CN102580687A - Polyamine-modified chitosan base expanded bed adsorbing medium and preparation method thereof - Google Patents
Polyamine-modified chitosan base expanded bed adsorbing medium and preparation method thereof Download PDFInfo
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Abstract
The invention provides a polyamine-modified chitosan base expanded bed adsorbing medium and a preparation method of the polyamine-modified chitosan base expanded bed adsorbing medium, and relates to an adsorbing medium and a preparation method of the adsorbing medium. The matrix is a chitosan/stainless steel powder composite micro-ball, and the genin is induced phenyl by activating benzaldehyde and the re-coupled polyethylene polyamine by activating epichlorohydrin. The preparation method comprises the following steps of: adopting a reversed phase suspension crosslinking method to prepare the chitosan/stainless steel powder composite micro-ball which is taken as a blank matrix; and adopting a method that two genins are designed in a dividing way to firstly react chitosan unit molecule C2 site amino in the micro-ball matrix with benzaldehyde, build the hydrophobic interacted genin, activate matrix chitosan unit molecule C6 site hydroxyl by epichlorohydrin, and couple polyethylene polyamine, so as to obtain the expanded bed adsorbing medium which takes the phenyl and the polyethylene polyamine as the genins. The expanded bed adsorbing medium provided by the invention has the ion exchange genin and the hydrophobic genin, and the absorbing and separating efficiency and the selectivity of target matter in feed liquid can be improved, so that the biological substance such as the protein and the like can be separated and purified on a large scale.
Description
Technical field
The present invention relates to a kind of adsorbing medium and preparation method thereof, particularly relating to a kind of is that aglucon is used for expanded bed adsorption medium and the preparation method that biological substance separates with phenyl and polyamines, belongs to expanded bed adsorption isolation technics in the chromatography.
Background technology
Bioseparation technology is the important step of biotechnology; Improve bioseparation technology; Low-cost, high yield, purification of target product expeditiously; And the content of controlling objectionable impurities effectively, be the matter of utmost importance that can bioactivator change the product with practical value and competitiveness into.To the expanded bed adsorption medium, increase new medium simultaneously, expand bigger purposes, improving reaction mass and efficient also is very important problem.
(expanded bed adsorption is a kind of novel Separation of Proteins operating technology EBA) to expanded bed adsorption, is formed at the nineties in 20th century, has obtained using widely at bioengineering field in recent years.Expanded bed operation can be integrated into a unit operations with centrifugal, initial gross separation and concentration process, thereby has simplified the step of Separation of Proteins, and can improve the yield of destination protein, reduces the operating time.The quality of adsorbing medium performance directly influences separating effect, develops well behaved adsorbent matrix, is the key that this technology is able to extensive use.Should be easy to fluidisation; Make separating effect approach fixed bed again; Must guarantee that absorbent particles can appropriateness expand in expanded bed, and stable classification, rest on certain location separately; Therefore need carry out special design to the expanded bed adsorption agent, such as density, particle diameter and distribution thereof, functional group kind and quantity thereof etc.Mostly expanded bed adsorption agent host material is agarose, cellulose, silica gel etc. at present, does not also see the report of using shitosan.And mostly commercialization expanded bed adsorption medium is the Streamline series medium that GE Healthcare company provides, and adopts quartz sand to make the weightening finish kernel, and skin is Sepharose, the outward appearance sphere, and particle diameter but costs an arm and a leg between 100-300 μ m.In addition, the Streamline series plastics still exists the narrow partially deficiency of operated in flow rate scope.In order to improve the disposal ability of EBA, the expanded bed adsorption agent of developing low-cost, high density, high-adsorption-capacity is an inexorable trend.
Make up the aglucon of suitable chemical structure, highly selective is caught target product, and impurity such as particle are then taken out of bed, for expanded bed adsorption chromatography process, are very important.Mixed mode adsorption chromatography (mixed-mode chromatography) is the bio-separation new method of developing in recent years; Its function aglucon contains hydrophobic grouping and ion-exchange group simultaneously, has hydrophobic effect and electrostatic interaction concurrently, can through static inhale mutually strengthen absorption or Coulomb repulsion assist desorb; (BURTON S C has broad application prospects; Et al.Journal of Biochemical and Biophysical Methods, 2001,49:275).Simultaneously, aglucon density has typical salt tolerant absorption property usually than higher; Target protein is had very strong capture ability, can be from the system of low concentration the enrichment target protein, be particularly suitable for requirement (the GHOSE S that biological object just separates; Et al.Biotechnology Progress, 2005,21:498. BURTON S C; Et al.Biotechnology and Bioengineering, 1997,56:45).Expanded bed adsorption and mixed mode adsorption chromatography are organically combined, realize the mixed mode expanded bed adsorption, can give full play to the advantage of the two, reach the separate targets of low ingredient requirement, format high throughput and high selectivity.
Summary of the invention
The object of the present invention is to provide chitosan-based expanded bed adsorption medium of a kind of modified polyamine and preparation method thereof; With phenyl and polyethylene polyamine is the expanded bed adsorption medium and the preparation method of aglucon; Reach ion-exchange aglucon and hydrophobic effect aglucon synergy; Raising forms a kind of efficient integrated bio-separation method to the adsorbing separation efficient and the selectivity of target substance in the feed liquid, effectively promotes the scale separation and purification of biomolecule such as protein.
The objective of the invention is to realize through following technical scheme:
The chitosan-based expanded bed adsorption medium of a kind of modified polyamine, the composition mesostroma of medium are shitosan/stainless steel powder complex microsphere, and aglucon is shitosan C
2Phenyl that bit amino is introduced after the benzaldehyde activation and shitosan C
6Position hydroxyl polyethylene polyamine of coupling again after the epoxychloropropane activation.
The chitosan-based expanded bed adsorption medium of described a kind of modified polyamine; The skeleton of its described shitosan/stainless steel powder complex microsphere is a shitosan; Weighting agent is a stainless steel powder, and the mass percent that weighting agent accounts for wet bulb is 6% ~ 40%, and the particle diameter of shitosan/stainless steel powder complex microsphere is 50 ~ 300 μ m.
The chitosan-based expanded bed adsorption medium of described a kind of modified polyamine, its described aglucon is phenyl and polyethylene polyamine.
The chitosan-based expanded bed adsorption medium of described a kind of modified polyamine, its described phenyl density is 20-100 μ mol/ml medium, amino density is the 0.5-1.5mmol/ml medium.
The preparation method of the chitosan-based expanded bed adsorption medium of a kind of modified polyamine; This method comprises following process: be that 5% chitosan solution fully mixes with stainless steel powder, PEG2000 with mass fraction at first; PEG2000 and the stainless steel powder quality percentage composition in mixture is respectively 3% and 6% ~ 40%; Add and isopyknic atoleine of chitosan solution and an amount of emulsifying agent span-80 again; High-speed stirred 30min makes system become emulsion form, adds the formaldehyde of 0.12 times of chitosan solution volume then, pours emulsion into 10 ﹪ NaOH/ absolute ethyl alcohols behind the stirring 1h and (leaves standstill 1h in the mixed liquor of V (NaOH): V (absolute ethyl alcohol)=1:1); Fully wash with xylenes, absolute ethyl alcohol, deionized water, shitosan/stainless steel powder complex microsphere that screening obtains particle diameter 50 ~ 300 μ m is as matrix; Secondly complex microsphere, methyl alcohol, benzaldehyde are mixed, the volume that benzaldehyde adds is 0.3 ~ 1 times of complex microsphere volume, reacts 8 ~ 16h in 60 ℃ of following 200rpm shaking tables, adds boron Cymag room temperature reaction 12h again, and suction filtration gets benzaldehyde matrix after the washing; Epoxychloropropane with benzaldehyde matrix, NaOH solution, 2 ~ 5 times of matrix microspheres volumes mixes then, 50 ℃ of reaction 4h, and suction filtration, washing get epoxy-activated matrix; At last the polyethylene polyamine of epoxy-activated matrix, 2 ~ 10 times of epoxy-activated matrix quality, an amount of sodium hydroxide solution are mixed; In 55 ℃ of following 200rpm shaking tables, react 6h; Suction filtration, washing is to neutral, and obtaining with phenyl and polyethylene polyamine is the expanded bed adsorption medium of aglucon.
Course of reaction on the shitosan unit molecule is:
The novel expanded bed adsorption medium that the present invention developed; With shitosan/stainless steel powder complex microsphere is matrix; Have ion-exchange aglucon and hydrophobic effect aglucon simultaneously; Raising can be used for the scale separation and purification of biomolecule such as protein to the adsorbing separation efficient and the selectivity of target substance in the feed liquid.
Advantage of the present invention and effect are:
The present invention has prepared the composite expanded bed substrate of a kind of novel chitosan/stainless steel powder.Adopt " inverse suspension crosslinking method ", as matrix scaffold, stainless steel powder is a weighting agent with shitosan, and shitosan/stainless steel powder complex microsphere that preparation has proper density and particle diameter is as blank matrix.Take the separately method of design of two kinds of aglucons, make distribution and the controllable density of two kinds of aglucons on matrix, to improve adsorption efficiency.Earlier with microballoon glycan substrate C
2Bit amino and benzaldehyde reaction make up a hydrophobic effect aglucon, use epoxychloropropane activated substrate C again
6The position hydroxyl, the coupling polyethylene polyamine, having prepared with phenyl and polyethylene polyamine is the mixed mode shitosan/stainless steel powder expanded bed adsorption medium of aglucon.Reach ion-exchange aglucon and hydrophobic effect aglucon synergy, improve adsorbing separation efficient and selectivity, form a kind of efficient integrated bio-separation new method, effectively promote the scale separation and purification of biomolecule such as protein target substance in the feed liquid.
The specific embodiment
Below through embodiment the present invention is further described.
Embodiment 1
Be that 5% chitosan solution fully mixes with 20g stainless steel powder, 3gPEG2000 with the 100ml mass fraction; Add the 100ml atoleine again; 15 span-80; High-speed stirred 30min makes system become emulsion form, adds 11ml formaldehyde then, pours emulsion into 10 ﹪ NaOH/ absolute ethyl alcohols behind the stirring 1h and (leaves standstill 1h in the mixed liquor of V (NaOH): V (absolute ethyl alcohol)=1:1); Fully wash with xylenes, absolute ethyl alcohol, deionized water, shitosan/stainless steel powder complex microsphere that screening obtains particle diameter 50 ~ 300 μ m is as matrix; The complex microsphere of draining, 50ml methyl alcohol, 5ml benzaldehyde are mixed, react 12h in 60 ℃ of following 200rpm shaking tables, add boron Cymag room temperature reaction 12h again, suction filtration gets benzaldehyde matrix after the washing; NaOH solution, 40ml epoxychloropropane with benzaldehyde matrix, 100ml0.4mol/L mixes then, 50 ℃ of reaction 4h, and suction filtration, washing get epoxy-activated matrix; At last epoxy-activated matrix, sodium hydroxide solution, 50g polyethylene polyamine are mixed, in 55 ℃ of following 200rpm shaking tables, react 6h, suction filtration is washed till neutrality, and obtaining with phenyl and polyethylene polyamine is the expanded bed adsorption medium of aglucon.The wet true density of medium is 1.26g/ml, and phenyl density is 60 μ mol/ml media, and amino content is the 1.4mmol/ml medium.
Embodiment 2
Be that 5% chitosan solution fully mixes with 35g stainless steel powder, 3gPEG2000 with the 100ml mass fraction; Add the 100ml atoleine again; 15 span-80; High-speed stirred 30min makes system become emulsion form, adds 11ml formaldehyde then, pours emulsion into 10 ﹪ NaOH/ absolute ethyl alcohols behind the stirring 1h and (leaves standstill 2h in the mixed liquor of V (NaOH): V (absolute ethyl alcohol)=1:1); Fully wash with xylenes, absolute ethyl alcohol, deionized water, shitosan/stainless steel powder complex microsphere that screening obtains particle diameter 50 ~ 300 μ m is as matrix; The complex microsphere of draining, 50ml methyl alcohol, 5ml benzaldehyde are mixed, react 8h in 60 ℃ of following 200rpm shaking tables, add boron Cymag room temperature reaction 12h again, suction filtration gets benzaldehyde matrix after the washing; NaOH solution, 25ml epoxychloropropane with benzaldehyde matrix, 100ml0.4mol/L mixes then, 50 ℃ of reaction 4h, and suction filtration, washing get epoxy-activated matrix; At last epoxy-activated matrix, sodium hydroxide solution, 30g polyethylene polyamine are mixed, in 55 ℃ of following 200rpm shaking tables, react 4h, suction filtration is washed till neutrality, and obtaining with phenyl and polyethylene polyamine is the expanded bed adsorption medium that aglucon is used for Separation of Proteins.The wet true density of medium is 1.39g/ml, and phenyl density is 45 μ mol/ml media, and amino content is the 1.0mmol/ml medium.
Claims (5)
1. the chitosan-based expanded bed adsorption medium of modified polyamine is characterized in that, the composition mesostroma of medium is shitosan/stainless steel powder complex microsphere, and aglucon is the polyethylene polyamine of coupling again after the phenyl introduced after the benzaldehyde activation and epoxychloropropane activation.
2. the chitosan-based expanded bed adsorption medium of a kind of modified polyamine according to claim 1; It is characterized in that; The skeleton of described shitosan/stainless steel powder complex microsphere is a shitosan; Weighting agent is a stainless steel powder, and the mass percent that weighting agent accounts for wet bulb is 6% ~ 40%, and the particle diameter of shitosan/stainless steel powder complex microsphere is 50 ~ 300 μ m.
3. the chitosan-based expanded bed adsorption medium of a kind of modified polyamine according to claim 1 is characterized in that described aglucon is phenyl and polyethylene polyamine.
4. the chitosan-based expanded bed adsorption medium of a kind of modified polyamine according to claim 1 is characterized in that, described phenyl density is 20-100 μ mol/ml medium, and amino density is the 0.5-1.5mmol/ml medium.
5. the preparation method of the chitosan-based expanded bed adsorption medium of modified polyamine; It is characterized in that: this method comprises following process: be that 5% chitosan solution fully mixes with stainless steel powder, PEG2000 with mass fraction at first; PEG2000 and the stainless steel powder quality percentage composition in mixture is respectively 3% and 6% ~ 40%; Add and isopyknic atoleine of chitosan solution and an amount of emulsifying agent span-80 again; High-speed stirred 30min makes system become emulsion form, adds the formaldehyde of 0.12 times of chitosan solution volume then, pours emulsion into 10 ﹪ NaOH/ absolute ethyl alcohols behind the stirring 1h and (leaves standstill 1h in the mixed liquor of V (NaOH): V (absolute ethyl alcohol)=1:1); Fully wash with xylenes, absolute ethyl alcohol, deionized water, shitosan/stainless steel powder complex microsphere that screening obtains particle diameter 50 ~ 300 μ m is as matrix; Secondly complex microsphere, methyl alcohol, benzaldehyde are mixed, the volume that benzaldehyde adds is 0.3 ~ 1 times of complex microsphere volume, reacts 8 ~ 16h in 60 ℃ of following 200rpm shaking tables, adds boron Cymag room temperature reaction 12h again, and suction filtration gets benzaldehyde matrix after the washing; Epoxychloropropane with benzaldehyde matrix, NaOH solution, 2 ~ 5 times of matrix microspheres volumes mixes then, 50 ℃ of reaction 4h, and suction filtration, washing get epoxy-activated matrix; At last the polyethylene polyamine of epoxy-activated matrix, 2 ~ 10 times of epoxy-activated matrix quality, an amount of sodium hydroxide solution are mixed; In 55 ℃ of following 200rpm shaking tables, react 6h; Suction filtration; Washing is to neutral, and obtaining with phenyl and polyethylene polyamine is that aglucon is used for the biochemical expanded bed adsorption medium that separates.
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Cited By (6)
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| CN103074322A (en) * | 2013-01-04 | 2013-05-01 | 沈阳化工大学 | Method for preparing immobilized lipase by utilizing amphipathy chitosan microsphere carrier |
| CN109647361A (en) * | 2019-02-22 | 2019-04-19 | 北京石油化工学院 | A kind of preparation method of composite polymer chromatography media |
| CN113070045A (en) * | 2021-04-29 | 2021-07-06 | 临沂海普新材料科技有限公司 | Preparation method of adsorbent for removing nitrate in industrial wastewater |
| CN113772759A (en) * | 2021-09-16 | 2021-12-10 | 安徽庞海科技股份有限公司 | Efficient purifying material for floating liquid in space capsule and production process thereof |
| CN115108806A (en) * | 2022-05-17 | 2022-09-27 | 西卡德高(广州)企业管理有限公司 | Waterproof protective layer material and preparation method thereof |
| CN115254008A (en) * | 2022-08-15 | 2022-11-01 | 大连工业大学 | Mesoporous silica material modified based on mixed mode, preparation method and application thereof |
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Cited By (9)
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| CN103074322A (en) * | 2013-01-04 | 2013-05-01 | 沈阳化工大学 | Method for preparing immobilized lipase by utilizing amphipathy chitosan microsphere carrier |
| CN109647361A (en) * | 2019-02-22 | 2019-04-19 | 北京石油化工学院 | A kind of preparation method of composite polymer chromatography media |
| CN113070045A (en) * | 2021-04-29 | 2021-07-06 | 临沂海普新材料科技有限公司 | Preparation method of adsorbent for removing nitrate in industrial wastewater |
| CN113070045B (en) * | 2021-04-29 | 2022-05-24 | 临沂海普新材料科技有限公司 | Preparation method of adsorbent for removing nitrate in industrial wastewater |
| CN113772759A (en) * | 2021-09-16 | 2021-12-10 | 安徽庞海科技股份有限公司 | Efficient purifying material for floating liquid in space capsule and production process thereof |
| CN115108806A (en) * | 2022-05-17 | 2022-09-27 | 西卡德高(广州)企业管理有限公司 | Waterproof protective layer material and preparation method thereof |
| CN115108806B (en) * | 2022-05-17 | 2023-08-25 | 西卡德高(广州)企业管理有限公司 | Waterproof protective layer material and preparation method thereof |
| CN115254008A (en) * | 2022-08-15 | 2022-11-01 | 大连工业大学 | Mesoporous silica material modified based on mixed mode, preparation method and application thereof |
| CN115254008B (en) * | 2022-08-15 | 2023-06-13 | 大连工业大学 | Mesoporous silica material modified based on mixed mode, preparation method and application thereof |
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