CN106632806B - Lipase cooperates with the Pickering lotions of recessed soil stabilization and using Pickering lotion as the preparation method of matrix polymerization microballoon containing enzyme - Google Patents
Lipase cooperates with the Pickering lotions of recessed soil stabilization and using Pickering lotion as the preparation method of matrix polymerization microballoon containing enzyme Download PDFInfo
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- CN106632806B CN106632806B CN201610843389.5A CN201610843389A CN106632806B CN 106632806 B CN106632806 B CN 106632806B CN 201610843389 A CN201610843389 A CN 201610843389A CN 106632806 B CN106632806 B CN 106632806B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/12—Esters of monohydric alcohols or phenols
- C08F220/14—Methyl esters, e.g. methyl (meth)acrylate
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- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
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Abstract
The Pickering lotions of recessed soil stabilization are cooperateed with the invention discloses lipase and using this Pickering lotion as the preparation method of matrix polymerization microballoon containing enzyme, recessed soil is scattered in phosphate buffer and forms dispersion liquid, dispersion liquid is mixed with lipase enzyme solution again, obtains water phase；With 2,2 azodiisobutyronitrile of initiator（AIBN）, methyl methacrylate (MMA) and ethylene glycol dimethacrylate (EGDMA) be dissolved in toluene, obtain oil phase；After oil phase is mixed with water conjunction, high speed is beaten, that is, forms oil-in-water type (O/W) Pickering lotions that enzyme is stablized with recessed earth cooperation；After Pickering lotion letting nitrogen in and deoxidizing, thermostat polymerization is moved to；After reaction, it is cooled to room temperature, it is multiple with phosphate buffer washed product, obtain microballoon containing enzyme.The present invention by the solidification process of enzyme it is synchronous with emulsion polymerization process carry out, biocatalyst lipase not only plays intrinsic catalytic action, and play emulsifier in lotion is formed, thus obtained microsphere catalytic performance is imparted, a new approach is provided to enzyme immobilizatio.
Description
Technical field
Natural minerals and biocatalyst collaboration emulsification and the preparation for aggregating into microballoon are utilized the present invention relates to a kind of
Method belongs to natural mineral resource and utilizes technical field.
Background technology
Pickering lotions be it is a kind of by solid particle instead of traditional organic surface active agent stabilized emulsion system
New Emulsion, more general lotion are more stable.Three-dimensional net structure is often presented when forming lotion in the emulsifier of clay class, stablizes
Property higher；Since clay is natural mineral, environmental protection, large-scale application cost performance higher；It is in layer compared to the fixed particle of spherical shape
The only seldom amount of clay of shape and threadiness can be covered in spherome surface, it is possible to reduce the dosage of emulsifier, it is cost-effective.
Enzyme is the protein for having catalytic activity, but when enzyme is directly catalyzed, affected by environment larger, fixation techniques for enzyme
Therefore grow up.The application of immobilised enzymes can be improved the stability of enzyme and recycle number.
Invention content
Present invention aims at：A kind of lipase is provided and cooperates with the Pickering lotions of recessed soil stabilization and with this
Pickering lotions be matrix polymerization microballoon containing enzyme preparation method, using this protein with catalytic activity of enzyme with it is recessed
Earth cooperation prepares Pickering lotions, and is matrix polymerization into microballoon using this Pickering lotion, enzyme is directly fixed on micro-
Ball surface so that the enzyme in system not only plays emulsifier, while also assigning microballoon catalytic performance, is provided to enzyme immobilizatio
New approach.
Technical solution of the invention is that the preparation method of the Pickering lotions includes the following steps：
（1）Recessed soil is scattered in phosphate buffer and forms dispersion liquid, dispersion liquid again with certain density lipase enzyme
Liquid mixes, and obtains water phase, also known as recessed native dispersion liquid；The lipase enzyme solution refers to that lipase is separately dissolved in phosphate buffer
The solution of formation, abbreviation enzyme solution.
（2）With initiator 2,2- azodiisobutyronitriles（AIBN）, methyl methacrylate (MMA) and ethylene glycol dimethyl third
Olefin(e) acid ester (EGDMA) is dissolved in toluene, obtains oil phase；
（3）By step（2）Oil phase and step（1）Water phase mixing after, high speed be beaten, that is, form enzyme and recessed earth cooperation be steady
Fixed oil-in-water type (O/W) Pickering lotions.
Wherein, a concentration of 1.5-2.5w/v% of recessed native dispersion liquid, a concentration of 0.065-0.135wt% of enzyme solution, in water phase point
Dispersion liquid is 4 with enzyme solution mixed volume ratio:1.
Wherein, in 2.0-4.0mL toluene, 0.015-0.025g initiators 2,2- azodiisobutyronitriles is added（AIBN）、
0.65-2.35mL methyl methacrylates (MMA) and 0.35-0.65mL ethylene glycol dimethacrylates (EGDMA), fill
Divide mixing, obtains oil phase.
Wherein, it is by the preparation method of matrix polymerization microballoon containing enzyme of Pickering lotions：Pickering lotions lead to nitrogen
After deoxygenation, thermostat polymerization is moved to；After reaction, it is cooled to room temperature, it is multiple with phosphate buffer washed product, it must contain
Enzyme microballoon.
Wherein, polymerisation 1.0-3.0h in 50 DEG C of oil bath pan.
The advantage of the invention is that:
1, enzyme immobilizatio process is synchronous with emulsion polymerization carries out, this provides a new way to enzyme immobilizatio method
Diameter；
2, enzyme generally only cooks biocatalyst, but the enzyme in the present invention not only plays catalyst, while also in breast
Liquid plays emulsifier in stablizing, and gained lotion is made more to stablize；
3, thus obtained microsphere catalysis is assigned, the application range of Pickering lotions has been widened.
Specific implementation mode
The technical solution further illustrated the present invention with reference to embodiment, these embodiments should not be understood as pair
The limitation of technical solution.
Embodiment 1：0.15g recessed soil ultrasonic disperse 5min in the phosphate buffer that 8mL pH value is 7.4 are weighed, are added
The lipase enzyme solution 2mL of 0.065wt% is uniformly mixed (its concave soil is 1.5w/v%), obtains water phase；It is burnt in the round bottom of a 50mL
4.0mL toluene, 0.015g AIBN, 0.65mL MMA and 0.35mL EGDMA are sequentially added in bottle, so that it is sufficiently mixed, obtains
Oil phase；Water phase and oil phase are poured into round-bottomed flask, are beaten with 10000 revs/min of speed with the adjustable high speed homogenate machines of FS-1
3min obtains Pickering lotions.
Pickering lotion letting nitrogen in and deoxidizing 30min, round-bottomed flask is transferred in 50 DEG C of oil bath pan and carries out polymerisation
3.0h；After reaction, wait for that it is cooled to room temperature, it is multiple with phosphate buffer washed product, obtain microballoon containing enzyme i.e. sample S1.
Embodiment 2：The phosphate buffer ultrasonic disperse 5min that the recessed soil of 0.20g is 7.4 in 8mL pH value is weighed, is added
The enzyme solution 2mL of 0.100wt% is uniformly mixed (its concave soil is 2.0w/v%), obtains water phase；In the round-bottomed flask of a 50mL according to
Secondary addition 3.0mL toluene, 0.020g AIBN, 1.5mL MMA and 0.5mL EGDMA, make it be sufficiently mixed, obtain oil phase；By water
It is mutually poured into the round-bottomed flask with oil phase, 3min is beaten with 10000 revs/min of speed with FS-1 adjustable high speed homogenate machines, is obtained
To Pickering lotions.
Pickering lotion letting nitrogen in and deoxidizing 30min, round-bottomed flask is transferred in 50 DEG C of oil bath pan and carries out polymerisation
2h；After reaction, wait for that it is cooled to room temperature, it is multiple with phosphate buffer washed product, obtain microballoon containing enzyme i.e. sample S2.
Embodiment 3：The phosphate buffer ultrasonic disperse 5min that the recessed soil of 0.25g is 7.4 in 8mL pH value is weighed, is added
The enzyme solution 2mL of 0.135wt% is uniformly mixed (its concave soil is 2.5w/v%), obtains water phase；In the round-bottomed flask of a 50mL according to
Secondary addition 2.0mL toluene, 0.025g AIBN, 2.35mL MMA and 0.65mL EGDMA, make it be sufficiently mixed, obtain oil phase；It will
Water phase and oil phase pour into the round-bottomed flask, and 3min is beaten with 10000 revs/min of speed with the adjustable high speed homogenate machines of FS-1,
Obtain Pickering lotions.
Pickering lotion letting nitrogen in and deoxidizing 30min, round-bottomed flask is transferred in 50 DEG C of oil bath pan and carries out polymerisation
1h；After reaction, wait for that it is cooled to room temperature, it is multiple with phosphate buffer washed product, obtain microballoon containing enzyme i.e. sample S3.
The enzyme activity determination of above-mentioned sample selects Copoloid method：1.7mL phosphate buffer solutions and 0.6mL olive oil are taken, is put
Enter and preheat 5min in 37 °C of constant temperature water bath magnetic stirring apparatus, 0.03g microballoons containing enzyme are added, magnetic agitation 10min is added immediately
4.5mL toluene stirs 2min, terminates reaction, while extracting the aliphatic acid of generation；Above-mentioned solution is moved in centrifuge tube,
4000r/min centrifuges 10min, organic phase and water phase layering clarification；Upper organic phase 4mL is taken, 1mL color developing agents are added, magnetic force stirs
It is moved in centrifuge tube after mixing 3min, 4000r/min centrifuges 10min, takes supernatant, it is surveyed in 710nm wavelength with spectrophotometer
Light absorption value；It is reference to prepare the blank solution without lipase in the same way, compares aliphatic acid absorbance working curve, that is, asks
Obtain the concentration of aliphatic acid.
Survey the enzyme activity of lipase as Copoloid method investigate the enzyme activity of the microballoon containing enzyme obtained by embodiment 1-3, thermal stability,
Storage stability and recycling situation.Thermal stability is will to contain enzyme microballoon to be placed in 50 °C of environment, it is surveyed every a hour
Enzyme activity compares 70% time used below that enzyme activity is down to initial enzyme activity.Storage stability is will to contain enzyme microballoon to be placed in 4 °C of environment
In, its enzyme activity is every other day surveyed, 70% time used below that enzyme activity is down to initial enzyme activity is compared.It recycles and mainly compares
Use the opposite enzyme activity after 1 time.The experimental results are shown inthe following table：
。
Claims (5)
1. lipase cooperates with the Pickering lotions of recessed soil stabilization, it is characterized in that the preparation method of the Pickering lotions includes
Following steps：
（1）Recessed soil is scattered in phosphate buffer and forms dispersion liquid, dispersion liquid is mixed with lipase enzyme solution again, obtains water phase,
Also known as recessed native dispersion liquid；The lipase enzyme solution refers to that lipase is separately dissolved in the solution formed in phosphate buffer, letter
Claim enzyme solution；
（2）With initiator 2,2- azodiisobutyronitriles AIBN, methyl methacrylate MMA and ethylene glycol dimethacrylate
EGDMA is dissolved in toluene, obtains oil phase；
（3）Step（2）Oil phase and step（1）Water phase mixing after, high speed is beaten, that is, forms the water that enzyme and recessed earth cooperation are stablized
Oil-in Pickering lotions.
2. lipase according to claim 1 cooperates with the Pickering lotions of recessed soil stabilization, it is characterized in that：Recessed soil dispersion
A concentration of 1.5-2.5w/v% of liquid, a concentration of 0.065-0.135wt% of enzyme solution, dispersion liquid is with enzyme solution mixed volume ratio in water phase
4:1。
3. lipase according to claim 1 cooperates with the Pickering lotions of recessed soil stabilization, it is characterized in that：In the step
Suddenly（2）In, the preparation process of the oil phase is as follows：In 2.0-4.0mL toluene, 0.015-0.025g initiators 2 are added, 2- is even
Nitrogen bis-isobutyronitrile AIBN, 0.65-2.35mL methyl methacrylate MMA and 0.35-0.65mL ethyleneglycol dimethacrylate
Ester EGDMA, is sufficiently mixed, and obtains oil phase.
4. using the Pickering lotions obtained by claim 1 as the preparation method of matrix polymerization microballoon containing enzyme, it is characterized in that：
After Pickering lotion letting nitrogen in and deoxidizing, thermostat polymerization is moved to；After reaction, it is cooled to room temperature, uses phosphate buffer
Washed product is multiple, obtains microballoon containing enzyme.
5. according to claim 4 using Pickering lotions as the preparation method of matrix polymerization microballoon containing enzyme, feature
It is：Polymerisation 1.0-3.0h in 50 DEG C of oil bath pan.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610843389.5A CN106632806B (en) | 2016-09-23 | 2016-09-23 | Lipase cooperates with the Pickering lotions of recessed soil stabilization and using Pickering lotion as the preparation method of matrix polymerization microballoon containing enzyme |
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| CN201610843389.5A CN106632806B (en) | 2016-09-23 | 2016-09-23 | Lipase cooperates with the Pickering lotions of recessed soil stabilization and using Pickering lotion as the preparation method of matrix polymerization microballoon containing enzyme |
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| CN106632806B true CN106632806B (en) | 2018-09-07 |
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| CN103204966B (en) * | 2013-04-11 | 2015-06-10 | 江苏大学 | Method for preparing magnetic/hollow double-shell layer print adsorbent by emulsion polymerization |
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Effective date of registration: 20191213 Address after: 310017 room 701, building 2, Xizi international building, east station, Jianggan District, Hangzhou City, Zhejiang Province Patentee after: Hangzhou Shizi Hede Biotechnology Co., Ltd Address before: 223005 Huaian city of Jiangsu Province, Mei Cheng Road No. 1 Patentee before: Huaijin Polytechnical College |