CN106987579B - A method of microcapsules are prepared based on natural true protein and improve interfacial catalysis reaction efficiency - Google Patents
A method of microcapsules are prepared based on natural true protein and improve interfacial catalysis reaction efficiency Download PDFInfo
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- CN106987579B CN106987579B CN201710214104.6A CN201710214104A CN106987579B CN 106987579 B CN106987579 B CN 106987579B CN 201710214104 A CN201710214104 A CN 201710214104A CN 106987579 B CN106987579 B CN 106987579B
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Abstract
A method of microcapsules are prepared based on natural true protein and improve interfacial catalysis reaction efficiency.The invention belongs to biocatalysis to synthesize field, and in particular to a method of microcapsules are prepared based on natural true protein and improve interfacial catalysis reaction efficiency.The present invention is to solve the problems, such as biocompatibility poor in existing water-in-oil system and reduce native protein enzyme stability.Method: the one, preparation of natural true protein solution；Two, using Pickering micro-emulsion process, the oil-in-water microemulsion using native protein as stabilizer is prepared；Three, 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, avoids the complicated processes of synthesis stabilizer, realizes the efficient catalytic of water-oil phase.
Description
Technical field
The invention belongs to biocatalysis to synthesize field, and in particular to one kind prepares microcapsules raising based on natural true protein
The method of interfacial catalysis reaction efficiency.
Background technique
Enzyme is the natural true protein with catalytic activity, and catalysis reaction has many advantages, as reaction rate is fast, vertical
Body is selectively good, reaction condition is mild etc..Therefore, the synthesis of substance is carried out using enzymic catalytic reaction and conversion is biosynthesis
Learn the hot fields with crossing research in chemical synthesis.In real reaction system, how during the reaction can
The activity of enzyme is enough kept, and the catalytic efficiency that can be improved enzyme becomes the research emphasis in enzymic catalytic reaction.Due to enzymatic activity
Dependent on medium or microenvironment existing for it, therefore, explores the catalytic activity for being suitable for keeping enzyme and improve enzyme and reactant
Between contact area to improve catalytic efficiency be always scientist research the problem of.Compared to traditional single organic solvent
Catalysis reaction, while scientists find that water-in-oil microemulsion can be used as enzymic catalytic reaction medium, by reactant in molecule
Uniform dispersion is realized in level, to realize efficient catalysis.But this water-in-oil microemulsion has poor biology
Compatibility, to reduce application of the multiple proteins enzyme in biomaterial synthesis；And a large amount of organic solvent will lead to
Native protein enzyme functionally inactive.Therefore, how microemulsion method is not only utilized, but also can be improved biocompatibility, at
In order to which the present invention will solve the problems, such as.
The present invention, which is successfully realized, prepares stable oil-in-water microemulsion using a variety of amphipathic natural true proteins, and
Based on microemulsion template, native protein microcapsules are prepared on the surface of microemulsion, it is same with good describing property of biology
When, it still can be improved contact area between hydrophobic substrate and amphipathic protease, improve catalytic efficiency；Oil-in-water microemulsion
Traditional a large amount of oil is mutually also changed into water phase, not only ensure that the stability of enzyme, also reduces the ratio of enzyme inactivation；This
Outside, the native protein capsule prepared based on microemulsion template is not only had stable structure, and has good life
Object compatibility, the new model provided for the following simulation for carrying out biological cell.
Summary of the invention
The present invention is to solve biocompatibility poor in existing water-in-oil system and reduce native protein enzyme
The problem of stability, and provide a kind of side that microcapsules raising interfacial catalysis reaction efficiency is prepared based on natural true protein
Method.
It is a kind of that the method for microcapsules raising interfacial catalysis reaction efficiency is prepared specifically by following based on natural true protein
What step carried out:
One, the preparation of PBS buffer solution: by NaH2PO4And Na2HPO4It is slow that mixed dissolution is configured to PBS in deionized water
Rush solution；The NaH2PO4With Na2HPO4Mass ratio be 1:(25~46)；The concentration of the PBS buffer solution is 45mmol/
L~55mmol/L, pH value are 8.0~8.2；
Two, natural true protein the preparation of natural true protein solution: is dissolved in the PBS buffer solution that step 1 obtains
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 of lysozyme, lipase and urease or in which several mixtures, when the natural true protein
When for mixture, wherein various substances are equal mass mixings；
Three, 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 the substrate, the substrate of lipase and the substrate of urease of lysozyme
A kind of or in which several mixture, when the substrate of the enzyme is mixture, wherein various substances are to wait mass mixings；And institute
Select the substrate of enzyme corresponding with the enzyme selected in step 2；Oil used is isooctanol or dodecane；
Four, it is prepared using Pickering microemulsion method using natural true protein solution as the oil-in-water microemulsion of stabilizer
Liquid: after the natural true protein solution that step 2 obtains and the oil-phase solution that step 3 obtains are mixed, 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 and step 3 that the step 2 obtains obtained
The volume ratio of oil-phase solution is 1:(5~15).
Beneficial effects of the present invention:
The present invention, which is successfully realized, prepares stable oil-in-water microemulsion using a variety of amphipathic natural true proteins, and
Based on microemulsion template, native protein microcapsules are prepared on the surface of microemulsion, it is same with good describing property of biology
When, it still can be improved contact area between hydrophobic substrate and amphipathic protease, improve catalytic efficiency；Oil-in-water microemulsion
Traditional a large amount of oil is mutually also changed into water phase, not only ensure that the stability of enzyme, also reduces the ratio of enzyme inactivation；This
Outside, the native protein capsule prepared based on microemulsion template is not only had stable structure, and has good life
Object compatibility, the new model provided for the following simulation for carrying out biological cell.
Detailed description of the invention
Fig. 1 is the exterior appearance figure for the native protein oil-in-water microcapsules that embodiment one obtains；
Fig. 2 is the electron scanning micrograph for the native protein oil-in-water microcapsules that embodiment one obtains；
Fig. 3 is the transmission electron microscope photo for the native protein oil-in-water microcapsules that embodiment one obtains；
Fig. 4 is the atomic force microscopy for the native protein oil-in-water microcapsules that embodiment one obtains；
Fig. 5 is the catalytic efficiency for the native protein oil-in-water microcapsules that embodiment one obtains and pair of common catalyzed efficiency
Than figure, wherein the catalytic efficiency of the 1 native protein oil-in-water microcapsules obtained for embodiment one, 2 urging for common microcapsule
Change efficiency.
Specific embodiment
Specific embodiment 1: one kind of present embodiment, which is based on natural true protein, prepares microcapsules raising interfacial catalysis
What the method for reaction efficiency specifically carried out according to the following steps:
One, the preparation of PBS buffer solution: by NaH2PO4And Na2HPO4It is slow that mixed dissolution is configured to PBS in deionized water
Rush solution；The NaH2PO4With Na2HPO4Mass ratio be 1:(25~46)；The concentration of the PBS buffer solution is 45mmol/
L~55mmol/L, pH value are 8.0~8.2；
Two, natural true protein the preparation of natural true protein solution: is dissolved in the PBS buffer solution that step 1 obtains
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 of lysozyme, lipase and urease or in which several mixtures, when the natural true protein
When for mixture, wherein various substances are equal mass mixings；
Three, 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 the substrate, the substrate of lipase and the substrate of urease of lysozyme
A kind of or in which several mixture, when the substrate of the enzyme is mixture, wherein various substances are to wait mass mixings；And institute
Select the substrate of enzyme corresponding with the enzyme selected in step 2；Oil used is isooctanol or dodecane；
Four, it is prepared using Pickering microemulsion method using natural true protein solution as the oil-in-water microemulsion of stabilizer
Liquid: after the natural true protein solution that step 2 obtains and the oil-phase solution that step 3 obtains are mixed, 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 and step 3 that the step 2 obtains obtained
The volume ratio of oil-phase solution is 1:(5~15).
The microcapsules prepared using the natural true protein capsule of preparation, can substrate in efficient catalytic oil phase, side
What method specifically carried out according to the following steps:
The PBS solution of natural true protein is added in the oil-phase solution containing substrate, is shaken using turbula shaker
After swinging, the microcapsule structure of formation is placed under fluorescence microscope and is observed, and has measured fluorescence intensity change, thus
Determine catalytic efficiency.
Specific embodiment 2: the present embodiment is different from the first embodiment in that: NaH described in step 12PO4
With Na2HPO4Mass ratio be 1:40.Other steps and parameter are same as the specific embodiment one.
Specific embodiment 3: the present embodiment is different from the first and the second embodiment in that: PBS described in step 1
The concentration of buffer solution is 50mmol/L.Other steps and parameter are the same as one or two specific embodiments.
Specific embodiment 4: unlike one of present embodiment and specific embodiment one to three: pH in step 1
Value is 8.1.Other steps and parameter are identical as one of specific embodiment one to three.
Specific embodiment 5: unlike one of present embodiment and specific 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 as one of specific embodiment one to four.
Specific embodiment 6: unlike one of present embodiment and specific 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 as one of specific embodiment one to five.
Specific embodiment 7: unlike one of present embodiment and specific embodiment one to six: will in step 4
Oil-in-water microemulsion stands 40min.Other steps and parameter are identical as one of specific embodiment one to six.
Specific embodiment 8: unlike one of present embodiment and specific embodiment one to seven: institute in step 4
The volume ratio for stating the natural true protein solution that step 2 obtains and the oil-phase solution that step 3 obtains is 1:10.Other steps and
Parameter is identical as one of specific embodiment one to seven.
Beneficial effects of the present invention are verified by following embodiment:
Embodiment one: a kind of method preparing microcapsules raising interfacial catalysis reaction efficiency based on natural true protein is specific
It carries out according to the following steps:
One, the preparation of PBS buffer solution: by NaH2PO4And Na2HPO4It is slow that mixed dissolution is configured to PBS in deionized water
Rush solution；The NaH2PO4With Na2HPO4Mass ratio be 1:30；The concentration of the PBS buffer solution is 45mmol/L, pH value
It is 8.0；
Two, the natural true protein of 0.5mg the preparation of natural true protein solution: is dissolved in what 1.0mL step 1 obtained
In PBS buffer solution, 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 substances are to wait mass mixings；
Three, 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 the mixture of NAG-NAM, 4-methyl umbelliferone ester and urea, wherein respectively
Kind substance is equal mass mixings；Oil used is isooctanol or dodecane；
Four, it is prepared using Pickering microemulsion method using natural true protein solution as the oil-in-water microemulsion of stabilizer
Liquid: after the natural true protein solution that step 2 obtains and the oil-phase solution that step 3 obtains are mixed, 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 and step that the step 2 obtains
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 obtains；As can be seen from the figure
Uniformly (size is between 20~30 μm), translucency is good, illustrates it with apparent Capsules for protein capsule size distribution
Structure, and it is with good stability.
Fig. 2 is the electron scanning micrograph for the native protein oil-in-water microcapsules that embodiment one obtains；From figure
It can be seen that the membrane structure of native protein capsule, thickness is uniform, and illustrates that the stability after its drying is fine.
Fig. 3 is the transmission electron microscope photo for the native protein oil-in-water microcapsules that embodiment one obtains；From figure
It can be seen that the hollow structure of the protein capsule, and there is good membrane structure, the stabilization after equally also illustrating its drying
Property is fine.
Fig. 4 is the atomic force microscopy for the native protein oil-in-water microcapsules that embodiment one obtains；It can from figure
With find out the thickness of the protein capsule in 20 rans, it is with good stability.
Fig. 5 is the catalytic efficiency for the native protein oil-in-water microcapsules that embodiment one obtains and pair of common catalyzed efficiency
Than figure；The wherein catalytic efficiency of the 1 native protein oil-in-water microcapsules obtained for embodiment one, 2 urging for common microcapsule
Change efficiency；It can use efficiency of the catalytic efficiency than no protein microcapsules structure of protein microcapsules structure realization from figure
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 be based on
Natural true protein prepares what the method that microcapsules improve interfacial catalysis reaction efficiency specifically carried out according to the following steps:
One, the preparation of PBS buffer solution: by NaH2PO4And Na2HPO4It is molten that mixed dissolution is configured to PBS buffering in deionized water
Liquid；The NaH2PO4With Na2HPO4Mass ratio be 1:(25~46)；The concentration of the PBS buffer solution be 45mmol/L~
55mmol/L, pH value are 8.0~8.2；
Two, the preparation of natural true protein solution: natural true protein being dissolved in the PBS buffer solution that step 1 obtains,
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 the mixture of lysozyme, lipase and urease, wherein various substances are to wait mass mixings；
Three, 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 of the substrate of lysozyme, the substrate of lipase and substrate of urease
Or in which several mixtures, when the substrate of the enzyme is mixture, wherein various substances are to wait mass mixings；And selected enzyme
Substrate it is corresponding with the enzyme selected in step 2；Oil used is isooctanol or dodecane；
Four, it is prepared using Pickering microemulsion method using natural true protein solution as the oil-in-water microemulsion of stabilizer: will
After the oil-phase solution mixing that the natural true protein solution and step 3 that step 2 obtains obtain, 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 lotion；The oily phase that the natural true protein solution that the step 2 obtains is obtained with step 3
The volume ratio of solution is 1:(5~15).
It a kind of microcapsules is 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 12PO4With Na2HPO4Mass ratio be 1:40.
It a kind of microcapsules is 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 buffer solution described in step 1 is 50mmol/L.
It a kind of microcapsules is 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 1.
It a kind of microcapsules is 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.
It a kind of microcapsules is 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 are urea.
It a kind of microcapsules is 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.
It a kind of microcapsules is 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 and step 3 that step 2 described in step 4 obtains obtain
Volume ratio be 1:10.
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| 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 |
| 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 |
| CN111671727B (en) * | 2020-05-22 | 2022-03-29 | 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 |
| CN104940995A (en) * | 2015-06-29 | 2015-09-30 | 哈尔滨工业大学 | Method for preparing protein hydrogel microspheres by taking bovine serum albumin as element reversed-phase microemulsion |
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| CN1953735A (en) * | 2004-05-18 | 2007-04-25 | 雀巢技术公司 | Oil-in-water emulsion for delivery |
| CN104940995A (en) * | 2015-06-29 | 2015-09-30 | 哈尔滨工业大学 | Method for preparing protein hydrogel microspheres by taking bovine serum albumin as element reversed-phase microemulsion |
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