CN106188042B - The method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method - Google Patents
The method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method Download PDFInfo
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Abstract
The invention discloses a kind of method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, this method mainly isolates and purifies to obtain PQQ using PQQ molecularly imprinted polymer from rich in PQQ zymotic fluid.The present invention is using silica as base material, and PQQ is template molecule, and methacrylic acid is function monomer, and ethylene glycol dimethacrylate is crosslinking agent, and PQQ molecularly imprinted polymer is prepared using thermal polymerization technology.Absorption property experiment shows that PQQ molecularly imprinted polymer prepared by the present invention has the performance of preferably special selection adsorbed target thing PQQ, and maximum adsorption capacity is up to 10mg/mg (molecularly imprinted polymer).SPE is carried out to target product PQQ using the polymer as solid phase extraction filler, realizes the purpose that purifying PQQ is efficiently separated from zymotic fluid.
Description
Technical field
The present invention relates to a kind of method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method,
Belong to technical field of biochemical industry.
Background technology
PQQ (PQQ) is a kind of water-soluble quinones, is widely present in food and animal, is glucose
The coenzyme of dehydrogenase and alcohol dehydrogenase.Because PQQ has critically important physiological function, therefore PQQ is also considered as one kind newly
B family vitamin.Research shows that PQQ can prevent the formation of amyloid, suppresses the anomaly cynapse core egg that C- ends are shortened
White cytotoxicity, therefore PQQ can be used for the treatment of nerve and moral ataxia disease.In addition, PQQ can also be prevented effectively
Nerve retrograde affection caused by oxidative stress.In simple terms, function related to nervous system PQQ has following four kinds：(1) resist
Oxidation, remove free radical；(2) respiratory chain function is influenceed, safeguards mitochondrial；(3) point of nerve growth factor is stimulated
Secrete, repair and promote nerve growth；(4) delay the deposition of α-synuclein albumen, prevent nerve cell fibrosis.Therefore, have
Guan researcher thinks that PQQ has potential treatment valency to a variety of neurodegenerative diseases such as Parkinson's and senile dementia
Value.So finding simple and easy acquisition PQQ method, carry out industrialization production and be significant.
Currently used PQQ synthetic methods are mainly chemical method synthesis, but chemical synthesis PQQ steps are more, and low yield is different
The removal of structure body and byproduct needs more purification, and needs to use a variety of toxic reagents, pollutes environment.Therefore, study such as
What obtains PQQ using non-chemical method has more industrialization meaning.PQQ is widely present in gram-negative bacteria, but synthetic quantity
Different, some bacterium only produce trace PQQ, for normal physiological metabolism demand, such as pseudomonas putida；Also some bacterium but can
Excessive PQQ is produced, and is secreted into extracellular.The wild mushroom that can produce excessive PQQ found so far includes bacillus of oxidizing glucose
Belong to (Gluconobacter), achromobacter (Achromobacter), Methanomonas (Methanomonas), methyl bacterium
Belong to (Methy-lobacillus), methylomonas (Methylomonas), thermophilic Methylobacillus (Methy-lophilus) and
Flavobacterium (Xanthobacter) etc..At present, there is the correlative study that PQQ is produced using the method for microbial fermentation,
For used original strain PQQ yield in 0.07-7mg/L, fermentation time is 2-5 days or so, but in these researchs not
There is provided it is simple and easy PQQ is isolated and purified from microbial fermentation solution, PQQ preparation is realized the side of industrial-scale production
Method.
Molecularly imprinted polymer (MIPs) is a kind of polymer to target molecule with specific adsorption capacity.From 1972
Since Wulff etc. successfully prepares MIPs, engram technology research and development is rapid.MIPs extracts in chromatographic stationary phases, solid phase at present
Take, sensor, UF membrane, the field such as enzymatic are used widely.Using MIPs as solid phase extraction filler, extraction can be strengthened
Selectivity and adsorption capacity, there is good application prospect.
The content of the invention
The technical problems to be solved by the invention are to provide one kind and isolate and purify fermentation using molecular engram solid phase extraction method
The method of PQQ in liquid, this method can simply, efficiently, isolate and purify PQQ on a large scale, be more suitable for pyrroles
The industrialized development of quinoline quinone.
To achieve these goals, the technical solution adopted in the present invention is：
The method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, is comprised the following steps：
(1) prepare：The zymotic fluid containing PQQ is prepared using PQQ production bacterium；
(2) extract and separate：The zymotic fluid containing PQQ is extracted using molecular engram solid phase extraction method, will
Zymotic fluid containing PQQ is splined in the molecular engram extraction column for being filled with molecularly imprinted polymer, is then used successively
Ethanol and ethanol-organic acid mixed liquor elution, collect ethanol-organic acid mixing eluent, are concentrated under reduced pressure, obtain PQQ
Crude product；
(3) purify：PQQ crude product is dissolved with ultra-pure water, adjusts pH to 3-4, ethanol is added, is stood after stirring,
Obtain PQQ.
Described molecularly imprinted polymer is using silica as base material, and PQQ is template molecule, metering system
Acid is function monomer, and ethylene glycol dimethacrylate is crosslinking agent, is prepared using thermal polymerization technology.
The preparation method of described molecularly imprinted polymer is：By 100-300mg PQQs, 0.1-0.5mL methyl
Acrylic acid, it is added in 50-200mL acetonitrile solutions, room temperature mechanical stirring 6-20h；Then it is micro- to add 100-800mg silica
Ball, ultrasonic wave disperse；0.6-1.0mL ethylene glycol dimethacrylates and 50-150mg azodiisobutyronitriles are added, is passed through
Nitrogen 5min, sealing, 50-70 DEG C, mechanic whirl-nett reaction 8-20h is heated to, separation, obtains polymeric material；Polymeric material is used
After ethanol washs 5-10 times, 50 DEG C of vacuum drying 8-16h；With volume ratio 6-10:1 ethanol/acetic acid mixed solution elutes dry repeatedly
Polymeric material after dry, the template molecule PQQ in polymeric material is removed, until can't detect template point in eluent
Sub- PQQ, finally with the polymeric material after distillation water washing elution to neutrality, 50 DEG C of vacuum drying 12h, obtain molecule
Imprinted polymer, it is standby.
The particle diameter of described silicon dioxide microsphere is 100-200 mesh.
The solid-to-liquid ratio 1 of described molecularly imprinted polymer and the zymotic fluid containing PQQ:100-500(g/ml).
The amount of PQQ is inhaled with the maximum no more than molecularly imprinted polymer in zymotic fluid containing PQQ
Attached amount is advisable.Preferably, the concentration of PQQ is 100-200mg/L in the zymotic fluid containing PQQ.
The volume ratio of ethanol and organic acid is 8-10 in described ethanol-organic acid mixed liquor:1-2；Described organic acid
For any one in formic acid, acetic acid or trifluoroacetic acid.
The described vacuum being concentrated under reduced pressure is ﹣ 0.9MPa, and rotating speed is 150-180 revolutions per seconds.
PQQ crude product is dissolved to final concentration of 9-12g/L with ultra-pure water in step (3).
The volume ratio of ultra-pure water and ethanol is 3-5 in step (3):1.
Regulation pH agents useful for same is hydrochloric acid in step (3).
Whipping temp is 20-25 DEG C, mixing time 5-6h, time of repose 12-24h in step (3).
Described PQQ production bacterium bag includes torsional demethylation coli (Methylobacterium extorquens),
Friedlander's bacillus (Klebsiella Pneumonia), Gluconobacter oxvdans (Gluconobacter oxidans),
Pseudomonas aeruginosa (Pseudomonas aeruginosa), streptomycete (Streptomyces rochei).
The principle of the method for molecular engram solid phase extraction separation PQQ of the present invention is shown in Fig. 1.
Beneficial effect
1st, for the present invention using silica as base material, PQQ is template molecule, and methacrylic acid is function monomer, second
Diol dimethacrylate is crosslinking agent, and PQQ molecularly imprinted polymer is prepared using thermal polymerization technology.Should
PQQ molecularly imprinted polymer has the performance of preferably special selection adsorbed target thing PQQ, and maximum is inhaled
Attached capacity is up to 10mg/mg (molecularly imprinted polymer).It is solid phase extraction filler to target product pyrroles's quinoline using the polymer
Quinoline quinone carries out SPE, realizes the purpose that purifying PQQ is efficiently separated from zymotic fluid.
2nd, PQQ molecular engram solid phase extraction is a kind of new extract and separate system, and the present invention uses pyrroles's quinoline
Quinone molecule is template molecule, forms the polymer with PQQ molecular engram, the polymer can be with selective absorption pyrrole
Cough up quinoline quinone, without adsorbing other water-solubility impurities, so as to efficiently, isolate and purify PQQ with high selectivity.Test table
Bright, the isolated PQQ purity of the present invention is up to more than 99%, and the rate of recovery is up to more than 90%.
3rd, method technological operation of the invention is simple, cost is cheap, is easy to large-scale industrial production, to promoting pyrroles's quinoline
The industrialized development of quinoline quinone is significant.
Brief description of the drawings
The embodiment of the present invention is described in further detail below in conjunction with accompanying drawing.
Fig. 1 is the schematic diagram for the method that molecular engram solid phase extraction of the present invention separates PQQ.
Fig. 2 is the ultraviolet spectrogram of PQQ products of the present invention, wherein, A is PQQ standard items, and B is PQQ products of the present invention.
Fig. 3 is the mass spectrogram of PQQ products of the present invention.
Fig. 4 is present invention extraction eluent HPLC chromatogram under 230nm.
Fig. 5 is PQQ products of the present invention HPLC chromatogram under 230nm.
Embodiment
The embodiment of the present invention is described in further detail with reference to embodiments.
Culture medium compound method used in the present invention is as follows：
Fermentation medium：Contain 40g sorbierites, 20g yeast extracts, 5g (NH in every liter of culture medium4)2SO4、2g
KH2PO4、5g MgSO4·H2O。
Enriched medium：Contain 80g sorbierites, 40g yeast extracts, 10g (NH in every liter of culture medium4)2SO4、4g
KH2PO4、10g MgSO4·H2O。
The present invention equipment used that is concentrated under reduced pressure is rotary evaporator.
Embodiment 1
A kind of method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, including following step
Suddenly：
(1) prepare：The zymotic fluid containing PQQ is prepared using PQQ production bacterium, specific method is：Will
Gluconobacter oxvdans are inoculated in fermentation medium, concussion and cultivate 3d, is obtained under the conditions of 28 DEG C according to 5% inoculum concentration
To seed liquor；Then seed liquor is inoculated in enriched medium, the concussion and cultivate under the conditions of 28 DEG C according to 10% inoculum concentration
5d, 5 DEG C of nutrient solution, 9000r/min centrifugation 15min, collects supernatant, it is 100-200mg/L's to obtain PQQ concentration
Zymotic fluid rich in PQQ；
(2) extract and separate：The zymotic fluid containing PQQ is extracted using molecular engram solid phase extraction method, will
Zymotic fluid containing PQQ is splined in the molecular engram extraction column for being filled with molecularly imprinted polymer, and molecular engram gathers
The solid-to-liquid ratio of compound and the zymotic fluid containing PQQ is 1:100 (g/ml), then successively with the ethanol of 10 times of column volumes
With ethanol-formic acid mixed liquor elution of 10 times of column volumes, ethanol-formic acid mixing eluent is collected, is concentrated under reduced pressure, obtains pyrroles's quinoline
Quinoline quinone crude product；The vacuum being concentrated under reduced pressure is ﹣ 0.9MPa, and rotating speed is 170 revolutions per seconds；
The volume ratio of ethanol and formic acid is 8 in described ethanol-formic acid mixed liquor:1；
The preparation method of described molecularly imprinted polymer is：By 100mg PQQs, 0.1mL methacrylic acids, add
Enter into 50mL acetonitrile solutions, room temperature mechanical stirring 6h；Then 100mg silicon dioxide microspheres are added, ultrasonic wave disperses；Add
0.6mL ethylene glycol dimethacrylates and 50mg azodiisobutyronitriles, nitrogen 5min is passed through, sealed, be heated to 50 DEG C, machinery
Stirring reaction 20h, separation, obtains polymeric material；After polymeric material is washed into 5 times with ethanol, 50 DEG C of vacuum drying 8h；Use volume
Than 6:1 ethanol/acetic acid mixed solution elutes dried polymeric material repeatedly, removes template molecule pyrroles's quinoline in polymeric material
Quinoline quinone, until template molecule PQQ is can't detect in eluent, finally with the polymeric material after distillation water washing elution
To neutrality, 12h is dried under 50 DEG C of vacuum, obtains molecularly imprinted polymer, it is standby；
The particle diameter of described silicon dioxide microsphere is 100-200 mesh；
(3) purify：PQQ crude product is dissolved to the final concentration of 9g/L of PQQ with ultra-pure water, adds hydrochloric acid
The volume ratio of regulation pH to 3, addition ethanol, ultra-pure water and ethanol is 3:1,24h is stood after stirring 5h under the conditions of 20 DEG C, is obtained
The crystal of PQQ.
Embodiment 2
A kind of method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, including following step
Suddenly：
(1) prepare：The zymotic fluid containing PQQ is prepared using PQQ production bacterium, specific method is：Will
Gluconobacter oxvdans are inoculated in fermentation medium, concussion and cultivate 3d, is obtained under the conditions of 28 DEG C according to 5% inoculum concentration
To seed liquor；Then seed liquor is inoculated in enriched medium, the concussion and cultivate under the conditions of 28 DEG C according to 10% inoculum concentration
5d, 5 DEG C of nutrient solution, 9000r/min centrifugation 15min, collects supernatant, it is 100-200mg/L's to obtain PQQ concentration
Zymotic fluid rich in PQQ；
(2) extract and separate：The zymotic fluid containing PQQ is extracted using molecular engram solid phase extraction method, will
Zymotic fluid containing PQQ is splined in the molecular engram extraction column for being filled with molecularly imprinted polymer, and molecular engram gathers
The solid-to-liquid ratio 1 of compound and the zymotic fluid containing PQQ:200 (g/ml), then successively with the ethanol of 10 times of column volumes and
The Ethanol-Acetic Acid mixed liquor elution of 10 times of column volumes, collects Ethanol-Acetic Acid mixing eluent, is concentrated under reduced pressure, obtains pyrroles's quinoline
Quinone crude product；The vacuum being concentrated under reduced pressure is ﹣ 0.9MPa, and rotating speed is 160 revolutions per seconds；
The volume ratio of ethanol and acetic acid is 10 in described Ethanol-Acetic Acid mixed liquor:2；
The preparation method of described molecularly imprinted polymer is：By 200mg PQQs, 0.3mL methacrylic acids, add
Enter into 100mL acetonitrile solutions, room temperature mechanical stirring 10h；Then 300mg silicon dioxide microspheres are added, ultrasonic wave disperses；Again plus
Enter 0.8mL ethylene glycol dimethacrylates and 80mg azodiisobutyronitriles, be passed through nitrogen 5min, seal, be heated to 60 DEG C, machine
Tool stirring reaction 10h, separation, obtains polymeric material；After polymeric material is washed into 8 times with ethanol, 50 DEG C of vacuum drying 10h；With
Volume ratio 8:1 ethanol/acetic acid mixed solution elutes dried polymeric material repeatedly, removes the template molecule pyrrole in polymeric material
Quinoline quinone is coughed up, until template molecule PQQ is can't detect in eluent, finally with the polymerization after distillation water washing elution
Material dries 12h, obtains molecularly imprinted polymer to neutrality under 50 DEG C of vacuum, standby；
The particle diameter of described silicon dioxide microsphere is 100-200 mesh；
(3) purify：PQQ crude product is dissolved to the final concentration of 10g/L of PQQ with ultra-pure water, adds salt
Acid for adjusting pH adds ethanol to 4, and the volume ratio of ultra-pure water and ethanol is 3:1,20h is stood after stirring 6h under the conditions of 21 DEG C, is obtained
To the crystal of PQQ.
Embodiment 3
A kind of method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, including following step
Suddenly：
(1) prepare：The zymotic fluid containing PQQ is prepared using PQQ production bacterium, specific method is：Will
Gluconobacter oxvdans are inoculated in fermentation medium, concussion and cultivate 3d, is obtained under the conditions of 28 DEG C according to 5% inoculum concentration
To seed liquor；Then seed liquor is inoculated in enriched medium, the concussion and cultivate under the conditions of 28 DEG C according to 10% inoculum concentration
5d, 5 DEG C of nutrient solution, 9000r/min centrifugation 15min, collects supernatant, it is 100-200mg/L's to obtain PQQ concentration
Zymotic fluid rich in PQQ；
(2) extract and separate：The zymotic fluid containing PQQ is extracted using molecular engram solid phase extraction method, will
Zymotic fluid containing PQQ is splined in the molecular engram extraction column for being filled with molecularly imprinted polymer, and molecular engram gathers
The solid-to-liquid ratio 1 of compound and the zymotic fluid containing PQQ:400 (g/ml), then successively with the ethanol of 10 times of column volumes and
The ethanol of 10 times of column volumes-trifluoroacetic acid mixed liquor elution, collects ethanol-trifluoroacetic acid mixing eluent, is concentrated under reduced pressure, obtains
PQQ crude product；The vacuum being concentrated under reduced pressure is ﹣ 0.9MPa, and rotating speed is 180 revolutions per seconds；
The volume ratio of ethanol and trifluoroacetic acid is 9 in described ethanol-trifluoroacetic acid mixed liquor:2；
The preparation method of described molecularly imprinted polymer is：By 250mg PQQs, 0.4mL methacrylic acids, add
Enter into 150mL acetonitrile solutions, room temperature mechanical stirring 15h；Then 500mg silicon dioxide microspheres are added, ultrasonic wave disperses；Again plus
Enter 0.9mL ethylene glycol dimethacrylates and 100mg azodiisobutyronitriles, be passed through nitrogen 5min, seal, be heated to 60 DEG C,
Mechanic whirl-nett reaction 15h, separation, obtains polymeric material；After polymeric material is washed into 10 times with ethanol, 50 DEG C of vacuum drying 12h；
With volume ratio 9:1 ethanol/acetic acid mixed solution elutes dried polymeric material repeatedly, removes the template molecule in polymeric material
PQQ, until template molecule PQQ is can't detect in eluent, finally with poly- after distillation water washing elution
Condensation material dries 12h, obtains molecularly imprinted polymer to neutrality under 50 DEG C of vacuum, standby；
The particle diameter of described silicon dioxide microsphere is 100-200 mesh；
(3) purify：PQQ crude product is dissolved to the final concentration of 11g/L of PQQ with ultra-pure water, adds salt
Acid for adjusting pH adds ethanol to 3.2, and the volume ratio of ultra-pure water and ethanol is 4:1,16h is stood after stirring 5h under the conditions of 23 DEG C,
Obtain the crystal of PQQ.
Embodiment 4
A kind of method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, including following step
Suddenly：
(1) prepare：The zymotic fluid containing PQQ is prepared using PQQ production bacterium, specific method is：Will
Gluconobacter oxvdans are inoculated in fermentation medium, concussion and cultivate 3d, is obtained under the conditions of 28 DEG C according to 5% inoculum concentration
To seed liquor；Then seed liquor is inoculated in enriched medium, the concussion and cultivate under the conditions of 28 DEG C according to 10% inoculum concentration
5d, 5 DEG C of nutrient solution, 9000r/min centrifugation 15min, collects supernatant, it is 100-200mg/L's to obtain PQQ concentration
Zymotic fluid rich in PQQ；
(2) extract and separate：The zymotic fluid containing PQQ is extracted using molecular engram solid phase extraction method, will
Zymotic fluid containing PQQ is splined in the molecular engram extraction column for being filled with molecularly imprinted polymer, and molecular engram gathers
The solid-to-liquid ratio 1 of compound and the zymotic fluid containing PQQ:500 (g/ml), then successively with the ethanol of 10 times of column volumes and
The ethanol of 10 times of column volumes-formic acid mixed liquor elution, collects ethanol-formic acid mixing eluent, is concentrated under reduced pressure, obtains pyrroles's quinoline
Quinone crude product；The vacuum being concentrated under reduced pressure is ﹣ 0.9MPa, and rotating speed is 150 revolutions per seconds；
The volume ratio of ethanol and formic acid is 9 in described ethanol-formic acid mixed liquor:1；
The preparation method of described molecularly imprinted polymer is：By 300mg PQQs, 0.5mL methacrylic acids, add
Enter into 200mL acetonitrile solutions, room temperature mechanical stirring 20h；Then 800mg silicon dioxide microspheres are added, ultrasonic wave disperses；Again plus
Enter 1.0mL ethylene glycol dimethacrylates and 150mg azodiisobutyronitriles, be passed through nitrogen 5min, seal, be heated to 70 DEG C,
Mechanic whirl-nett reaction 8h, separation, obtains polymeric material；After polymeric material is washed into 10 times with ethanol, 50 DEG C of vacuum drying 16h；
With volume ratio 10:1 ethanol/acetic acid mixed solution elutes dried polymeric material repeatedly, removes the template point in polymeric material
Sub- PQQ, until template molecule PQQ is can't detect in eluent, finally with after distillation water washing elution
Polymeric material dries 12h, obtains molecularly imprinted polymer to neutrality under 50 DEG C of vacuum, standby；
The particle diameter of described silicon dioxide microsphere is 100-200 mesh；
(3) purify：PQQ crude product is dissolved to the final concentration of 12g/L of PQQ with ultra-pure water, adds salt
Acid for adjusting pH adds ethanol to 3.6, and the volume ratio of ultra-pure water and ethanol is 5:1,12h is stood after stirring 6h under the conditions of 25 DEG C,
Obtain the crystal of PQQ.
Experimental example
1st, product analysis
1.1st, ultraviolet spectral analysis
The crystallized product of the present invention is detected simultaneously with PQQ standard items using ultraviolet spectrometer, as a result sees Fig. 2.Knot
Fruit shows that crystallized product of the invention is consistent with the UV adsorption results of PQQ standard items.
1.2nd, mass spectral analysis
The crystallized product of the present invention is detected using mass spectrograph, as a result sees Fig. 3.By the crystallized product mass spectrum of the present invention
Data contrast with known PQQ mass spectrometric datas, the results showed that, crystallized product of the invention is consistent with known PQQ mass spectrometric datas.
1.3rd, HPLC methods detect
Detection method：Using Waters Symmetry 300C18Liquid-phase chromatographic column, with acetonitrile：(acetonitrile and water contain water
2% formic acid) it is mobile phase, flow velocity 1mL/min, gradient elution (30-90%, 30min), Detection wavelength 230nm.
Detection object：Extract eluent (ethanol-organic acid mixing eluent), PQQ crystallized products.
Interpretation of result：HPLC analysis results are shown in Fig. 4,5.Fig. 4 shows, extracts the object for having high level in eluent,
Extraction has compared with high selectivity；Fig. 5 shows that, by recrystallization, crystallized product purity reaches more than 99%.
1.4th, the rate of recovery
Using the zymotic fluid of embodiment 1-4 PQQs as sample solution, the molecular engram solid phase extraction is studied to pyrroles
The separating effect of quinoline quinone, PQQ in ethanol-organic acid mixing eluent is detected using high-efficient liquid phase chromatogram technology
Content, calculate the rate of recovery (table 1).
The interpretation of result of the PQQ rate of recovery of the present invention of table 1
The result of table 1 shows that molecular engram solid phase extraction method of the present invention isolates and purifies the effect of PQQ in zymotic fluid
Very good, the rate of recovery is up to more than 90%.
2nd, the measure of molecularly imprinted polymer adsorption capacity
Weigh in 10mg molecularly imprinted polymer adsorption tubes, then respectively add 10mL mass concentrations be respectively 2,4,8,9,
10th, 11,12g/L PQQ standard liquids, at room temperature, Static Adsorption 12h, centrifugation, the molecular engram polymerization after adsorption saturation is separated
Thing is with the solution after absorption, and PQQ mass concentration is using high performance liquid chromatography measure (table 2) in the solution after absorption.
Interpretation of result (the mass concentration unit of the molecularly imprinted polymer adsorption capacity of the present invention of table 2：g/L)
The result of table 2 shows that molecularly imprinted polymer prepared by the present invention has preferably special, selection adsorbed target thing pyrrole
The performance of quinoline quinone is coughed up, maximum adsorption capacity is up to 10mg/mg (molecularly imprinted polymer).
Claims (9)
1. the method that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method, it is characterised in that including
Following steps：
（1）Prepare：The zymotic fluid containing PQQ is prepared using PQQ production bacterium；
（2）Extract and separate：The zymotic fluid containing PQQ is extracted using molecular engram solid phase extraction method, will be contained
The zymotic fluid of PQQ is splined in the molecular engram extraction column for being filled with molecularly imprinted polymer, then uses ethanol successively
With ethanol-organic acid mixed liquor elution, ethanol-organic acid mixing eluent is collected, is concentrated under reduced pressure, it is thick to obtain PQQ
Product；
（3）Purifying：PQQ crude product is dissolved with ultra-pure water, adjusts pH to 3-4, ethanol is added, stands, obtain after stirring
PQQ；
Described molecularly imprinted polymer is using silica as base material, and PQQ is template molecule, and methacrylic acid is
Function monomer, ethylene glycol dimethacrylate are crosslinking agent, are prepared using thermal polymerization technology.
2. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that the preparation method of described molecularly imprinted polymer is：By 100-300mg PQQs, 0.1-
0.5mL methacrylic acids, it is added in 50-200mL acetonitrile solutions, room temperature mechanical stirring 6-20h；Then 100-800mg is added
Silicon dioxide microsphere, ultrasonic wave disperse；Add 0.6-1.0mL ethylene glycol dimethacrylates and 50-150mg azos two are different
Butyronitrile, nitrogen 5min is passed through, sealed, be heated to 50-70 DEG C, mechanic whirl-nett reaction 8-20h, separation, obtain polymeric material；Will be poly-
After condensation material is washed 5-10 times with ethanol, 50 DEG C of vacuum drying 8-16h；With volume ratio 6-10:1 ethanol/acetic acid mixed solution is anti-
After backwashing takes off dried polymeric material, removes the template molecule PQQ in polymeric material, until being detected not in eluent
To template molecule PQQ, finally with the polymeric material after distillation water washing elution to neutrality, 50 DEG C are dried in vacuo 12h,
Molecularly imprinted polymer is obtained, it is standby；
The particle diameter of described silicon dioxide microsphere is 100-200 mesh.
3. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that the solid-to-liquid ratio 1 of described molecularly imprinted polymer and the zymotic fluid containing PQQ:100-500
（g/ml）.
4. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that the volume ratio of ethanol and organic acid is 8-10 in described ethanol-organic acid mixed liquor:1-2；Described
Organic acid is any one in formic acid, acetic acid or trifluoroacetic acid.
5. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that the described vacuum being concentrated under reduced pressure is ﹣ 0.9MPa, and rotating speed is 150-180 revolutions per seconds.
6. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that step（3）Middle PQQ crude product is dissolved to final concentration of 9-12g/L with ultra-pure water.
7. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that step（3）The volume ratio of middle ultra-pure water and ethanol is 3-5:1.
8. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that step（3）Middle regulation pH agents useful for same is hydrochloric acid.
9. the side according to claim 1 that PQQ in zymotic fluid is isolated and purified using molecular engram solid phase extraction method
Method, it is characterised in that step（3）Middle whipping temp is 20-25 DEG C, mixing time 5-6h, time of repose 12-24h.
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