CN109266636A - A kind of nano enzyme and its preparation method and application - Google Patents
A kind of nano enzyme and its preparation method and application Download PDFInfo
- Publication number
- CN109266636A CN109266636A CN201811119308.2A CN201811119308A CN109266636A CN 109266636 A CN109266636 A CN 109266636A CN 201811119308 A CN201811119308 A CN 201811119308A CN 109266636 A CN109266636 A CN 109266636A
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- Prior art keywords
- lipase
- phosphoric acid
- polyethylene glycol
- acid polyethylene
- nano enzyme
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- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- 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)
- C12N9/16—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
-
- 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
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- C—CHEMISTRY; METALLURGY
- 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)
Abstract
The invention belongs to enzyme engineering field, a kind of nano enzyme and its preparation method and application is refered in particular to.The present invention is formed in phosphoric acid polyethylene glycol-lipase mixt that oily phase and water phase can disperse using phosphoric acid polyethylene glycol and lipase, then phosphoric acid polyethylene glycol-lipase is coated with metal organic frame (MOF) structure, free lipase is coated on generation nanometer lipase in MOF shell, the nano enzyme energy efficient catalytic grease and organic ester hydrolysis, and nano enzyme is easily recycled after reacting, it can reuse, the rate of recovery is high, greatly enhances catalytic performance of the lipase in industrial application.
Description
Technical field
The invention belongs to enzyme engineering field, refer in particular to a kind of metal framework coated fat enzyme-phosphoric acid polyethylene glycol nano enzyme and
Its method and the utilization in grease hydrolysis.
Background technique
As the application of bioengineering, the continuous development of enzyme engineering research, lipase is constantly explored.Lipase is not only
Structure enantiospecific and Substratspezifitaet with higher, and lipase itself is also a kind of efficient environmentally friendly urge
Agent (Mitragotri S., Burke P.A.and Langer R.Overcoming the challenges in
administering biopharmaceuticals:formulation and delivery
strategies.Nation.Review,Drug Discover.,2014,13(20):655–672.).Lipase is due to itself
Selectivity is high, catalytic activity is high and is widely applied.But the protein that the essence of lipase is made of amino acid, activity are held
Vulnerable to pH, temperature, the factors such as substrate specificity influence, and in strong acid, highly basic is easy deactivation under conditions of high temperature, is adding
The purification that the enzyme that entering lipase reverse should dissociate afterwards is difficult to recycle and enzyme is easy to be mixed into product to product causes to be stranded
It is difficult.Catalytic activity and tolerance that lipase is improved using the method for immobilized lipase become the hot spot of development.Magnetism is received
Rice corpuscles is often used in fatty enzyme immobilizatio, reaches raising catalytic activity by the way that lipase is fixed on nano grain surface
Purpose (Chiriac H, Moga A E, Iacob G.Amorphous magnetic micropheres for
Biomedical applications, J.Magn.Magn.Mater., 2005,293:28-32.).But with the reaction time
Increase, lipase and organic phase constantly contact, and the lipase of nanoparticle surface constantly reduces sharp to the circulation of lipase
With impacting.Therefore, it is badly in need of the Ke Xunhuanliyong high-activity nano lipase of research.
Summary of the invention
The present invention devises a kind of new nanometer lipase preparation method.Using metal organic frame as protective layer, in water-soluble
In liquid, using the method for the binding force coated fat enzyme between 2-methylimidazole and metal ion, stable tool is successfully synthesized
There is the nanometer lipase (Lipase-mPEG@ZIF-8) of greater activity.This nanometer lipase (Lipase-mPEG@ZIF-8)
Size is 10 μm.It can be used for grease hydrolysis.
The present invention is formed in the phosphoric acid polyethylene glycol-that oily phase and water phase can disperse using phosphoric acid polyethylene glycol and lipase
Then lipase mixt coats phosphoric acid polyethylene glycol-lipase with metal organic frame (MOF) structure, will swim
From lipase be coated in MOF shell generation nanometer lipase, the nano enzyme energy efficient catalytic grease and organic ester hydrolysis,
And nano enzyme is easily recycled after reacting, and can be reused, the rate of recovery is high, therefore our method greatly enhances rouge
Catalytic performance of the fat enzyme in industrial application.
The specific technical solution that the present invention uses is as follows:
A kind of preparation method of metal framework coated fat enzyme-phosphoric acid polyethylene glycol nano enzyme carries out as steps described below:
In aqueous solution, lipase and phosphoric acid polyethylene glycol is added, first time magnetic agitation mixes solution equal at room temperature
It is even, then above-mentioned mixed solution is slowly added into 2-methylimidazole solution, carries out second of magnetic agitation；Add acetic acid
Zinc solution carries out third time magnetic agitation, forms metal-organic framework in reaction solution；After third time stirs, room
Temperature is stood, and the nano enzyme of metal-organic framework cladding can be obtained.
Wherein the mass ratio of the lipase and phosphoric acid polyethylene glycol is 1:1-3:1, optimum quality ratio 2:1.
The time of the magnetic agitation of first time at room temperature is 30-90min, preferably 60min.
Described second and the third time magnetic agitation time be 30min.
The wherein zinc acetate and 2-methylimidazole molar ratio 1:3-1:5, optimum mole ratio 1:4.
The wherein zinc acetate and lipase mass ratio 3:1-6:1, optimum quality ratio 4.9:1.
In reaction solution after forming metal-organic framework, lipase concentration 0.4-1mgmL-1, optium concentration
For 0.6mgmL-1。
Wherein the time of repose is 10-20h, and best time of repose is 12h.
Here we report a kind of completely new metal framework coated fat enzyme-phosphoric acid polyethylene glycol nano enzyme preparation side
Phosphoric acid polyethylene glycol is added in method, our first passages, and main purpose is the control to nano enzyme pattern, protection nanometer fat
The activity of enzyme and the intermiscibility for increasing ester.Reduce requirement of the nano enzyme to solvent；Furthermore the present invention has machine frame using metal for the first time
Frame (ZIF-8) coated fat enzyme-phosphoric acid polyethylene glycol forms nano enzyme, and the nano enzyme is by introducing phosphoric acid polyethylene glycol to pattern
Regulated and controled, is combined with being coordinated between 2-methylimidazole and metal ion, obtain novel using ZIF-8 as shell, the poly- second two of phosphoric acid
Alcohol-lipase is the nanometer lipase (Fig. 1) of core, and this lipase active is higher, has higher hydrolysis effect to grease, and free
Lipase is compared, active higher (table 1).In addition, by being ground to porcine pancreatic lipase and lipase from candida sp nanometer immobilization
Study carefully, it is presumed that, present invention can apply to the nanometer immobilizations of different types of lipase.
Detailed description of the invention
Fig. 1 is the scanning electron microscope (SEM) photograph that lipase-mPEG@ZIF-8 is prepared in embodiment 1.Uniform particle sizes are hollow knot
Structure, compared with Lipase@ZIF-8, specific surface area increases.
Specific embodiment
The synthesis of Lipase-mPEG@ZIF-8
Embodiment 1 (the best preparation method of Lipase-mPEG@ZIF-8): in 10mL aqueous solution, it is poly- that 15mg phosphoric acid is added
Ethylene glycol and 30mg lipase (lipase is lipase from candida sp, lipase and phosphoric acid polyethylene glycol mass ratio 2:1), room temperature
Lower magnetic agitation 60min is uniformly mixed solution.At room temperature, the mixed solution of lipase and phosphoric acid polyethylene glycol is added to
3.2mmol 2-methylimidazole solution, after magnetic agitation 30min, by 0.8mmol Zn (OAc)2Solution is added to lipase-phosphorus
(wherein Zn (OAc) in the 2-methylimidazole mixed solution of acid polyethylene glycol2Molar ratio with 2-methylimidazole is 1:4, Zn
(OAc)2It is 4.9:1 with lipase mass ratio, the concentration of lipase is 0.6mgmL-1), after magnetic agitation 30min, in room temperature
The nano enzyme of metal-organic framework cladding can be obtained in static placement 12h.
Embodiment 2: in 10mL aqueous solution, (lipase is false silk for addition 10mg phosphoric acid polyethylene glycol and 30mg lipase
Yeast-lipase, lipase and phosphoric acid polyethylene glycol mass ratio 3:1), magnetic agitation 60min is uniformly mixed solution at room temperature.
At room temperature, the mixed solution of lipase and phosphoric acid polyethylene glycol is added to 3.2mmol 2-methylimidazole solution, magnetic force stirs
After mixing 30min, by 0.8mmol Zn (OAc)2Solution is added to lipase-phosphoric acid polyethylene glycol 2-methylimidazole mixed solution
In (wherein Zn (OAc)2Molar ratio with 2-methylimidazole is 1:4, Zn (OAc)2It is 4.9:1, lipase with lipase mass ratio
Concentration be 0.6mgmL-1), in the static placement 12h of room temperature after magnetic agitation 30min, metal-organic framework packet can be obtained
The nano enzyme covered.
Embodiment 3: in 10mL aqueous solution, (lipase is false silk for addition 15mg phosphoric acid polyethylene glycol and 30mg lipase
Yeast-lipase, lipase and phosphoric acid polyethylene glycol mass ratio 2:1), magnetic agitation 60min is uniformly mixed solution at room temperature.
The mixed solution of lipase and phosphoric acid polyethylene glycol is added to 3.2mmol 2-methylimidazole solution, after magnetic agitation 30min,
By 0.8mmol Zn (OAc)2Solution is added to (wherein Zn in lipase-phosphoric acid polyethylene glycol 2-methylimidazole mixed solution
(OAc)2Molar ratio with 2-methylimidazole is 1:4, Zn (OAc)2It is 4.9:1 with lipase mass ratio, the concentration of lipase is
1mg·mL-1), in the static placement 12h of room temperature after magnetic agitation 30min, the nanometer of metal-organic framework cladding can be obtained
Enzyme.
Embodiment 4: in 10mL aqueous solution, (lipase is false silk for addition 15mg phosphoric acid polyethylene glycol and 30mg lipase
Yeast-lipase, lipase and phosphoric acid polyethylene glycol mass ratio 2:1), magnetic agitation 60min is uniformly mixed solution at room temperature.
At room temperature, the mixed solution of lipase and phosphoric acid polyethylene glycol is added to 3.2mmol 2-methylimidazole solution, magnetic agitation
After 30min, by 0.64mmol Zn (OAc)2Solution is added to lipase-phosphoric acid polyethylene glycol 2-methylimidazole mixed solution
In (wherein Zn (OAc)2Molar ratio with 2-methylimidazole is 1:5, Zn (OAc)2It is 4:1 with lipase mass ratio, lipase
Concentration is 0.6mgmL-1), in the static placement 12h of room temperature after magnetic agitation 30min, metal-organic framework cladding can be obtained
Nano enzyme.
Embodiment 5: in 10mL aqueous solution, (lipase is false silk for addition 24mg phosphoric acid polyethylene glycol and 48mg lipase
Yeast-lipase, lipase and phosphoric acid polyethylene glycol mass ratio 2:1), magnetic agitation 60min is uniformly mixed solution at room temperature.
At room temperature, the mixed solution of lipase and phosphoric acid polyethylene glycol is added to 3.2mmol 2-methylimidazole solution, magnetic force stirs
After mixing 30min, by 0.8mmol Zn (OAc)2Solution is added to lipase-phosphoric acid polyethylene glycol 2-methylimidazole mixed solution
In (wherein Zn (OAc)2Molar ratio with 2-methylimidazole is 1:4, Zn (OAc)2It is 3:1 with lipase mass ratio, lipase
Concentration is 0.96mgmL-1), after magnetic agitation 30min, in the static placement 12h of room temperature, metal-organic framework packet can be obtained
The nano enzyme covered.
Embodiment 6: in 10mL aqueous solution, 24.5mg phosphoric acid polyethylene glycol and 24.5mg lipase is added, and (lipase is
Lipase from candida sp, lipase and phosphoric acid polyethylene glycol mass ratio 1:1), magnetic agitation 60min keeps solution mixing equal at room temperature
It is even.At room temperature, the mixed solution of lipase and phosphoric acid polyethylene glycol is added to 3.2mmol 2-methylimidazole solution, magnetic force
After stirring 30min, by 0.8mmol Zn (OAc)2It is molten that solution is added to the mixing of lipase-phosphoric acid polyethylene glycol 2-methylimidazole
(wherein Zn (OAc) in liquid2Molar ratio with 2-methylimidazole is 1:4, Zn (OAc)2It is 6:1, lipase with lipase mass ratio
Concentration be 0.49mgmL-1), after magnetic agitation 30min, in the static placement 12h of room temperature, metal-organic framework can be obtained
The nano enzyme of cladding.
The research of embodiment 7:Lipase-mPEG@ZIF-8 hydrolysis property
The preparation gained Lipase-mPEG@ZIF-8 of step 1 Example 1, is made into 1mgmL-1Lipase-mPEG@
The aqueous solution of ZIF-8, is put in reactor.
Grease is added in reactor by step 2.
Step 3 at room temperature, magnetic agitation 10min.
Step 4 observes grease hydrolysis situation, weighs the quality of fatty acid after hydrolysis.
Table 1 is the hydrolysis property summary sheet of Lipase-mPEG@ZIF-8 and free-fat enzyme.It is obtained from gained oleic acid quality
Know, compared with free-fat enzyme, grease hydrolysis performance gets a promotion the hydrolysis property of Lipase-mPEG@ZIF-8.
Table 1
Claims (7)
1. a kind of nano enzyme, which is characterized in that prepare with the following method: being formed in oil using phosphoric acid polyethylene glycol and lipase
Phosphoric acid polyethylene glycol-the lipase mixt that mutually can disperse with water phase, it is then poly- to phosphoric acid with metal-organic framework
Ethylene glycol-lipase is coated, and free lipase is coated on generation nanometer lipase in MOF shell.
2. a kind of nano enzyme as described in claim 1, which is characterized in that the size of the nano enzyme is 10 μm.
3. a kind of nano enzyme as described in claim 1, which is characterized in that the nano enzyme is with metal organic frame ZIF-8
For shell, phosphoric acid polyethylene glycol-lipase is the nanometer lipase of core.
4. a kind of purposes of nano enzyme as described in claim 1, which is characterized in that hydrolyzed for grease.
5. a kind of preparation method of nano enzyme as described in claim 1, which is characterized in that utilize phosphoric acid polyethylene glycol and fat
Enzyme is formed in the oily phosphoric acid polyethylene glycol-lipase mixt that mutually can disperse with water phase, then uses metal organic frame knot
Structure coats phosphoric acid polyethylene glycol-lipase, and free lipase is coated on generation nanometer lipase in MOF shell,
Specific step is as follows:
In aqueous solution, lipase and phosphoric acid polyethylene glycol is added, solution is uniformly mixed by first time magnetic agitation at room temperature, so
Above-mentioned mixed solution is slowly added into 2-methylimidazole solution afterwards, carries out second of magnetic agitation；It is molten to add zinc acetate
Liquid carries out third time magnetic agitation, forms metal-organic framework in reaction solution；After third time stirs, room temperature is quiet
It sets, the nano enzyme of metal-organic framework cladding can be obtained.
6. a kind of preparation method of nano enzyme as claimed in claim 5, which is characterized in that the lipase and the poly- second of phosphoric acid
The mass ratio of glycol is 1:1-3:1；The time of the magnetic agitation of first time at room temperature is 30-90min；Described second and the
The magnetic agitation time is 30min three times；The zinc acetate and 2-methylimidazole molar ratio is 1:3-1:5；The zinc acetate
It is 3:1-6:1 with lipase mass ratio；In reaction solution after forming metal-organic framework, lipase concentration 0.4-
1mg·mL-1；The time of repose is 10-20h.
7. a kind of preparation method of nano enzyme as claimed in claim 6, which is characterized in that the lipase and the poly- second of phosphoric acid
The mass ratio of glycol is 2:1；The time of the magnetic agitation of first time at room temperature is 60min；The wherein zinc acetate and 2-
Methylimidazole molar ratio is 1:4；The zinc acetate and lipase mass ratio is 4.9:1；After forming metal-organic framework
Reaction solution in, lipase concentration 0.6mgmL-1；The time of repose is 12h.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111569053A (en) * | 2020-03-13 | 2020-08-25 | 中国科学院长春应用化学研究所 | Nano enzyme for enhancing immunotherapy by regulating and controlling tumor metabolic behaviors and preparation method and application thereof |
| CN111608623A (en) * | 2020-04-27 | 2020-09-01 | 夏文杰 | Biological nano preparation applied to oil and gas resource exploitation |
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| CN111569053A (en) * | 2020-03-13 | 2020-08-25 | 中国科学院长春应用化学研究所 | Nano enzyme for enhancing immunotherapy by regulating and controlling tumor metabolic behaviors and preparation method and application thereof |
| CN111608623A (en) * | 2020-04-27 | 2020-09-01 | 夏文杰 | Biological nano preparation applied to oil and gas resource exploitation |
| CN111608623B (en) * | 2020-04-27 | 2022-06-28 | 夏文杰 | Biological nano preparation applied to oil and gas resource exploitation |
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