CN110437491A - Nattokinase molecular imprinted polymer on surface and its preparation method and application - Google Patents

Nattokinase molecular imprinted polymer on surface and its preparation method and application Download PDF

Info

Publication number
CN110437491A
CN110437491A CN201910742958.0A CN201910742958A CN110437491A CN 110437491 A CN110437491 A CN 110437491A CN 201910742958 A CN201910742958 A CN 201910742958A CN 110437491 A CN110437491 A CN 110437491A
Authority
CN
China
Prior art keywords
nattokinase
gel
solution
adsorption
imprinted polymer
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
Application number
CN201910742958.0A
Other languages
Chinese (zh)
Other versions
CN110437491B (en
Inventor
张勋力
张迎庆
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Nabian Food Co Ltd
Original Assignee
Beijing Nabian Food Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Beijing Nabian Food Co Ltd filed Critical Beijing Nabian Food Co Ltd
Priority to CN201910742958.0A priority Critical patent/CN110437491B/en
Publication of CN110437491A publication Critical patent/CN110437491A/en
Application granted granted Critical
Publication of CN110437491B publication Critical patent/CN110437491B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/0066—Use of inorganic compounding ingredients
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
    • C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Materials Engineering (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Inorganic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The present invention provides a kind of Nattokinase molecular imprinted polymer on surface and its preparation method and application.The Nattokinase molecular imprinted polymer on surface is core-shell structure；Interior nucleome is the silica of Nattokinase trace, and outer shell is the konjak portuguese gansu polyose gel of Nattokinase trace.Nattokinase molecular imprinted polymer on surface adsorbance with higher of the invention and stability；There is selection characterization of adsorption energy to Nattokinase, the Nattokinase molecule isolated and purified has greater activity.

Description

Nattokinase surface molecularly imprinted polymer and preparation method and application thereof
Technical Field
The invention belongs to the technical field of protein bioseparation engineering, and relates to a nattokinase surface molecularly imprinted polymer, and a preparation method and application thereof.
Background
Molecular imprinting is a technique for preparing polymers having selective recognition properties for template molecules, and the prepared polymers are called imprinted polymers (MIPs). The principle of imprinting is similar to biological natural recognition systems, such as antigens and antibodies, enzymes and substrates, like locks and keys, MIPs have selective recognition properties for template molecules and their analogues. When the template molecule contacts with the functional monomer, a plurality of action points are formed, the action is memorized in the polymerization process, then the template molecule is removed by a certain method, and finally a cavity polymer matched with the spatial configuration of the template molecule, namely MIPs, is formed. It can identify specific template molecule and has certain tolerance to harsh environment, such as high temperature and high pressure. Therefore, the molecular imprinting has wide application prospect in the aspects of chemical sensing, chromatographic separation, solid phase extraction and the like.
At present, molecular imprinting technology is applied in a large number of small molecule fields, the technology is mature, and imprinting of biological macromolecules has a plurality of bottlenecks, so that the progress is slow. The biological macromolecular imprinting is a technology for preparing an imprinted polymer by using macromolecular substances (protein, DNA, RNA and the like) as template molecules. Because the biological macromolecules have larger specific surface area, the molecular surfaces have a large number of potential recognition sites, and different areas show different physicochemical properties, template molecules are difficult to enter and exit in the process of molecular recognition, and even cross reaction is caused; the addition of conventional non-physiological blotting conditions denatures the protein. Therefore, the preparation of the biomacromolecule imprinted polymer is difficult and the preparation conditions are more strict. The imprinting method of the biological macromolecules mainly comprises an embedding method, a surface imprinting method and an epitope method.
Nattokinase is a serine protease isolated from natto, and consists of 275 amino acids, has a molecular weight of 27.7kDa and a PI of 8.6, and shows a considerable fibrinolytic activity at pH 6-12, but lacks functional and structural stability in an acidic environment, and its activity is reduced or even inactivated at a temperature above 60 ℃. Researches find that the nattokinase not only has fibrinolytic activity and thrombolysis capacity, but also can be used for treating and preventing thrombotic diseases; it can also stimulate endothelial cell to produce t-PA, activate prourokinase in vivo to convert into urokinase, so as to realize human body self-physiological thrombolysis and enhance thrombolysis capacity.
Imprinting of macromolecules such as protein and the like always has some bottlenecks, and because the protein has large molecular weight, a complex structure and more non-specific binding sites, is easy to inactivate, needs to be carried out in a water phase and the like, and has great difference with an imprinting technology of small molecules, a matrix material suitable for a protein molecularly imprinted polymer and a preparation method thereof are in the direction of continuous exploration in the industry; particularly for the natto kinase imprinting technology, at present, no effective process technology for applying the molecular imprinting technology to natto kinase separation and purification has been reported.
Disclosure of Invention
Based on the defects in the prior art, the invention aims to provide a nattokinase surface molecularly imprinted polymer and a preparation method thereof; the preparation method takes the nattokinase as a template molecule, simultaneously takes silicon dioxide as a carrier and konjac glucomannan as a surface imprinting matrix, combines the nattokinase and the surface imprinting matrix together, and is rigid and flexible, so that the adsorption quantity and the stability of the prepared nattokinase surface molecularly imprinted polymer are improved. The invention also aims to provide the application of the nattokinase surface molecularly imprinted polymer in the separation and purification of nattokinase, and the nattokinase obtained by the separation and purification of the nattokinase surface molecularly imprinted polymer has higher activity.
The purpose of the invention is realized by the following technical means:
on one hand, the invention provides a nattokinase surface molecularly imprinted polymer which is of a core-shell structure; the inner core body is silicon dioxide imprinted by nattokinase, and the outer shell layer is konjac glucomannan gel imprinted by nattokinase.
The konjak fine powder, which contains Konjak Glucomannan (KGM) as a main component, is a neutral polysaccharide with a large number of hydroxyl structures, is easy to modify and form a film, and has more advantages by taking the KGM as an imprinting matrix: the good biocompatibility of the nattokinase creates a relatively mild environment for imprinting of the nattokinase, so that the nattokinase is not easy to denature; the easily-controlled net structure is beneficial to the in-and-out of the nattokinase and is convenient for the attachment and the elution of the nattokinase; the polyhydroxy structure can provide a large number of hydrogen bond binding sites, and the nonspecific adsorption of the charged nattokinase can be effectively avoided by virtue of the weak interaction. Silicon dioxide is used as a carrier, and in the silica gel imprinting process, the surface of the silica spheres can effectively reduce the embedding phenomenon, thereby being beneficial to the elution and recognition of nattokinase and further improving the imprinting efficiency; in addition, the silicon dioxide has stable chemical properties and good mechanical stability and thermal stability, so that the prepared nattokinase imprinted polymer shows high stability, and the service life of the material is prolonged; thirdly, the porous, light and soft nature of the silica makes the size and shape of the nattokinase imprinted polymer easy to control.
The nattokinase molecularly imprinted polymer is of a core-shell structure, silicon dioxide is used as a carrier, konjac glucomannan is used as a surface imprinted matrix, a cavity is formed in the silicon dioxide and konjac glucomannan gel through the surface molecular imprinting of the nattokinase, the silicon dioxide with the natto kinase imprinted on the inner core is obtained, the nattokinase molecularly imprinted polymer is on the surface of the nattokinase of the konjac glucomannan gel with the natto kinase imprinted on the outer shell, the cavity has the characteristic of selective recognition and combination of the nattokinase, the silicon dioxide and the konjac glucomannan are combined together and are rigid and flexible, and the adsorption quantity and the stability of the nattokinase surface molecularly imprinted polymer are improved.
In addition, in the invention, besides the nattokinase protein molecules contained in the gel shell layer, the nattokinase protein molecules are combined on the surface of the gel shell layer by utilizing the adsorption effect of silicon dioxide, so that the loading amount of the template nattokinase is increased, more recognition binding sites can be formed after the gel shell layer is combined, the defect of the molecular loading amount of the surface molecularly imprinted polymer nattokinase protein molecules is made up, and the imprinting recognition efficiency of the nattokinase is improved.
In another aspect, the invention provides a preparation method of a nattokinase surface molecularly imprinted polymer, which comprises the following steps:
mixing the konjac gel and nattokinase protein to carry out gel imprinting adsorption to obtain imprinted adsorbed konjac gel;
mixing silicon dioxide and nattokinase protein to carry out silica gel imprinting adsorption to obtain imprinted adsorbed silicon dioxide;
mixing the silicon dioxide adsorbed by the imprinting with the konjac gel adsorbed by the imprinting; mixing, adding the mixture into a dispersion medium, stirring and polymerizing, adding a dispersing agent, stirring, standing and layering, and collecting a polymer;
and fully eluting the polymer by an eluent to obtain the nattokinase surface molecularly imprinted polymer.
The preparation method is simple and convenient, has high efficiency, uses less organic solvent, and can obtain the polymer material with better biocompatibility and stability.
In the above method, preferably, the method further comprises a process of preparing konjac gel from konjac powder, wherein konjac gel is obtained by adding water to the konjac powder to swell the konjac powder to obtain a konjac swelling solution, and then adding a cross-linking agent or a high molecular compound to react;
in the above method, preferably, before adding the cross-linking agent, alkali is added to adjust the pH value of the konjac swelling solution to 7-11.
In the above method, preferably, the alkali may be sodium hydroxide.
In the above method, preferably, the konjak swelling solution has a mass concentration of 0.1 to 1.5 wt% of konjak powder.
In the above method, preferably, the crosslinking agent may include one or more of epichlorohydrin, N' -methylenebisacrylamide, ethylene glycol dimethacrylate, vinyl dimethacrylate, and the like in combination.
In the above method, preferably, the volume of the cross-linking agent is 12.5 to 22.5 vol% of the volume of the konjac swelling solution.
In the above method, the temperature for adding the cross-linking agent or the high molecular compound to carry out the reaction is preferably 20 ℃ to 60 ℃; the reaction time is 2-7 h.
In the above method, preferably, the polymer compound may include one or more of xanthan gum, carrageenan, sodium alginate, gelatin, soy protein isolate, carboxymethyl cellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, polyethylene glycol, and the like.
In the above method, the concentration of the polymer compound is preferably 0.1 to 100% (mass concentration of the prepared aqueous solution), and the volume ratio of the polymer compound to the konjac swollen solution is preferably 1 (0.02 to 50).
In the above method, the konjac fine powder is swollen with water to form a gel, but the gel is not stable enough and is stabilized by adding the above cross-linking agent for cross-linking, or the polymer compound is added to the gel to form a blended gel for increasing stability.
In the method, preferably, in the gel blotting adsorption process, the nattokinase is mixed with the konjac gel in the form of solution, and the concentration of the nattokinase solution is 0.5-50.0 mg/mL; the volume usage ratio of the konjac gel to the nattokinase protein solution is 1: (0.1-10).
In the above method, the time for performing gel blotting adsorption stirring is preferably 0.5 to 20 hours.
In the method, preferably, in the process of silica gel blotting adsorption, the nattokinase protein is mixed with silicon dioxide in a solution form, and the concentration of the nattokinase protein solution is 0.5-50.0 mg/mL; the dosage ratio of the silicon dioxide to the nattokinase protein solution is 1.0g: (1-50) mL.
In the method, the silica gel blotting adsorption standing time is preferably 0.5-20 h.
In the above method, preferably, before the silica gel blotting adsorption, the silica is repeatedly subjected to acid-base soaking, water washing to neutrality, and drying.
In the above method, preferably, the amount ratio of the silica to the konjac gel adsorbed by the blot is 1.0g (0.5 to 100) mL during the stirring polymerization.
In the above method, preferably, the dispersion medium may include one or a combination of more of liquid paraffin, toluene, xylene, benzene, cyclohexane, n-hexane, n-pentane, petroleum ether, and the like.
In the above method, preferably, the dispersant may include one or more of methanol, ethanol, acetone, tween-80, span-60, span-80 and the like.
In the method, the specific dispersion medium and the dispersing agent are selected by utilizing a reversed phase suspension method, combining the properties of the konjac glucomannan matrix of the shell layer and the properties of the loaded template protein nattokinase, thereby ensuring that the polymer forms a proper form and is not adhered and the activity of the nattokinase is not damaged.
In the above method, preferably, the eluent may include one or a combination of more of SDS-HAc solution, NaCl solution, phosphate buffer solution, and boric acid buffer solution; further preferably, the concentration of the SDS-HAc solution is 2-15 wt% SDS-2-15 vol% HAc; the concentration of the NaCl solution is 2-30% (w/v).
In another aspect, the invention also provides application of the nattokinase surface molecularly imprinted polymer in separation and purification of nattokinase.
In the above application, preferably, the application includes:
adding physiological saline into fresh natto, stirring, leaching and filtering, adding the nattokinase surface molecularly imprinted polymer into filtrate, mixing uniformly, standing for adsorption, centrifuging to remove supernatant, adding an eluant for elution, centrifuging to obtain eluted supernatant, dialyzing or performing gel filtration to remove salt, and freeze-drying to obtain the nattokinase.
Further preferably, the application comprises:
adding 50-1000 mL of physiological saline into every 50g of fresh natto, stirring and leaching for 10 min-24 h, filtering by using a 80-300 mesh filter screen, adding 1-5 g of nattokinase surface molecularly imprinted polymer into filtrate, uniformly mixing, standing at 4 ℃ for adsorbing for 1-24 h, centrifuging, pouring out supernatant, adding an eluent of 0.1-2.0 mol/L NaCl-PBS buffer solution with the pH of 7-10 into the centrifuged precipitate, placing on a shaking table for eluting for 1-24 h, centrifuging to obtain eluted supernatant, dialyzing or carrying out gel filtration for desalting, and freeze-drying to obtain the nattokinase.
In the application of the invention, the nattokinase obtained by separating and purifying the molecular imprinting polymer on the surface of the nattokinase has higher activity.
The natto kinase surface molecularly imprinted polymer combines a flexible net structure of konjac glucomannan and a porous rigid structure of silicon dioxide, improves the adsorption capacity and stability of the polymer while protecting natto kinase molecules, has selective adsorption property on natto kinase, and has high activity of the eluted natto kinase molecules.
Drawings
FIG. 1 is a transmission electron microscope image of a molecularly imprinted polymer on the surface of nattokinase in example 1 of the present invention.
FIG. 2 is a comparison graph of adsorption kinetics curves of a molecularly imprinted polymer on the surface of nattokinase and a non-imprinted polymer in example 1 of the invention.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
The raw materials of the reagents used in the following examples are commercially available unless otherwise specified.
Example 1:
the embodiment provides a preparation method of a nattokinase surface molecularly imprinted polymer, which comprises the following steps:
weighing konjac powder, adding 100mL of distilled water for swelling to obtain 0.9 wt% konjac swelling solution, and adding sodium hydroxide to adjust the pH value to 10; adding 17.5mL (17.5 vol%) of epichlorohydrin into the konjac swelling solution, and stirring and crosslinking at 40 ℃ for 4.5h to obtain konjac gel;
taking 10mL of konjac gel, adding 5mL of 5.0mg/mL nattokinase protein solution, stirring, and carrying out gel blotting adsorption for 1h to obtain konjac gel with blotting adsorption;
repeatedly soaking silicon dioxide in acid and alkali, then washing with water to neutrality and drying for later use, taking 1.0g of silicon dioxide, adding 5mL of 1.0mg/mL nattokinase protein solution, shaking uniformly, standing and imprinting for 12h to obtain imprinted adsorbed silicon dioxide;
fully mixing the silicon dioxide adsorbed by the blot and the konjac gel adsorbed by the blot, adding the mixture into 150mL of liquid paraffin, stirring at a high speed for 5min, adding 20mL of 40 vol% ethanol solution, continuously stirring for 5min, standing for layering, and collecting a polymer;
and (3) repeatedly eluting the polymer with 30mL of 10 wt% SDS-10 vol% HAc eluent under stirring for 30min each time, washing with deionized water after three times of elution until no absorption peak exists at 265.5nm of the filtrate, and collecting the nattokinase surface molecularly imprinted polymer which is recorded as MIP.
The transmission electron microscope image of the nattokinase surface molecularly imprinted polymer is shown in figure 1, and as can be seen from figure 1, the nattokinase surface molecularly imprinted polymer is of a core-shell structure; the inner core body is silicon dioxide imprinted by nattokinase, and the outer shell layer is konjac glucomannan gel imprinted by nattokinase.
Example 2:
the embodiment provides a preparation method of a nattokinase surface molecularly imprinted polymer, which comprises the following steps:
weighing konjac fine powder, adding 100mL of distilled water for swelling to obtain 0.7 wt% konjac swelling solution, adding 1.0 wt% xanthan gum 15mL into 0.7 wt% konjac swelling solution 35mL, mixing to obtain 50mL solution, and stirring and mixing at 40 ℃ for 3h to obtain konjac gel;
taking 30mL of konjac gel, adding 15mL of 20.0mg/mL nattokinase protein solution, stirring, and carrying out gel blotting adsorption for 5h to obtain konjac gel with blotting adsorption;
repeatedly soaking silicon dioxide in acid and alkali, then washing with water to neutrality and drying for later use, taking 1.0g of silicon dioxide, adding 5mL of 50.0mg/mL nattokinase protein solution, shaking uniformly, standing and imprinting for 20h to obtain imprinted adsorbed silicon dioxide;
fully mixing the silicon dioxide adsorbed by the blot and the konjac gel adsorbed by the blot, then adding the mixture into 200mL of normal hexane, stirring at a high speed for 10min, adding 30mL of Tween-80 solution, continuously stirring for 5min, standing for layering, and collecting a polymer;
and (3) repeatedly eluting the polymer with 30mL of 1.0mol/L NaCl eluent under stirring for 30min each time, washing with deionized water after three times of elution until no absorption peak exists in the filtrate at 265.5nm, and collecting the nattokinase surface molecularly imprinted polymer which is recorded as MIP.
Comparative experiment:
the adsorption performance of the polymers prepared by different imprinting preparation methods is compared and researched:
the experimental method comprises the following steps:
swollen and crosslinked konjac gel was prepared as in example 1, and imprinted gel and unimpressed gel were prepared as follows:
blotting gel: to 50mL of swollen and crosslinked konjac gel, 25mL of a 5.0mg/mL nattokinase solution prepared in a pH 7.0PBS buffer solution was added, and the mixture was mechanically stirred for 1 hour to prepare a blotting gel.
Non-blotted gel: adding 25mL of PBS buffer solution with pH value of 7.0 into 50mL of swollen and cross-linked konjac gel, and uniformly stirring to obtain non-imprinted gel;
preparation of blot adsorbed silica gel and non-blot adsorbed silica gel:
silica gel adsorbed by blotting: 1mg/mL nattokinase solution prepared by adding 5mL pH 7.0PBS buffer solution into 1g of silicon dioxide and mixing uniformly to be used as blotting adsorption silica gel.
Non-imprinted adsorbed silica gel: adding 1g of silicon dioxide into 5mL of PBS buffer solution with pH value of 7.0, and uniformly mixing to obtain silica gel which is not subjected to imprinting adsorption;
preparation of different core-shell imprinted polymers:
the gel was mixed well with silica gel according to the following protocol:
scheme 1: mixing silica gel adsorbed without imprinting and konjac gel adsorbed without imprinting
Scheme 2: mixing silica gel without blotting adsorption and konjac gel with blotting adsorption.
Scheme 3: mixing silica gel adsorbed by imprinting and konjac gel not adsorbed by imprinting.
Scheme 4: the blot-adsorbed silica gel and the blot-adsorbed konjac gel were mixed (example 1).
Respectively adding the mixture into 150mL of liquid paraffin, stirring at a high speed for 5min, adding 20mL of 40 vol% ethanol solution, continuously stirring for 5min, standing for layering, and collecting respective polymers;
and (3) repeatedly eluting the polymer with 30mL of 10 wt% SDS-10 vol% HAc eluent for 30min each time, washing with deionized water after three times of elution until no absorption peak exists in the filtrate at 265.5nm, and collecting the nattokinase surface molecularly imprinted polymer to obtain the polymer prepared by different imprinting methods.
The selective adsorption performance test experiment results of the polymers obtained by different imprinting preparation methods are as follows:
preparing 0.1-0.5 mg/mL nattokinase solution by using PBS solution with pH of 7.0, measuring absorbance value under the maximum absorption wavelength, and drawing a nattokinase solution standard curve. The maximum absorption wavelength of the nattokinase solution is determined to be 265.5nm through experiments. Under the maximum absorption wavelength, the linear relation is better in the range of 0.1-0.5 mg/mL, and the linear equation is that y is 1.102x-0.009, R2＝0.9979。
0.05g of each polymer is added into 4mL of 1.0mg/mL nattokinase solution, and after the mixture is uniformly mixed, the mixture is kept stand and adsorbed for 24 hours at the temperature of 4 ℃. And then centrifuging at 5000rpm for 5min, taking supernatant after the imprinted polymer reabsorption, filtering by using a 0.45-um microporous filter membrane, measuring an absorbance value, and calculating according to a nattokinase standard curve to obtain the concentration of the solution protein so as to obtain the adsorption quantity. And calculating the imprinting efficiency according to the ratio of the adsorption amount of the imprinted polymer to the adsorption amount of the non-imprinted polymer.
The results of measuring the adsorption capacity of the nattokinase surface molecularly imprinted polymer obtained by different preparation methods on nattokinase are shown in table 1.
TABLE 1 adsorption capacity results for various blotting methods
Comparing the adsorption amounts of the polymers obtained by the four imprinting schemes, it can be seen from table 1 that the adsorption amount of the imprinted polymer prepared by the imprinting scheme 4, i.e., the method of imprinting both silica and konjac gel and then polymerizing is the largest, and the average adsorption amount is 31.155 mg/g; secondly, scheme 2, polymerizing the konjac gel adsorbed by imprinting and silicon dioxide not adsorbed by imprinting to obtain imprinted polymer with average adsorption capacity of 28.550 mg/g; scheme 3 the adsorption amount of the imprinted polymer polymerized by the konjac gel without imprinting adsorption and the silica with imprinting adsorption is also increased to 27.670 mg/g. The result shows that compared with the non-imprinted polymer (NIP) prepared in the scheme 1, the imprinted polymer prepared in the scheme 4 can selectively adsorb the template protein nattokinase, and the imprinting of the template molecules is performed from the two aspects of 'shell layer' and 'core' by adopting gel and silica gel (the scheme 4), so that the imprinting is better than the imprinting by only adopting gel (the scheme 2) and silica gel (the scheme 3), and the imprinting adsorption amount and the imprinting efficiency can be effectively improved to a certain extent.
Polymer specific adsorption performance test experiment:
preparing nattokinase surface Molecularly Imprinted Polymers (MIPs) according to example 1, weighing 0.05g of MIP, respectively adding 1.0mg/mL of nattokinase, trypsin and bovine serum albumin solution 4mL, uniformly mixing, standing at 4 ℃ for adsorption for 24h, measuring the protein concentration of supernatant, calculating the adsorption quantity according to the standard curve of each protein solution, measuring the adsorption quantity of non-imprinted polymers (NIP) by the same method, and calculating the imprinting efficiency. The results of the experiment are shown in table 2 below:
TABLE 2 Nattokinase surface molecularly imprinted polymer and non-imprinted polymer specific adsorption performance
As can be seen from Table 2, the molecularly imprinted polymer on the surface of nattokinase and the non-imprinted polymer show different adsorption amounts to nattokinase, trypsin and bovine serum albumin, and the imprinting efficiency of each protein is different, which shows that the adsorption amount to nattokinase is the maximum (Q)MIP：31.155±0.882mg/g，QNIP: 26.139 + -1.064 mg/g); minor (Q) trypsinMIP：25.561±0.355mg/g；QNIP: 23.555. + -. 0.257mg/g), bovine serum albumin minimum (Q)MIP：5.687±0.937mg/g；QNIP: 8.466 +/-2.326 mg/g), which shows that the natto kinase surface molecularly imprinted polymer has better specific adsorption performance on natto kinase.
Performance testing experiments of polymer adsorption kinetics:
respectively weighing 0.05g of the nattokinase surface Molecularly Imprinted Polymer (MIP) prepared according to the embodiment 1, adding 4mL of 1.0mg/mL nattokinase solution, uniformly mixing, standing and adsorbing at 4 ℃, respectively standing and adsorbing for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 and 26 hours, taking supernate, and determining the concentration of the adsorbed supernatant protein. The adsorption kinetics experiment determination of the non-imprinted polymer is carried out by the same method. The results of the experiment are shown in FIG. 2.
As can be seen from the adsorption kinetics curve of FIG. 2, the adsorption amount of the molecularly imprinted polymer on the surface of the nattokinase is slightly higher than that of the non-imprinted polymer, and the adsorption amount rising process of the molecularly imprinted polymer on the surface of the nattokinase and the non-imprinted polymer is relatively smooth after 2 hours in the adsorption process. The adsorption time is about 20h, the adsorption process of the molecular imprinted polymer on the surface of the nattokinase to the nattokinase is basically balanced with that of the non-imprinted polymer, and after the adsorption time is prolonged, the molecular imprinted polymer on the surface of the nattokinase is basically stable and the non-imprinted polymer has the tendency of desorption. When adsorption reaches equilibrium, namely the ratio of the average value of 5 data points of the molecular imprinted polymer on the surface of the nattokinase and the non-imprinted polymer for 18 h-26 h is taken as equilibrium adsorption quantity, and the imprinting efficiency is 1.10.
Application example:
the nattokinase surface molecularly imprinted polymer prepared in the embodiment 1 of the invention is used for separating and purifying nattokinase. Comprises two processes of adsorption and elution, and the enzyme activity is respectively used as an index for determination.
(1) Adsorption
Adding 50g of fresh natto into 100mL of normal saline, stirring and leaching for 30min, filtering with a 300-mesh sieve, and freeze-drying to obtain natto mucilage freeze-dried powder. Weighing natto mucus freeze-dried powder, and preparing 10mL of natto mucus solution with the concentration of 50mg/mL by using normal saline. Weighing 0.1g of the nattokinase surface Molecularly Imprinted Polymer (MIP) prepared in the example 1, adding 3mL of natto mucus solution, mixing uniformly, standing at 4 ℃, adsorbing for 20h, centrifuging, taking supernate and measuring the enzyme activity (IU/mL) of the adsorbed solution. The enzyme activity (IU/mL) of the solution after the non-imprinted polymer (NIP) is adsorbed is determined by the same method.
Sample enzyme activity adsorption capacity (IU/g) ═ enzyme activity of solution before adsorption-enzyme activity after adsorption (IU/mL) × constant volume (mL)/weighing sample amount (g)
The adsorption rate (%) of the enzyme activity of the sample is (solution enzyme activity before adsorption-enzyme activity after adsorption)/enzyme activity before adsorption is multiplied by 100%
The method for measuring and calculating the activity of the natto kinase comprises the following steps:
the activity of the solution natto kinase is measured by adopting an agar-fibrin plate method and taking urokinase as a standard substance.
The agar-fibrin plate preparation and determination method comprises the following steps: 40mL of 1.5% agarose gel at 55 ℃ was added with 40mL of 1.5mg/mL fibrinogen solution and 4mL of 1BP/mL bovine thrombin, poured onto a plate, and cooled at 4 ℃ for 30min for further use. Sucking 10uL of sample liquid to sample on a fibrin plate, culturing at 37 deg.C for 18h, and determining product S (mm) of two vertical diameters of transparent ring2)。
Preparation of urokinase standard curve: preparing 25.6, 51.2, 102.4, 204.8, 409.6IU/mL urokinase standard solution, spotting according to the above method, culturing at 37 deg.C for 18h, and measuring the product S (mm) of two vertical diameters of transparent ring2) The logarithm of urokinase concentration and the logarithm of the area of the transparent circle were plotted to prepare a standard curve.
Spotting protocol: in order to reduce errors, samples of each sample to be tested and a urokinase standard solution are applied on the same plate, 3 samples are added respectively, and the average value is calculated. The test results are shown in tables 3 and 4.
TABLE 3 area S (mm) of transparent circle of urokinase solution with different enzyme activities2)
TABLE 4 area S (mm) of transparent circles of different solutions before and after adsorption2)
The results show that: in the experiment, 18h urokinase standard substance cultured at the constant temperature of 37 ℃ is used as a standard curve, the obtained linear equation is lgS-0.236 lgU +1.9155, and R20.9341. Within the range of 25.6-409.6 IU/mL, the logarithm of the activity of the urokinase and the logarithm of the product S of the two vertical diameters of the transparent ring present a good linear relationship.
The areas of 18h transparent rings of the natto mucus solution before adsorption, the supernatant after adsorption of the natto kinase surface Molecularly Imprinted Polymer (MIP) and the supernatant after adsorption of the non-imprinted polymer (NIP) are substituted into a urokinase standard curve to obtain solutions with enzyme activities of 1167.00IU, 256.47IU and 336.12IU respectively, and the enzyme activities of the Molecularly Imprinted Polymer (MIP) adsorbed on the natto kinase surface and the non-imprinted polymer (NIP) are 910.53IU and 830.88IU respectively, so that the adsorption amounts are 9105.30IU/g and 8308.80IU/g respectively, the adsorption rates are 78.02% and 71.20% respectively according to the enzyme activities, and the imprinting efficiency is 1.10. The enzyme activity shows that the adsorption capacity of the imprinted polymer and the non-imprinted polymer to the nattokinase is larger, and the adsorption capacity of the imprinted polymer is larger than that of the non-imprinted polymer due to the specific binding space conformation.
(2) Elution is carried out
After the above adsorption, 4mL of 0.5mol/L NaCl-PBS buffer solution with pH 10 was added to the nattokinase blotting and non-blotting precipitates from which the supernatant was removed, and the mixture was placed on a shaker and eluted at a constant rate for 2 hours. Centrifuging to obtain the supernatant after elution, and performing three groups of parallel experiments. Measuring enzyme activity change by agar-fibrin plate method, and spotting three samples of eluent, washing and removing liquid on the molecularly imprinted polymer on the surface of natto kinase and washing and removing liquid on the non-imprinted polymer on the plate, wherein the three samples are parallel for 3 times, and the area of the transparent ring after 18h culture is measured. The enzyme activities of the supernatants after the elution of the nattokinase surface Molecularly Imprinted Polymer (MIP) and the non-imprinted polymer (NIP) are calculated, and the elution rate is calculated, and the result is shown in Table 5.
The elution rate (%). ratio (enzyme activity of eluent/enzyme activity adsorbed on polymer) × 100%
TABLE 5 area S (mm) of transparent circles for different solutions before and after elution2)
The results show that: the eluent has no transparent ring and does not have thrombolytic activity, and the measured results of other two samples are substituted into a urokinase standard curve to obtain the nattokinase surface molecularly imprinted polymer washing and removing liquid with the enzyme activity of 135.99IU and 67.37IU respectively and the elution rate of 14.94 percent and 8.11 percent respectively, which indicates that the nattokinase with high activity can be obtained by single elution for 2 hours.

Claims (10)

1. A nattokinase surface molecularly imprinted polymer is a core-shell structure; the inner core body is silicon dioxide imprinted by nattokinase, and the outer shell layer is konjac glucomannan gel imprinted by nattokinase.
2. A preparation method of a nattokinase surface molecularly imprinted polymer comprises the following steps:
mixing the konjac gel and nattokinase protein to carry out gel imprinting adsorption to obtain imprinted adsorbed konjac gel;
mixing silicon dioxide and nattokinase protein to carry out silica gel imprinting adsorption to obtain imprinted adsorbed silicon dioxide;
mixing the silicon dioxide adsorbed by the imprinting with the konjac gel adsorbed by the imprinting; mixing, adding the mixture into a dispersion medium, stirring and polymerizing, adding a dispersing agent, stirring, standing and layering, and collecting a polymer;
and fully eluting the polymer by an eluent to obtain the nattokinase surface molecularly imprinted polymer.
3. The method according to claim 2, further comprising a step of preparing konjak gel from the konjak powder, wherein the konjak powder is swollen with water to obtain a konjak swelling solution, and then a crosslinking agent or a high molecular compound is added to the solution to react to obtain konjak gel; wherein,
optionally adding alkali before adding the cross-linking agent to adjust the pH value of the konjac swelling solution to 7-11; preferably, the base is sodium hydroxide;
preferably, the mass concentration of the konjac powder in the konjac swelling solution is 0.1-1.5 wt%.
4. The process of claim 3, wherein the crosslinker comprises a combination of one or more of epichlorohydrin, N' -methylenebisacrylamide, ethylene glycol dimethacrylate, and vinyl dimethacrylate;
preferably, the volume consumption of the cross-linking agent is 12.5-22.5 vol% of the volume of the konjak swelling solution;
preferably, the temperature for adding the cross-linking agent or the high molecular compound for reaction is 20-60 ℃; the reaction time is 2-7 h.
5. The method of claim 3, wherein the high molecular compound comprises one or more of xanthan gum, carrageenan, sodium alginate, gelatin, soy protein isolate, carboxymethyl cellulose, sodium carboxymethyl cellulose, polyvinyl alcohol, and polyethylene glycol;
preferably, the concentration of the macromolecular compound is 0.1-100%, and the volume ratio of the macromolecular compound to the konjak swelling solution is 1 (0.02-50).
6. The method according to claim 2, wherein in the gel blotting adsorption process, the nattokinase protein is mixed with the konjac gel in a solution form, and the concentration of the nattokinase protein solution is 0.5-50.0 mg/mL; the volume usage ratio of the konjac gel to the nattokinase protein solution is 1: (0.1 to 10);
preferably, the time for carrying out gel blotting adsorption stirring is 0.5-20 h.
7. The method according to claim 2, wherein in the silica gel blotting adsorption process, the nattokinase protein is mixed with silicon dioxide in a solution form, and the concentration of the nattokinase protein solution is 0.5-50.0 mg/mL; the dosage ratio of the silicon dioxide to the nattokinase protein solution is 1.0g: (1-50) mL;
preferably, the silica gel blotting adsorption standing time is 0.5-20 h;
preferably, before the silica gel imprinting adsorption, the silica is repeatedly soaked in acid and alkali, washed to be neutral and dried.
8. The method according to claim 2, wherein the amount ratio of the silica to the gel of konjak adsorbed on the blot in the stirring polymerization is 1.0g: (0.5-100) mL;
preferably, the dispersion medium comprises a combination of one or more of liquid paraffin, toluene, xylene, benzene, cyclohexane, n-hexane, n-pentane, and petroleum ether;
preferably, the dispersant comprises a combination of one or more of methanol, ethanol, acetone, tween-80, span-60 and span-80;
preferably, the eluent comprises a combination of one or more of SDS-HAc solution, NaCl solution, phosphate buffer solution and boric acid buffer solution.
9. Use of the nattokinase surface molecularly imprinted polymer of claim 1 for separating and purifying nattokinase.
10. The application of claim 9, wherein the application comprises:
adding physiological saline into fresh natto, stirring, leaching and filtering, adding the nattokinase surface molecularly imprinted polymer into filtrate, mixing uniformly, standing, adsorbing, centrifuging to remove supernatant, adding an eluant for elution, centrifuging to obtain eluted supernatant, dialyzing or performing gel filtration to remove salt, and freeze-drying to obtain nattokinase;
preferably, the application comprises:
adding 50-1000 mL of physiological saline into every 50g of fresh natto, stirring and leaching for 10 min-24 h, filtering by using a 80-300 mesh filter screen, adding 1-5 g of nattokinase surface molecularly imprinted polymer into filtrate, uniformly mixing, standing at 4 ℃ for adsorbing for 1-24 h, centrifuging, pouring out supernatant, adding an eluent of 0.1-2.0 mol/L NaCl-PBS buffer solution with the pH of 7-10 into the centrifuged precipitate, placing on a shaking table for eluting for 1-24 h, centrifuging to obtain eluted supernatant, dialyzing or carrying out gel filtration for desalting, and freeze-drying to obtain the nattokinase.
CN201910742958.0A 2019-08-13 2019-08-13 Nattokinase surface molecularly imprinted polymer and preparation method and application thereof Active CN110437491B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910742958.0A CN110437491B (en) 2019-08-13 2019-08-13 Nattokinase surface molecularly imprinted polymer and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910742958.0A CN110437491B (en) 2019-08-13 2019-08-13 Nattokinase surface molecularly imprinted polymer and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN110437491A true CN110437491A (en) 2019-11-12
CN110437491B CN110437491B (en) 2021-09-21

Family

ID=68434962

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910742958.0A Active CN110437491B (en) 2019-08-13 2019-08-13 Nattokinase surface molecularly imprinted polymer and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN110437491B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113209946A (en) * 2021-04-20 2021-08-06 湖北工业大学 Aristolochic acid core-shell molecularly imprinted polymer of konjac glucomannan matrix and preparation method and application thereof
CN115336755A (en) * 2022-08-17 2022-11-15 北京纳百恩食品有限公司 Fibrinolysis-promoting active substance and preparation method and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6379599B1 (en) * 2000-01-10 2002-04-30 Council Of Scientific And Industrial Research Process for the preparation of molecularly imprinted polymers useful for separation of enzymes
CN101940916A (en) * 2009-07-03 2011-01-12 湖北工业大学 Konjac glucomannan stroma protein molecularly imprinted membrane as well as preparation method and application thereof
CN104231143A (en) * 2013-06-14 2014-12-24 中国科学院大连化学物理研究所 Protein surface molecular imprinting material based on RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy as well as preparation method and application thereof
CN106076288A (en) * 2016-06-17 2016-11-09 江苏大学 A kind of preparation method of porous ion/molecularly imprinted polymer
CN109675531A (en) * 2018-12-21 2019-04-26 洛阳理工学院 A kind of preparation method of magnetic metal ion surface molecular engram material

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6379599B1 (en) * 2000-01-10 2002-04-30 Council Of Scientific And Industrial Research Process for the preparation of molecularly imprinted polymers useful for separation of enzymes
CN101940916A (en) * 2009-07-03 2011-01-12 湖北工业大学 Konjac glucomannan stroma protein molecularly imprinted membrane as well as preparation method and application thereof
CN104231143A (en) * 2013-06-14 2014-12-24 中国科学院大连化学物理研究所 Protein surface molecular imprinting material based on RAFT (Reversible Addition-Fragmentation Chain Transfer) strategy as well as preparation method and application thereof
CN106076288A (en) * 2016-06-17 2016-11-09 江苏大学 A kind of preparation method of porous ion/molecularly imprinted polymer
CN109675531A (en) * 2018-12-21 2019-04-26 洛阳理工学院 A kind of preparation method of magnetic metal ion surface molecular engram material

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
XIANGZHI DONG: "Preparation of pH and temperature dual‐...or recognition of bovine serum albumin", 《POLYM INT》 *
吴振岳: "分子印迹CS@SiO2聚合物吸附性能评价", 《福建师范大学学报》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113209946A (en) * 2021-04-20 2021-08-06 湖北工业大学 Aristolochic acid core-shell molecularly imprinted polymer of konjac glucomannan matrix and preparation method and application thereof
CN115336755A (en) * 2022-08-17 2022-11-15 北京纳百恩食品有限公司 Fibrinolysis-promoting active substance and preparation method and application thereof

Also Published As

Publication number Publication date
CN110437491B (en) 2021-09-21

Similar Documents

Publication Publication Date Title
EP0764047B2 (en) Polymeric microbeads and method of preparation
Guo et al. Adsorptive separation of hemoglobin by molecularly imprinted chitosan beads
Glad et al. Use of silane monomers for molecular imprinting and enzyme entrapment in polysiloxane-coated porous silica
Burow et al. Molecular imprinting: synthesis of polymer particles with antibody-like binding characteristics for glucose oxidase
Tuncel et al. Cibacron Blue F3G-A-attached monosize poly (vinyl alcohol)-coated polystyrene microspheres for specific albumin adsorption
AU2007227827B2 (en) Composite sorbent material, its preparation and its use
Shi et al. Functionalized anodic aluminum oxide (AAO) membranes for affinity protein separation
US7122381B2 (en) Selective affinity material, preparation thereof by molecular imprinting, and use of the same
CN110437491B (en) Nattokinase surface molecularly imprinted polymer and preparation method and application thereof
Uzun et al. Poly (ethylene dimethacrylate-glycidyl methacrylate) monolith as a stationary phase in dye-affinity chromatography
Li et al. Biocompatible polymeric monoliths for protein and peptide separations
Yang et al. Coated hydrophilic polystyrene-based packing materials
Hajizadeh Application of composite cryogels in downstream processing-A review
Deni̇zli̇ et al. Dye‐ligand column chromatography: Albumin adsorption from aqueous media and human plasma with dye‐affinity microbeads
Arrua et al. Preparation of macroporous monoliths based on epoxy-bearing hydrophilic terpolymers and applied for affinity separations
Kobos et al. A novel fluorocarbon-based immobilization technology
Yu et al. RECENT DEVELOPMENT AND APPLICATION OF MONOLITHIC COLUMNS.
Zhao et al. Sorption of unconjugated bilirubin by means of novel immobilized β-cyclodextrin polymers
Karakoç et al. Affinity adsorption of recombinant human interferon-α on a porous dye-affinity adsorbent
Kaçar et al. Procion Green H-E4BD-immobilized porous poly (hydroxyethylmethacrylate) ion-exchange membrane: preparation and application to lysozyme adsorption
KR20020084672A (en) A microsphere and process for producting thereof using polyfructose and its derivatives
Denizli et al. Affinity microspheres and their application to lysozyme adsorption: Cibacron Blue F3GA and Cu (II) with poly (HEMA‐EGDMA)
JPH0623258B2 (en) Hydrophilic porous particles
CN117866214A (en) Macroporous frozen gel medium and preparation method and application thereof
Garipcan et al. Poly (hydroxyethyl methacrylate-co-methacrylamidoalanine) membranes and their utilization as metal-chelate affinity adsorbents for lysozyme adsorption

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant