption mode is forward desorption or reverse desorption.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114134067A (en) * | 2021-10-19 | 2022-03-04 | 山东睿智医药科技有限公司 | Escherichia coli and application thereof |
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