CN105944686A - Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres - Google Patents
Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres Download PDFInfo
- Publication number
- CN105944686A CN105944686A CN201610328568.5A CN201610328568A CN105944686A CN 105944686 A CN105944686 A CN 105944686A CN 201610328568 A CN201610328568 A CN 201610328568A CN 105944686 A CN105944686 A CN 105944686A
- Authority
- CN
- China
- Prior art keywords
- agarose
- agarose gel
- glucosamine
- solution
- microsphere
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28016—Particle form
- B01J20/28019—Spherical, ellipsoidal or cylindrical
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
- B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
- B01J20/28047—Gels
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
Abstract
The invention discloses agarose gel microspheres containing a glucosamine group and a preparation method of the agarose gel microspheres. An agarose solid is taken as a substrate and reacts with glucosamine salt after being activated with a cross-linking agent, glucosamine-based agarose is obtained, and the agarose gel microspheres are prepared from the glucosamine-based agarose; or agarose microspheres are taken as a substrate and coupled with glucosamine salt after being activated with the cross-linking agent, and the agarose gel microspheres are prepared. The obtained agarose gel microspheres as an anion exchange chromatography medium can be used for separation and purification of bioactive ingredients such as protein, nucleic acid and the like, and the agarose gel microspheres have the advantages that the agarose gel microspheres are safe and non-toxic to the bioactive ingredients, high in ligand density, high in mechanical strength, uniform in pore sizes, high in adsorption capacity and high in purification efficiency, the preparation method is simple, the cost is low, little pollution is caused and the like.
Description
Technical field
The invention belongs to isolated and purified media technology field, be specifically related to a kind of agarose gel containing aminoglucose glycosyl
Microsphere and preparation method thereof.
Background technology
In recent years, state key puts into development biomedicine field and living marine resources development field, along with upstream is each
Class biotechnology flourish, the separating and purifying technology of downstream biomacromolecule becomes the weight of bio-medical material research field
Wanting problem, its strong point has been intended to separating medium of good performance.Natural polysaccharide has excellent biocompatibility and degradable
Property, therefore have more more advantage than synthetic polymer using it as the separating medium of biomacromolecule, thus the most both at home and abroad
The emphasis of scholar's research.Agarose is isolated a kind of natural marine from the large ocean algae such as Eucheuma gelatinosum, Thallus Porphyrae, Gracilaria tenuistipitata
Biological polyoses, has various biological activity.And agarose gel microsphere because of have good biocompatibility, function base characteristic,
High-hydrophilic, porous, the advantage such as non-specific and become the separation of biopolymer medium of a kind of high-quality, it is mainly used in
The biomolecule such as albumen, antibody, enzyme isolated and purified.
Agarose gel microsphere is modified, makes agarose connect and there is different bioactive aglucon, i.e. can be used for
Different biochemical substances isolated and purified.But existing agarose gel microsphere modification technology exist complicated process of preparation, wayward,
The problems such as ligand density is low, cost is high, seriously polluted, and be typically all agarose is first prepared as microsphere after, then by chemistry
Microsphere is modified by reaction, so easily affects microsphere mechanical strength, surface flatness and pore size.
Glucosamine is the compound that a hydroxyl of glucose is replaced by an amino, glucosamine on the market
It is broadly divided into hydrochloric acid D-glucosamine and sulphuric acid D-glucosamine two kinds.Glucosamine is the sugar of unique a kind of band amino in nature, to albumen
Matter safety non-toxic, does not interferes with the activity of protein, and its amino carried can be used for the sub-anion-exchange chromatography of albumen.
Visible, by aminoglucose glycosyl in agarose gel microsphere coupling, it is prepared as the agarose containing aminoglucose glycosyl
Gel micro-ball, can be used as a kind of good anion-exchange chromatography medium, the biomolecule such as isolated and purified protein, nucleic acid.
At present, the report utilizing glucosamine to be modified agarose gel microsphere not yet occurs.Therefore, develop one to prepare
Simple for process, low cost, ligand density are high, pollute the little base agarose gel microsphere containing glucosamine and preparation thereof
Method is significant.
Summary of the invention
It is an object of the invention to provide a kind of agarose gel microsphere containing aminoglucose glycosyl and preparation method thereof, its
Prepared agarose gel microsphere has the bioactive ingredients safety non-toxic such as protein, nucleic acid, and ligand density is high, mechanical
Intensity is high, aperture is homogeneous, adsorption capacity is big, and purification efficiency is high, and advantage, the solution such as preparation method is simple, low cost, pollution are little
Existing ion-exchange chromatography media impact little, easy on the bioactive ingredients adsorption capacity such as protein, nucleic acid activity, and existing
Have complicated process of preparation in agarose gel microsphere modification technology, wayward, ligand density is low, microsphere mechanical strength and aperture
The problems such as homogeneity is poor, seriously polluted.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of agarose gel microsphere containing aminoglucose glycosyl, is with agarose solid as substrate, crosslinked dose activation after with
Glucosamine reactant salt, obtains aminoglucose glycosyl agarose, then is prepared into described agarose gel microsphere；Wherein,
Described glucosamine salt is glucosamine hydrochloride, glucosamine sulphate, Glucosamine sulfate potassium chloride, amino Portugal
One or more in grape sugar sodium sulfate salt.
Its preparation method comprises the following steps:
1) in agarose solid, add a certain amount of ultra-pure water mix homogeneously, prepare agarose solution；
2) in step 1) gained agarose solution, it is slowly added to NaBH4, more persistently add NaOH solution with pump by certain flow rate,
Mechanical agitation mixes；
3) under agitation, persistently with pump by certain flow rate to step 2) gained solution adds cross-linking agent, in uniform temperature
Lower reaction certain time；During it, during question response to 0.3-6h, the addition flow velocity of cross-linking agent and NaOH solution is turned down；
4) in step 3) gained solution, adds glucosamine saline solution with pump by certain flow rate, and stop adding cross-linking agent and
NaOH solution, reacts certain time at a certain temperature；
5) it is dried after step 4) gained solution ultrafiltration membrance filter, concentration, prepares aminoglucose glycosyl agarose；
6) step 5) gained aminoglucose glycosyl agarose is prepared as microsphere.
Wherein, in step 1), the weight ratio of agarose solid used and ultra-pure water is 1:50 ~ 8:25, preferably 1:25 ~ 4:25.
Step 2) in NaBH4The 0.0001-0.02% that addition is agarose solid weight used, preferably 0.0005-
0.01%；The concentration of described NaOH solution is 1wt%-50wt%, preferably 20wt%-40wt%；Churned mechanically rotating speed is 50-
200rpm, preferably 80-150rpm；
Cross-linking agent described in step 3) is epoxychloropropane, epoxy bromopropane, 2,3-dibromo-propanol, Ethylene glycol diglycidyl ether
Or 1,4-butanediol diglycidyl ether；
Meanwhile, step 2) and step 3) in, the threshold speed that NaOH solution adds controls at 0.01mL ~ 5mL/min, preferably
0.05mL ~ 4mL/min, turns 0.05-0.5 times to threshold speed afterwards down, adds and controls total time at 0.5-12h, preferably 1-
8h；
The threshold speed that cross-linking agent adds controls at 0.01mL ~ 3mL/min, preferably 0.05mL ~ 2mL/min, turns down afterwards to rising
0.05-0.5 times of beginning flow velocity, adds and controls total time at 0.5-12h, preferably 1-8h；
In step 3), reaction temperature is 20-70 DEG C, preferably 35-65 DEG C, and the response time is 0.5-12h, preferably 1-8h.
In step 4), the consumption of glucosamine salt is the 0.01-10% of agarose solid weight used, preferably 0.05-8%；
The concentration of glucosamine saline solution used is 1wt%-80wt%, preferably 10wt%-70wt%；
Glucosamine saline solution add flow speed control at 0.01mL ~ 2mL/min, preferably 0.01mL ~ 1mL/min, add fashionable
Between control at 0.5-12h, preferably 1-6h；
Reaction temperature is 20-90 DEG C, preferably 45-85 DEG C, and the response time is 0.5-12h, preferably 1-6h.
In step 5), the model of ultrafilter membrane used is 8040 or 4040.
Aminoglucose glycosyl agarose is prepared by the agarose gel microsphere production system using uniform particle diameter in step 6)
Become microsphere；The agarose gel microsphere production system of described uniform particle diameter includes producing district and being positioned at the control producing district's side
District, is provided with rubber mill and premix heating kettle in described production district, described rubber mill and a microwave heating still are connected, described pre-
Mixed heating kettle and a ultrasound wave homogeneous froth breaking still are connected, and described microwave heating still and ultrasound wave homogeneous froth breaking still are all ultrasonic with one
Ripple homogenizing still is connected, and described ultrasound wave homogenizing still is connected with oil water separator, the outlet of the oil phase of described oil water separator with
The isolated and purified equipment of oil product is connected, and the isolated and purified equipment of described oil product is also connected with described premix heating kettle, described profit
The aqueous phase outlet of separator is connected with a microsphere washer, and described microsphere washer and a vortex mixer are connected, described mixing
Device and a metering and separate loading machine are connected；
During preparation, gained aminoglucose glycosyl agarose is configured to aqueous solution, after being processed by rubber mill, then through microwave
Heating kettle fully dissolves formation glue；Simultaneously by oil phase through premix heating kettle Hybrid Heating, then carry out through ultrasonic homogeneous froth breaking still
Still oil phase is formed after homogenizing pre-emulsification defoaming treatment；Still oil phase and glue are transported to ultrasound wave homogenizing still subsequently mix
Close, then mixed liquor is transported to oil water separator, after the aqueous phase input microsphere washer of isolated is carried out, will wash
Clean microsphere carries out mixing, packing.
The another kind of agarose gel microsphere containing aminoglucose glycosyl, is with agarose microbeads as substrate, crosslinked dose of work
After change, itself and glucosamine salt are carried out coupling, prepare described agarose gel microsphere；Wherein, described agarose microbeads
Particle diameter is 10-1000 micron, and it can use the agarose gel microsphere production system of above-mentioned uniform particle diameter to be prepared from；Described ammonia
Base glucosamine salt is glucosamine hydrochloride, glucosamine sulphate, Glucosamine sulfate potassium chloride, glucosamine sulfur
One or more in acid sodium-salt.
Its preparation method comprises the following steps:
1) drain after agarose microbeads being washed, add a certain amount of ultra-pure water mix homogeneously, prepare agarose microbeads solution；
2) in step 1) gained agarose microbeads solution, it is slowly added to NaBH4, more persistently add NaOH with pump by certain flow rate
Solution, low frequency ultrasound concussion mixing；
3) under the conditions of earthquake, persistently with pump by certain flow rate to step 2) gained solution adds cross-linking agent, in uniform temperature
Lower reaction certain time；During it, during question response to 0.3-6h, the addition flow velocity of cross-linking agent and NaOH solution is turned down；
4) in step 3) gained solution, adds glucosamine saline solution with pump by certain flow rate, and stop adding cross-linking agent and
NaOH solution, reacts certain time at a certain temperature；
5) by step 4) gained solution filter-cloth filtering, clean, obtain the agarose gel microsphere containing aminoglucose glycosyl, by institute
Obtain microsphere to store in 20wt% ethanol solution.
Wherein, in step 1), the weight ratio of agarose microbeads used and ultra-pure water is 1:50 ~ 1:5, preferably 1:25 ~ 1:10.
Step 2) in NaBH4The 0.0001-0.02% that addition is agarose microbeads weight used, preferably 0.0005-
0.01%；
The concentration of described NaOH solution is 1wt%-50wt%, preferably 20wt%-40wt%；The frequency of described low frequency ultrasound is 20-
40KHz, preferably 25-35KHz.
Cross-linking agent described in step 3) be epoxychloropropane, epoxy bromopropane, 2,3-dibromo-propanol, ethylene glycol bisthioglycolate shrink sweet
Oil ether or 1,4-butanediol diglycidyl ether；
Meanwhile, step 2) and step 3) in, the threshold speed that NaOH solution adds controls at 0.01mL ~ 5mL/min, preferably
0.05mL ~ 4mL/min, turns 0.05-0.5 times to threshold speed afterwards down, adds and controls total time at 0.5-12h, preferably 1-
8h；
The threshold speed that cross-linking agent adds controls at 0.01mL ~ 3mL/min, preferably 0.05mL ~ 2mL/min, turns down afterwards to rising
0.05-0.5 times of beginning flow velocity, adds and controls total time at 0.5-12h, preferably 1-8h；
Step 3) reaction temperature is 30-60 DEG C, preferably 40-50 DEG C, and the response time is 1-12h, preferably 2-8h.
In step 4), the consumption of glucosamine salt is the 0.01-10% of agarose microbeads weight used, preferably 0.05-8%；
The concentration of glucosamine saline solution used is 1wt%-80wt%, preferably 10wt%-70wt%；
Glucosamine saline solution add flow speed control at 0.01mL ~ 2mL/min, preferably 0.01mL ~ 1mL/min, add fashionable
Between control at 0.5-12h, preferably 1-6h；
Reaction temperature is 30-60 DEG C, preferably 40-50 DEG C, and the response time is 0.5-12h, preferably 1-6h.
The gained of the present invention agarose gel microsphere containing aminoglucose glycosyl can be used for the biological activity such as protein, nucleic acid
That divides is isolated and purified.
There is advantages that
1. the agarose gel microsphere containing aminoglucose glycosyl prepared by the present invention has protein, nucleic acid, biological activity
The characteristic of the bioactive ingredients safety non-toxics such as little molecule, it is ensured that its activity, and have that ligand density is high, mechanical strength high,
Aperture is homogeneous, good stability, adsorption capacity big, purification efficiency advantages of higher.
2. the invention provides the preparation method containing aminoglucose glycosyl agarose gel microsphere, its have simple to operate,
The advantages such as reaction condition gentleness, low cost, pollution are little, solve the preparation work that existing agarose gel microsphere modification technology exists
Skill is complicated, wayward, cost is high, seriously polluted, and exist easily affect microsphere mechanical strength, surface flatness and aperture
The problems such as size.Development in terms of China's Bio-pharmaceutical Industry separating and purifying technology is had certain impetus, has good
Good social and economic benefits.
Accompanying drawing explanation
Fig. 1 be the agarose gel microsphere production system of uniform particle diameter of the present invention schematic diagram (A be system entirety signal
Figure；B is microwave heating still organigram；C is ultrasound wave homogeneous froth breaking still organigram；D is ultrasound wave homogenizing still structure
Schematic diagram), wherein, 100-produces district, 200-control zone, 110-microwave heating still, 111-full-view camera, 112-microwave heating
Device, 113-limit stirring paddle, 114-dispersion wheel, 120-ultrasound wave homogeneous froth breaking still, 121-emulsifying head, 122-high-frequency ultrasonic are sent out
Raw, 130-ultrasound wave homogenizing still.
Fig. 2 is that aminoglucose glycosyl agarose (A) of embodiment 1 preparation separates nucleic acid with unmodified agarose (B)
Electrophoresis comparison diagram.
Fig. 3 is the microgram of the agarose gel microsphere containing aminoglucose glycosyl of embodiment 1 preparation.
Fig. 4 is that aminoglucose glycosyl agarose (A) of embodiment 2 preparation separates nucleic acid with unmodified agarose (B)
Electrophoresis comparison diagram.
Fig. 5 is the microgram of the agarose gel microsphere containing aminoglucose glycosyl of embodiment 2 preparation.
Fig. 6 is the microgram of the agarose gel microsphere containing aminoglucose glycosyl of embodiment 3 preparation.
Detailed description of the invention
In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention to of the present invention
Technical scheme is described further, but the present invention is not limited only to this.
The agarose gel microsphere production system of uniform particle diameter used, as it is shown in figure 1, include producing district and being positioned at production district
The control zone of side, is provided with rubber mill and premix heating kettle, described rubber mill and a microwave heating still phase in described production district
Connecting, described premix heating kettle and a ultrasound wave homogeneous froth breaking still are connected, described microwave heating still and the homogeneous froth breaking of ultrasound wave
Still is all connected with a ultrasound wave homogenizing still, and described ultrasound wave homogenizing still is connected with oil water separator, described oil water separator
Oil phase export equipment isolated and purified with oil product be connected, the isolated and purified equipment of described oil product is also connected with described premix heating kettle
Connecing, the aqueous phase outlet of described oil water separator is connected with a microsphere washer, and described microsphere washer and a vortex mixer are connected
Connecing, described vortex mixer and a metering and separate loading machine are connected.
Embodiment 1
Weigh 40g agarose solid, add 1000mL ultra-pure water, mix homogeneously, prepare agarose solution, be then slowly added into
NaBH40.0004g, more persistently add the NaOH solution of 20wt% with the flow velocity of 0.5mL/min with pump, mechanical agitation mixes, stirs
Mixing rotating speed is 80rpm；Under agitation, persistently add epoxychloropropane with pump with the flow velocity of 0.3mL/min, anti-at 50 DEG C
Answer 1h, react and progressively the flow velocity that epoxychloropropane adds is turned down to 0.03mL/min to during 0.3h, NaOH solution is added and becomes a mandarin
Velocity modulation is little to 0.05mL/min；In solution, 10wt% glucosamine sulphate is added with the flow velocity of 0.01mL/min again with pump
Solution, and stop adding epoxychloropropane and NaOH solution, react 1h at 55 DEG C；By gained solution with 4040 ultrafiltration membrance filters,
It is dried after concentration, prepares aminoglucose glycosyl agarose.
Fig. 2 is the electrophoresis pair that prepared aminoglucose glycosyl agarose (A) separates nucleic acid with unmodified agarose (B)
Than figure.From in figure, the band of aminoglucose glycosyl agarose separation nucleic acid is than the bar of unmodified agarose separation nucleic acid
Carry apparent bright, and each band separating degree is more preferable.
Use the agarose gel microsphere production system of uniform particle diameter, gained aminoglucose glycosyl agarose is prepared as micro-
Ball, gained aminoglucose glycosyl agarose is specifically configured to aqueous solution, after being processed by rubber mill, then through microwave by it
Heating kettle fully dissolves formation glue；Simultaneously by oil phase through premix heating kettle Hybrid Heating, then carry out through ultrasonic homogeneous froth breaking still
Still oil phase is formed after homogenizing pre-emulsification defoaming treatment；Still oil phase and glue are transported to ultrasound wave homogenizing still subsequently mix
Close, then mixed liquor is transported to oil water separator, after the aqueous phase input microsphere washer of isolated is carried out, will wash
Clean microsphere carries out mixing, packing.In gained agarose gel microsphere, aminoglucose glycosyl content is 3.05mmol/g.
Fig. 3 is the microgram of the prepared agarose gel microsphere containing aminoglucose glycosyl.It can be seen that institute
Obtain microsphere uniform pore diameter.
Embodiment 2
Weigh 80g agarose solid, add the ultra-pure water of 1000mL, mix homogeneously, prepare agarose solution, be slowly added to
NaBH40.008g, more persistently add 30wt%NaOH solution with pump with the flow velocity of 1mL/min, mechanical agitation mixes, speed of agitator
For 100rpm；Under agitation, persistently Ethylene glycol diglycidyl ether is added, at 55 DEG C with pump with the flow velocity of 0.5mL/min
Lower reaction 2h, reacts and progressively turns the flow velocity that Ethylene glycol diglycidyl ether adds to 0.05mL/min down to during 0.5h, will
NaOH solution adds flow velocity and turns down to 0.1mL/min；In solution, 20wt% amino Portugal is added with 0.02mL/min flow velocity again with pump
Grape sugar HCI solution, and stop adding Ethylene glycol diglycidyl ether and NaOH solution, react 1h at 65 DEG C；By gained solution
It is dried after 8040 ultrafiltration membrance filters, concentration, prepares aminoglucose glycosyl agarose.
Fig. 4 is the electrophoresis pair that prepared aminoglucose glycosyl agarose (A) separates nucleic acid with unmodified agarose (B)
Than figure.From in figure, the band of aminoglucose glycosyl agarose separation nucleic acid is than the band of unmodified agarose separation nucleic acid
Apparent bright, and each band separating degree is more preferable.
The agarose gel microsphere production system of uniform particle diameter as described in embodiment 1, by gained aminoglucose glycosyl agar
Sugar is prepared as microsphere.In gained agarose gel microsphere, aminoglucose glycosyl content is 3.75mmol/g.
Fig. 5 is the microgram of the prepared agarose gel microsphere containing aminoglucose glycosyl.It can be seen that institute
Obtain microsphere uniform pore diameter.
Embodiment 3
Drain after weighing the washing of 60g agarose microbeads, add 1000mL ultra-pure water mix homogeneously, prepare agarose microbeads solution,
It is slowly added to NaBH40.0012g, more persistently add 40wt%NaOH solution with pump with the flow velocity of 0.8mL/min, low frequency ultrasound shakes
Swinging mixing, its supersonic frequency is 25KHz；Under the conditions of earthquake, persistently add ethylene glycol bisthioglycolate contracting with pump with the flow velocity of 0.4mL/min
Water glycerin ether, at 40 DEG C react 8h, react to during 4h progressively by Ethylene glycol diglycidyl ether add flow velocity turn down to
0.03mL/min, adds NaOH solution flow velocity and turns down to 0.06mL/min；Use pump with the flow velocity of 0.05mL/min to solution again
Middle addition 15wt% glucosamine hydrochloride solution, and stop adding Ethylene glycol diglycidyl ether and NaOH solution, at 45 DEG C
Reaction 1.5h；By gained agarose microbeads solution filter-cloth filtering, clean, prepare the agarose gel containing aminoglucose glycosyl micro-
Ball, is placed in 20wt% ethanol solution storage by thus obtained microsphere.In gained agarose gel microsphere, aminoglucose glycosyl content is
2.55mmol/g。
Fig. 6 is the microgram of the prepared agarose gel microsphere containing aminoglucose glycosyl.It can be seen that institute
Obtain microsphere uniform pore diameter.
The foregoing is only presently preferred embodiments of the present invention, all impartial changes done according to scope of the present invention patent with
Modify, all should belong to the covering scope of the present invention.
Claims (10)
1. the agarose gel microsphere containing aminoglucose glycosyl, it is characterised in that: with agarose solid as substrate, crosslinked
With glucosamine reactant salt after agent activation, obtain aminoglucose glycosyl agarose, then be prepared into described agarose gel
Microsphere；Or with agarose microbeads as substrate, after crosslinked dose of activation, itself and glucosamine salt are carried out coupling, prepare described
Agarose gel microsphere；
Wherein, the particle diameter of described agarose microbeads is 10-1000 micron；
Described glucosamine salt is glucosamine hydrochloride, glucosamine sulphate, Glucosamine sulfate potassium chloride, ammonia
One or more in base glucosamine sulphate sodium salt.
2. the preparation method of the agarose gel microsphere containing aminoglucose glycosyl as claimed in claim 1, it is characterised in that:
Comprise the following steps:
1) in agarose solid, add a certain amount of ultra-pure water mix homogeneously, prepare agarose solution；
2) in step 1) gained agarose solution, it is slowly added to NaBH4, then add NaOH solution by certain flow rate, and with 50-
The rotating speed machinery stirring and evenly mixing of 200rpm；
3) under agitation, by certain flow rate to step 2) gained solution adds cross-linking agent, react one at a certain temperature
Fix time；During it, during question response to 0.3-6h, the addition flow velocity of cross-linking agent and NaOH solution is turned down；
4) in step 3) gained solution, add glucosamine saline solution by certain flow rate, and stop adding cross-linking agent and NaOH
Solution, reacts certain time at a certain temperature；
5) it is dried after step 4) gained solution ultrafiltration membrance filter, concentration, prepares aminoglucose glycosyl agarose；
6) step 5) gained aminoglucose glycosyl agarose is prepared as microsphere.
3. the preparation method of the agarose gel microsphere containing aminoglucose glycosyl as claimed in claim 1, it is characterised in that:
Comprise the following steps:
1) drain after agarose microbeads being washed, add a certain amount of ultra-pure water mix homogeneously, prepare agarose microbeads solution；
2) in step 1) gained agarose microbeads solution, it is slowly added to NaBH4, then by certain flow rate addition NaOH solution, and with
The concussion mixing of 20-40KHz low frequency ultrasound；
3) under the conditions of earthquake, by certain flow rate to step 2) gained solution adds cross-linking agent, react one at a certain temperature
Fix time；During it, during question response to 0.3-6h, the addition flow velocity of cross-linking agent and NaOH solution is turned down；
4) in step 3) gained solution, add glucosamine saline solution by certain flow rate, and stop adding cross-linking agent and NaOH
Solution, reacts certain time at a certain temperature；
5) by step 4) gained solution filter-cloth filtering, clean, obtain the agarose gel microsphere containing aminoglucose glycosyl, by institute
Obtain microsphere to store in 20wt% ethanol solution.
4., according to the preparation method of the agarose gel microsphere containing aminoglucose glycosyl described in Claims 2 or 3, its feature exists
In: in step 1), the weight ratio of agarose solid used or agarose microbeads and ultra-pure water is 1:50 ~ 8:25.
5., according to the preparation method of the agarose gel microsphere containing aminoglucose glycosyl described in Claims 2 or 3, its feature exists
In step 2) in NaBH4Addition be agarose solid used or the 0.0001-0.02% of agarose microbeads weight；Described
The concentration of NaOH solution is 1wt%-50wt%；
Cross-linking agent described in step 3) is epoxychloropropane, epoxy bromopropane, 2,3-dibromo-propanol, Ethylene glycol diglycidyl ether
Or 1,4-butanediol diglycidyl ether.
6., according to the preparation method of the agarose gel microsphere containing aminoglucose glycosyl described in Claims 2 or 3, its feature exists
In step 2) and step 3) in, the threshold speed that NaOH solution adds controls at 0.01mL ~ 5mL/min, turns down afterwards to initial
0.05-0.5 times of flow velocity, adds and controls at 0.5-12h total time；
The threshold speed that cross-linking agent adds controls, at 0.01mL ~ 3mL/min, to turn 0.05-0.5 times to threshold speed afterwards down,
Add and control at 0.5-12h total time.
7., according to the preparation method of the agarose gel microsphere containing aminoglucose glycosyl described in Claims 2 or 3, its feature exists
In: in step 3), reaction temperature is 20-70 DEG C, and the response time is 0.5-12h.
8., according to the preparation method of the agarose gel microsphere containing aminoglucose glycosyl described in Claims 2 or 3, its feature exists
In: in step 4), the consumption of glucosamine salt is agarose solid used or the 0.01-10% of agarose microbeads weight, used
The concentration of glucosamine saline solution is 1wt%-80wt%；
The flow speed control that glucosamine saline solution adds controlled at 0.5-12h in 0.01mL ~ 2mL/min, joining day；
Reaction temperature is 20-90 DEG C, and the response time is 0.5-12h.
The preparation method of the agarose gel microsphere containing aminoglucose glycosyl the most according to claim 2, it is characterised in that: step
Rapid 6) use the agarose gel microsphere production system of uniform particle diameter that aminoglucose glycosyl agarose is prepared as microsphere in；Described
The agarose gel microsphere production system of uniform particle diameter includes producing district and being positioned at the control zone producing district's side, described production district
Inside being provided with rubber mill and premix heating kettle, described rubber mill and a microwave heating still are connected, described premix heating kettle and one
Ultrasound wave homogeneous froth breaking still is connected, and described microwave heating still froth breaking homogeneous with ultrasound wave still is all connected with a ultrasound wave homogenizing still
Connecing, described ultrasound wave homogenizing still is connected with oil water separator, and the oil phase outlet of described oil water separator is isolated and purified with oil product
Equipment is connected, and the isolated and purified equipment of described oil product is also connected with described premix heating kettle, the aqueous phase of described oil water separator
Outlet is connected with a microsphere washer, and described microsphere washer and a vortex mixer are connected, described vortex mixer and a metering point
Installation is connected；
During preparation, gained aminoglucose glycosyl agarose is configured to aqueous solution, after being processed by rubber mill, then through microwave
Heating kettle fully dissolves formation glue；Simultaneously by oil phase through premix heating kettle Hybrid Heating, then carry out through ultrasonic homogeneous froth breaking still
Still oil phase is formed after homogenizing pre-emulsification defoaming treatment；Still oil phase and glue are transported to ultrasound wave homogenizing still subsequently mix
Close, then mixed liquor is transported to oil water separator, after the aqueous phase input microsphere washer of isolated is carried out, will wash
Clean microsphere carries out mixing, packing.
10. the agarose gel microsphere containing aminoglucose glycosyl as claimed in claim 1 separates pure at bioactive ingredients
Application in change, it is characterised in that: described bioactive ingredients includes protein, nucleic acid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610328568.5A CN105944686B (en) | 2016-05-18 | 2016-05-18 | A kind of agarose gel microsphere of the glycosyl containing aminoglucose and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610328568.5A CN105944686B (en) | 2016-05-18 | 2016-05-18 | A kind of agarose gel microsphere of the glycosyl containing aminoglucose and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105944686A true CN105944686A (en) | 2016-09-21 |
| CN105944686B CN105944686B (en) | 2018-04-20 |
Family
ID=56913175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610328568.5A Active CN105944686B (en) | 2016-05-18 | 2016-05-18 | A kind of agarose gel microsphere of the glycosyl containing aminoglucose and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105944686B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107754767A (en) * | 2017-10-31 | 2018-03-06 | 苏州博进生物技术有限公司 | A kind of high stability immobilization metal chelates affinity chromatography medium |
| CN108905977A (en) * | 2018-05-25 | 2018-11-30 | 江苏珐玛赛谱生物科技有限公司 | A kind of agarose gel microsphere and preparation method thereof |
| CN109806916A (en) * | 2019-03-15 | 2019-05-28 | 中科森辉微球技术（苏州）有限公司 | High performance anion exchange media and preparation method thereof |
| CN110038523A (en) * | 2019-03-20 | 2019-07-23 | 江西师范大学 | It is a kind of for isolating and purifying the preparation method of the affinity chromatography medium of hexokinase |
| CN113281505A (en) * | 2021-04-06 | 2021-08-20 | 浙江大学 | Method for rapidly detecting staphylococcus aureus in milk |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0132244A1 (en) * | 1983-07-19 | 1985-01-23 | Pharmacia Aktiebolag | Separation material and its preparation |
| CN1472002A (en) * | 2003-06-16 | 2004-02-04 | 天津大学 | Macropore high-capacity agarose gel media preparing method |
| CN103212377A (en) * | 2013-04-19 | 2013-07-24 | 哈尔滨益材新材料有限公司 | Preparation method of agarose immune magnetic microspheres and applications thereof |
| CN103769057A (en) * | 2012-10-25 | 2014-05-07 | 中国科学院过程工程研究所 | High-strength polysaccharide aerogel microsphere, and preparation method and application thereof |
| CN105498715A (en) * | 2016-01-15 | 2016-04-20 | 武汉菲恩生物科技有限公司 | Preparation method and application of nickel metal chromatography medium based on 6B agarose microspheres |
| CN105498701A (en) * | 2015-10-21 | 2016-04-20 | 中科森辉微球技术（苏州）有限公司 | High-load agarose chromatography media and preparation method thereof |
-
2016
- 2016-05-18 CN CN201610328568.5A patent/CN105944686B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0132244A1 (en) * | 1983-07-19 | 1985-01-23 | Pharmacia Aktiebolag | Separation material and its preparation |
| CN1472002A (en) * | 2003-06-16 | 2004-02-04 | 天津大学 | Macropore high-capacity agarose gel media preparing method |
| CN103769057A (en) * | 2012-10-25 | 2014-05-07 | 中国科学院过程工程研究所 | High-strength polysaccharide aerogel microsphere, and preparation method and application thereof |
| CN103212377A (en) * | 2013-04-19 | 2013-07-24 | 哈尔滨益材新材料有限公司 | Preparation method of agarose immune magnetic microspheres and applications thereof |
| CN105498701A (en) * | 2015-10-21 | 2016-04-20 | 中科森辉微球技术（苏州）有限公司 | High-load agarose chromatography media and preparation method thereof |
| CN105498715A (en) * | 2016-01-15 | 2016-04-20 | 武汉菲恩生物科技有限公司 | Preparation method and application of nickel metal chromatography medium based on 6B agarose microspheres |
Non-Patent Citations (2)
| Title |
|---|
| 张静飞等: "葡聚糖接枝型高载量金属螯合介质的制备与性能", 《过程工程学报》 * |
| 龚波林等: "单分散多孔交联聚甲基丙烯酸环氧丙酯微球基质高效亲合色谱填料的研究", 《分析化学明天将更美好国际分析化学研讨会论文集》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107754767A (en) * | 2017-10-31 | 2018-03-06 | 苏州博进生物技术有限公司 | A kind of high stability immobilization metal chelates affinity chromatography medium |
| CN107754767B (en) * | 2017-10-31 | 2020-04-21 | 苏州博进生物技术有限公司 | High-stability immobilized metal chelating affinity chromatography medium |
| CN108905977A (en) * | 2018-05-25 | 2018-11-30 | 江苏珐玛赛谱生物科技有限公司 | A kind of agarose gel microsphere and preparation method thereof |
| CN109806916A (en) * | 2019-03-15 | 2019-05-28 | 中科森辉微球技术（苏州）有限公司 | High performance anion exchange media and preparation method thereof |
| CN109806916B (en) * | 2019-03-15 | 2021-12-21 | 中科森辉微球技术（苏州）有限公司 | High-performance anion exchange medium and preparation method thereof |
| CN110038523A (en) * | 2019-03-20 | 2019-07-23 | 江西师范大学 | It is a kind of for isolating and purifying the preparation method of the affinity chromatography medium of hexokinase |
| CN110038523B (en) * | 2019-03-20 | 2022-01-25 | 江西师范大学 | Preparation method of affinity chromatography medium for separating and purifying hexokinase |
| CN113281505A (en) * | 2021-04-06 | 2021-08-20 | 浙江大学 | Method for rapidly detecting staphylococcus aureus in milk |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105944686B (en) | 2018-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105944686A (en) | Agarose gel microspheres containing glucosamine group and preparation method of agarose gel microspheres | |
| CN105504760B (en) | It is a kind of to be used to handle hydrogel material containing heavy metal containing sewage and preparation method thereof | |
| CN104387617B (en) | A kind of preparation method of corn straw modified cellulose gel | |
| CN102500328A (en) | Preparation method and application of adsorbent of organic and inorganic composite material | |
| CN101914229B (en) | Method for preparing kieselguhr/natural rubber composite material | |
| CN105214629A (en) | A kind of biomass-based nano lanthanum oxide dephosphorization compound adsorbent and preparation method thereof | |
| CN108905977A (en) | A kind of agarose gel microsphere and preparation method thereof | |
| CN107583624A (en) | A kind of graphene oxide/sodium alginate composite microsphere and its preparation method and application | |
| CN107262072A (en) | A kind of industrial production process for carrying zirconium nanocomposite adsorbent | |
| CN104163921A (en) | Mesoporous silicon composite lead ion imprinted polymer and its preparation method and use | |
| CN102019213B (en) | Method for preparing strongly-acid ion exchange medium | |
| CN103992487A (en) | Cellulose acetate propionate microspheres and preparation method thereof | |
| CN105692633A (en) | Modified calcium metasilicate and application of modified calcium metasilicate for removing heavy metal and phosphorus at same time | |
| CN102863646B (en) | Preparation method for rosin-based porous polymer microsphere | |
| CN206597488U (en) | A kind of micro-filtration or milipore filter regenerative agent preparation facilities | |
| CN102504076B (en) | Malachite green molecularly imprinted solid phase extraction filler and preparation method thereof | |
| CN210825712U (en) | Ammonia nitrogen wastewater and acid mist combined recovery treatment device | |
| CN103265720B (en) | Novel method for preparing porous crosslinked chitosan microsphere | |
| CN110041444B (en) | The preparation method of the modified xantham gum of oil and gas well fracturing fluid | |
| CN202322459U (en) | High-efficiency coagulation and precipitation device | |
| CN109399661A (en) | A kind of preparation method of Fe-SSZ-24 molecular sieve | |
| CN206069619U (en) | A kind of sewage water advanced treatment apparatus | |
| CN101768221B (en) | Method for preparing nano-crystal cellulose II by using basic anion-exchange resin | |
| CN115055157A (en) | Preparation and desorption method of zirconium modified zeolite-bentonite active particle dephosphorization filter material | |
| CN103694389A (en) | Sevelamer carbonate preparation technology |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20220113 Address after: 361028 units 501 and 502, building 12, collaborative innovation and entrepreneurship center, Xiamen biomedical port, No. 71, HouXiang Road, Haicang District, Xiamen City, Fujian Province Patentee after: Xiamen Sanji Technology Co.,Ltd. Address before: 361015 6 / F, building B, No. 3, Golden Bay wealth center, No. 1000 anling Road, Huli high tech park, Xiamen, Fujian Patentee before: LVQI (XIAMEN) MARINE BIOLOGICAL TECHNOLOGY CO.,LTD. |