CN106987579A - It is a kind of that the method that microcapsules improve interfacial catalysis reaction efficiency is prepared based on natural true protein - Google Patents
It is a kind of that the method that microcapsules improve interfacial catalysis reaction efficiency is prepared based on natural true protein Download PDFInfo
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Abstract
It is a kind of that the method that microcapsules improve interfacial catalysis reaction efficiency is prepared based on natural true protein.The invention belongs to living things catalysis synthesis field, and in particular to a kind of to prepare the method that microcapsules improve interfacial catalysis reaction efficiency based on natural true protein.The problem of present invention is to solve biocompatibility poor in existing water-in-oil system and reduce native protein enzyme stability.Method：First, the preparation of natural true protein solution；2nd, using Pickering micro-emulsion process, the oil-in-water microemulsion using native protein as stabilizer is prepared；3rd, it is that template prepares native protein microcapsules using microemulsion, using the catalytic activity of native protein, improves the catalytic efficiency of water-oil phase solution.The present invention serves as stabilizer using native protein, it is to avoid the complicated processes of synthesis stabilizer, realizes the efficient catalytic of water-oil phase.
Description
Technical field
The invention belongs to living things catalysis synthesis field, and in particular to a kind of to prepare microcapsules raising based on natural true protein
The method of interfacial catalysis reaction efficiency.
Background technology
Enzyme is the natural true protein with catalytic activity, and its catalytic reaction has many advantages, such as reaction rate is fast, vertical
Body is selectively good, reaction condition is gentle etc..Therefore, it is biosynthesis to carry out the synthesis of material and conversion using enzymic catalytic reaction
Learn the hot fields with crossing research in chemical synthesis.In real reaction system, how during the course of the reaction can
The activity of enzyme is enough kept, the catalytic efficiency of enzyme can be improved again becomes the research emphasis in enzymic catalytic reaction.Due to enzymatic activity
Dependent on the medium or microenvironment of its presence, therefore, explore the catalytic activity for being suitable for keeping enzyme and improve enzyme and reactant
Between contact area so as to which to improve catalytic efficiency be always the problem of scientist studies.Compared to traditional single organic solvent
Catalytic reaction, while scientists find that water-in-oil microemulsion can be as enzymic catalytic reaction medium, by reactant in molecule
Realize and uniformly disperse in level, it is achieved thereby that efficient catalysis.But, this water-in-oil microemulsion has poor biology
Compatibility, so as to reduce application of the multiple proteins enzyme in biomaterial synthesis；And substantial amounts of organic solvent can cause
Native protein enzyme functionally inactive.Therefore, how microemulsion method is both utilized, biocompatibility can be improved again, into
The problem of will being solved for the present invention.
The present invention is successfully realized and stable oil-in-water microemulsion is prepared using a variety of amphipathic natural true proteins, and
Based on microemulsion template, native protein microcapsules are prepared on the surface of microemulsion, with the same of good describing property of biology
When, contact area between hydrophobic substrate and amphipathic protease can be still improved, catalytic efficiency is improved；Oil-in-water microemulsion
Traditional substantial amounts of oil phase is also changed into aqueous phase, the stability of enzyme is not only ensure that, the ratio of enzyme inactivation is also reduced；This
Outside, the native protein capsule prepared based on microemulsion template, not only with stable structure, and with good life
Thing compatibility, the new model provided for the following simulation for carrying out biological cell.
The content of the invention
The present invention is to solve biocompatibility poor in existing water-in-oil system and to reduce native protein enzyme
The problem of stability, and there is provided a kind of side that microcapsules raising interfacial catalysis reaction efficiency is prepared based on natural true protein
Method.
A kind of method that microcapsules raising interfacial catalysis reaction efficiency is prepared based on natural true protein is specifically by following
What step was carried out：
First, the preparation of PBS cushioning liquid：By NaH2PO4And Na2HPO4Mixed dissolution is configured to PBS and delayed in deionized water
Rush solution；The NaH2PO4With Na2HPO4Mass ratio be 1:(25~46)；The concentration of the PBS cushioning liquid is 45mmol/
L~55mmol/L, pH value is 8.0~8.2；
2nd, the preparation of natural true protein solution：Natural true protein is dissolved in the PBS cushioning liquid that step one is obtained
In, obtain natural true protein solution；The concentration of the natural true protein solution is 0.5mg/mL~2mg/mL；It is described natural
True protein is one kind or wherein several mixtures in lysozyme, lipase and urease, when the natural true protein
During for mixture, wherein various materials are to wait mass mixing；
3rd, the preparation of oil-phase solution：The substrate of enzyme and oil are mixed, oil-phase solution is obtained；Enzyme in the oil-phase solution
The concentration of substrate is 10mg/mL；The substrate of the enzyme is in substrate, the substrate of lipase and the substrate of urease of lysozyme
A kind of or wherein several mixture, when the substrate of the enzyme is mixture, wherein various materials are to wait mass mixing；And institute
Select the substrate of enzyme corresponding with the enzyme selected in step 2；Oil used is isooctanol or dodecane；
4th, the oil-in-water microemulsion using natural true protein solution as stabilizer is prepared using Pickering microemulsion methods
Liquid：After the oil-phase solution mixing that the natural true protein solution and step 3 that step 2 is obtained are obtained, vortex concussion instrument is utilized
20s~30s is shaken, oil-in-water microemulsion is obtained；Oil-in-water microemulsion is stood into 30min~50min, in the oil-in-water
Microemulsion oil-water interfaces at obtain microcapsules；What the natural true protein solution that the step 2 is obtained was obtained with step 3
The volume ratio of oil-phase solution is 1:(5~15).
Beneficial effects of the present invention：
The present invention is successfully realized and stable oil-in-water microemulsion is prepared using a variety of amphipathic natural true proteins, and
Based on microemulsion template, native protein microcapsules are prepared on the surface of microemulsion, with the same of good describing property of biology
When, contact area between hydrophobic substrate and amphipathic protease can be still improved, catalytic efficiency is improved；Oil-in-water microemulsion
Traditional substantial amounts of oil phase is also changed into aqueous phase, the stability of enzyme is not only ensure that, the ratio of enzyme inactivation is also reduced；This
Outside, the native protein capsule prepared based on microemulsion template, not only with stable structure, and with good life
Thing compatibility, the new model provided for the following simulation for carrying out biological cell.
Brief description of the drawings
Fig. 1 is the exterior appearance figure for the native protein oil-in-water microcapsules that embodiment one is obtained；
Fig. 2 is the electron scanning micrograph for the native protein oil-in-water microcapsules that embodiment one is obtained；
Fig. 3 is the transmission electron microscope photo for the native protein oil-in-water microcapsules that embodiment one is obtained；
Fig. 4 is the atomic force microscopy for the native protein oil-in-water microcapsules that embodiment one is obtained；
Fig. 5 is the catalytic efficiency and pair of common catalyzed efficiency for the native protein oil-in-water microcapsules that embodiment one is obtained
Than figure, wherein 1 is the catalytic efficiency for the native protein oil-in-water microcapsules that embodiment one is obtained, 2 urging for common microcapsule
Change efficiency.
Embodiment
Embodiment one：One kind of present embodiment prepares microcapsules based on natural true protein and improves interfacial catalysis
What the method for reaction efficiency was specifically carried out according to the following steps：
First, the preparation of PBS cushioning liquid：By NaH2PO4And Na2HPO4Mixed dissolution is configured to PBS and delayed in deionized water
Rush solution；The NaH2PO4With Na2HPO4Mass ratio be 1:(25~46)；The concentration of the PBS cushioning liquid is 45mmol/
L~55mmol/L, pH value is 8.0~8.2；
2nd, the preparation of natural true protein solution：Natural true protein is dissolved in the PBS cushioning liquid that step one is obtained
In, obtain natural true protein solution；The concentration of the natural true protein solution is 0.5mg/mL~2mg/mL；It is described natural
True protein is one kind or wherein several mixtures in lysozyme, lipase and urease, when the natural true protein
During for mixture, wherein various materials are to wait mass mixing；
3rd, the preparation of oil-phase solution：The substrate of enzyme and oil are mixed, oil-phase solution is obtained；Enzyme in the oil-phase solution
The concentration of substrate is 10mg/mL；The substrate of the enzyme is in substrate, the substrate of lipase and the substrate of urease of lysozyme
A kind of or wherein several mixture, when the substrate of the enzyme is mixture, wherein various materials are to wait mass mixing；And institute
Select the substrate of enzyme corresponding with the enzyme selected in step 2；Oil used is isooctanol or dodecane；
4th, the oil-in-water microemulsion using natural true protein solution as stabilizer is prepared using Pickering microemulsion methods
Liquid：After the oil-phase solution mixing that the natural true protein solution and step 3 that step 2 is obtained are obtained, vortex concussion instrument is utilized
20s~30s is shaken, oil-in-water microemulsion is obtained；Oil-in-water microemulsion is stood into 30min~50min, in the oil-in-water
Microemulsion oil-water interfaces at obtain microcapsules；What the natural true protein solution that the step 2 is obtained was obtained with step 3
The volume ratio of oil-phase solution is 1:(5~15).
The microcapsules prepared using the natural true protein capsule of preparation, substrate that can be in efficient catalytic oil phase, its side
What method was specifically carried out according to the following steps：
The PBS solution of natural true protein is added in the oil-phase solution containing substrate, turbula shaker shake is being utilized
After swinging, the microcapsule structure of formation is placed under fluorescence microscope and observed, and measured fluorescence intensity change, so that
Determine catalytic efficiency.
Embodiment two：Present embodiment from unlike embodiment one：NaH described in step one2PO4
With Na2HPO4Mass ratio be 1:40.Other steps and parameter are identical with embodiment one.
Embodiment three：Present embodiment from unlike embodiment one or two：PBS described in step one
The concentration of cushioning liquid is 50mmol/L.Other steps and parameter are identical with embodiment one or two.
Embodiment four：Unlike one of present embodiment and embodiment one to three：PH in step one
It is worth for 8.1.Other steps and parameter are identical with one of embodiment one to three.
Embodiment five：Unlike one of present embodiment and embodiment one to four：Institute in step 2
The concentration for stating natural true protein solution is 1mg/mL.Other steps and parameter are identical with one of embodiment one to four.
Embodiment six：Unlike one of present embodiment and embodiment one to five：Institute in step 3
The substrate for stating lysozyme is NAG-NAM, and the substrate of lipase is 4-methyl umbelliferone ester, and the substrate of urease is urea.Other
Step and parameter are identical with one of embodiment one to five.
Embodiment seven：Unlike one of present embodiment and embodiment one to six：Will in step 4
Oil-in-water microemulsion stands 40min.Other steps and parameter are identical with one of embodiment one to six.
Embodiment eight：Unlike one of present embodiment and embodiment one to seven：Institute in step 4
The volume ratio for stating the oil-phase solution that the natural true protein solution that step 2 obtains is obtained with step 3 is 1:10.Other steps and
Parameter is identical with one of embodiment one to seven.
Beneficial effects of the present invention are verified by following examples：
Embodiment one：It is a kind of based on natural true protein prepare microcapsules improve interfacial catalysis reaction efficiency method it is specific
Carry out according to the following steps：
First, the preparation of PBS cushioning liquid：By NaH2PO4And Na2HPO4Mixed dissolution is configured to PBS and delayed in deionized water
Rush solution；The NaH2PO4With Na2HPO4Mass ratio be 1:30；The concentration of the PBS cushioning liquid is 45mmol/L, pH value
For 8.0；
2nd, the preparation of natural true protein solution：The natural true proteins of 0.5mg are dissolved in what 1.0mL steps one were obtained
In PBS cushioning liquid, natural true protein solution is obtained；The concentration of the natural true protein solution is 0.5mg/mL~2mg/
mL；The natural true protein is the mixture of lysozyme, lipase and urease, wherein various materials are to wait mass mixing；
3rd, the preparation of oil-phase solution：The substrate of enzyme and oil are mixed, oil-phase solution is obtained；Enzyme in the oil-phase solution
The concentration of substrate is 10mg/mL；The substrate of the enzyme is NAG-NAM, 4-methyl umbelliferone ester and urea mixture, wherein respectively
Material is planted to wait mass mixing；Oil used is isooctanol or dodecane；
4th, the oil-in-water microemulsion using natural true protein solution as stabilizer is prepared using Pickering microemulsion methods
Liquid：After the oil-phase solution mixing that the natural true protein solution and step 3 that step 2 is obtained are obtained, vortex concussion instrument is utilized
20s~30s is shaken, oil-in-water microemulsion is obtained；Oil-in-water microemulsion is stood into 30min, in the oil-in-water micro emulsion
Native protein oil-in-water microcapsules are obtained at the oil-water interfaces of liquid；The natural true protein solution that the step 2 is obtained and step
The volume ratio of rapid three obtained oil-phase solutions is 1:10.
Fig. 1 is the exterior appearance figure for the native protein oil-in-water microcapsules that embodiment one is obtained；As can be seen from the figure
Protein capsule size distribution is uniform (size is between 20~30 μm), and translucency is good, illustrates that it has obvious Capsules
Structure, and with good stability.
Fig. 2 is the electron scanning micrograph for the native protein oil-in-water microcapsules that embodiment one is obtained；From figure
It can be seen that the membrane structure of native protein capsule, thickness is uniform, and illustrate that its dried stability is fine.
Fig. 3 is the transmission electron microscope photo for the native protein oil-in-water microcapsules that embodiment one is obtained；From figure
It can be seen that the hollow structure of the protein capsule, and with good membrane structure, equally also illustrate its dried stabilization
Property is fine.
Fig. 4 is the atomic force microscopy for the native protein oil-in-water microcapsules that embodiment one is obtained；Can from figure
To find out the thickness of the protein capsule in 20 rans, with good stability.
Fig. 5 is the catalytic efficiency and pair of common catalyzed efficiency for the native protein oil-in-water microcapsules that embodiment one is obtained
Than figure；Wherein 1 is the catalytic efficiency for the native protein oil-in-water microcapsules that embodiment one is obtained, 2 urging for common microcapsule
Change efficiency；The catalytic efficiency that can be realized from figure using protein microcapsules structure is than the efficiency without protein microcapsules structure
It is higher by 6~7 times.
Claims (8)
1. a kind of prepare the method that microcapsules improve interfacial catalysis reaction efficiency based on natural true protein, it is characterised in that is based on
What the method that natural true protein prepares microcapsules raising interfacial catalysis reaction efficiency was specifically carried out according to the following steps：
First, the preparation of PBS cushioning liquid：By NaH2PO4And Na2HPO4It is molten that mixed dissolution is configured to PBS bufferings in deionized water
Liquid；The NaH2PO4With Na2HPO4Mass ratio be 1:(25~46)；The concentration of the PBS cushioning liquid be 45mmol/L~
55mmol/L, pH value is 8.0~8.2；
2nd, the preparation of natural true protein solution：Natural true protein is dissolved in the PBS cushioning liquid that step one is obtained,
Obtain natural true protein solution；The concentration of the natural true protein solution is 0.5mg/mL~2mg/mL；It is described natural pure
Protein is one kind or wherein several mixtures in lysozyme, lipase and urease, when the natural true protein is
During mixture, wherein various materials are to wait mass mixing；
3rd, the preparation of oil-phase solution：The substrate of enzyme and oil are mixed, oil-phase solution is obtained；The substrate of enzyme in the oil-phase solution
Concentration be 10mg/mL；The substrate of the enzyme is one kind in substrate, the substrate of lipase and the substrate of urease of lysozyme
Or wherein several mixture, when the substrate of the enzyme is mixture, wherein various materials is wait mass mixing；And selected enzyme
Substrate it is corresponding with the enzyme selected in step 2；Oil used is isooctanol or dodecane；
4th, the oil-in-water microemulsion using natural true protein solution as stabilizer is prepared using Pickering microemulsion methods：Will
After the oil-phase solution mixing that the natural true protein solution and step 3 that step 2 is obtained are obtained, shaken using vortex concussion instrument
20s~30s, obtains oil-in-water microemulsion；Oil-in-water microemulsion is stood into 30min~50min, described oil-in-water micro-
Microcapsules are obtained at the oil-water interfaces of emulsion；The oil phase that the natural true protein solution that the step 2 is obtained is obtained with step 3
The volume ratio of solution is 1:(5~15).
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 2. according to claim 1
Method, it is characterised in that NaH described in step one2PO4With Na2HPO4Mass ratio be 1:40.
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 3. according to claim 1
Method, it is characterised in that the concentration of PBS cushioning liquid described in step one is 50mmol/L.
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 4. according to claim 1
Method, it is characterised in that pH value is 8.1 in step one.
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 5. according to claim 1
Method, it is characterised in that the concentration of natural true protein solution described in step 2 is 1mg/mL.
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 6. according to claim 1
Method, it is characterised in that the substrate of lysozyme described in step 3 is NAG-NAM, and the substrate of lipase is 4-methyl umbelliferone
Ester, the substrate of urease is urea.
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 7. according to claim 1
Method, it is characterised in that oil-in-water microemulsion is stood into 40min in step 4.
A kind of microcapsules are prepared based on natural true protein improve interfacial catalysis reaction efficiency 8. according to claim 1
Method, it is characterised in that the oil-phase solution that the natural true protein solution that step 2 described in step 4 is obtained is obtained with step 3
Volume ratio be 1:10.
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Cited By (3)
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| CN109679114A (en) * | 2018-12-12 | 2019-04-26 | 青岛古高生物科技有限公司 | The preparation method and applications of the stable Pickering lotion of protein nano cage |
| CN111020004A (en) * | 2019-12-28 | 2020-04-17 | 哈尔滨工业大学 | Preparation method of urea sensor with Janus structure artificial cell model |
| CN111671727A (en) * | 2020-05-22 | 2020-09-18 | 哈尔滨工业大学 | Method for constructing protein/phospholipid/cholesterol multi-hybrid microscale vesicles based on hydrophobic interaction |
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| CN1953735A (en) * | 2004-05-18 | 2007-04-25 | 雀巢技术公司 | Oil-in-water emulsion for delivery |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679114A (en) * | 2018-12-12 | 2019-04-26 | 青岛古高生物科技有限公司 | The preparation method and applications of the stable Pickering lotion of protein nano cage |
| CN109679114B (en) * | 2018-12-12 | 2021-06-04 | 青岛古高生物科技有限公司 | Preparation method and application of Pickering emulsion with stable protein nanocages |
| CN111020004A (en) * | 2019-12-28 | 2020-04-17 | 哈尔滨工业大学 | Preparation method of urea sensor with Janus structure artificial cell model |
| CN111020004B (en) * | 2019-12-28 | 2022-09-30 | 哈尔滨工业大学 | Preparation method of urea sensor with Janus structure artificial cell model |
| CN111671727A (en) * | 2020-05-22 | 2020-09-18 | 哈尔滨工业大学 | Method for constructing protein/phospholipid/cholesterol multi-hybrid microscale vesicles based on hydrophobic interaction |
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