CN106542568A - A kind of immobilised enzymes, fixed enzyme vector and preparation method thereof - Google Patents
A kind of immobilised enzymes, fixed enzyme vector and preparation method thereof Download PDFInfo
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- CN106542568A CN106542568A CN201610964102.4A CN201610964102A CN106542568A CN 106542568 A CN106542568 A CN 106542568A CN 201610964102 A CN201610964102 A CN 201610964102A CN 106542568 A CN106542568 A CN 106542568A
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G9/00—Compounds of zinc
- C01G9/02—Oxides; Hydroxides
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01F—COMPOUNDS OF THE METALS BERYLLIUM, MAGNESIUM, ALUMINIUM, CALCIUM, STRONTIUM, BARIUM, RADIUM, THORIUM, OR OF THE RARE-EARTH METALS
- C01F7/00—Compounds of aluminium
- C01F7/78—Compounds containing aluminium and two or more other elements, with the exception of oxygen and hydrogen
- C01F7/784—Layered double hydroxide, e.g. comprising nitrate, sulfate or carbonate ions as intercalating anions
- C01F7/785—Hydrotalcite
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- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01G—COMPOUNDS CONTAINING METALS NOT COVERED BY SUBCLASSES C01D OR C01F
- C01G45/00—Compounds of manganese
- C01G45/02—Oxides; Hydroxides
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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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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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
- 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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- 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)
-
- C—CHEMISTRY; METALLURGY
- C01—INORGANIC CHEMISTRY
- C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
- C01P2004/00—Particle morphology
- C01P2004/80—Particles consisting of a mixture of two or more inorganic phases
Abstract
The invention discloses a kind of immobilised enzymes, fixed enzyme vector and preparation method thereof, fixed enzyme vector includes graphene oxide and modifies inorganic nanoparticles containing polyelectron group in the surface of graphene oxide；Fixed enzyme vector is fully to react to form inorganic nanoparticles and modify by the dressing agent in solvent and surfactant to be obtained in surface of graphene oxide, and immobilised enzymes then includes fixed enzyme vector and the lipase being fixed on fixed enzyme vector.On the one hand its surface has substantial amounts of oxygen-containing functional group higher with enzyme Percentage bound and enzyme can be firmly fixed to carrier surface fixed enzyme vector of the present invention, improves the operational stability of enzyme；On the other hand there is large specific surface area, is that immobilised enzymes provides sufficient catalysis interface environments and mass transfer in catalytic process；And, fixed enzyme vector inhibits reunion of the graphene oxide during Reusability, reduces enzyme and is covered by graphite linings, improves the repeat performance of enzyme.
Description
Technical field
The present invention relates to enzyme technique for fixing, more particularly, to a kind of immobilised enzymes, fixed enzyme vector and preparation method thereof.
Background technology
Enzyme as the biocatalyst with special catalysis, as which is catalyzed high efficiency, high selectivity and reaction bar
Part is gentle etc., and property is widely used in the fields such as bioengineering, food industry, medicine, fine chemical industry.Lipase is various
Biological enzyme reaction includes：Enantioselective hydrolysis and esterification, chiral resolution, enantiomer monomer are prepared and macromolecular polymeric reaction
Deng the enzyme of generally existing.But the application of resolvase still suffers from many problems, such as in storage and course of reaction enzyme easy in inactivation or
Denaturation and cause poor operational stability and reusability；In addition, enzyme-to-substrate and product are not readily separated after enzymatic reaction,
Cause product purity not high and increased production cost.In order to solve the problems, such as enzyme, enzyme is fixed on different carriers by people
In material, such as porous carrier materials (mesoporous silicon, mesoporous TiO2Deng), the carrier containing polyelectron group (hydrotalcite, polysaccharide, carbon
Material etc.).For porous material has a disadvantage in that, its limited hole size is easily restricted mass transfer, and this can cause reaction to be produced
The accumulation of thing and cause that the enzyme that is fixed in hole is more difficult to be contacted with substrate.For the carrier containing polyelectron group, the logical physics of enzyme is inhaled
Attached or covalent bond is fixed with the polyelectron Interaction of substituents of carrier surface.But the deficiency of this carrier material is limited
Specific surface area when excessive enzyme is fixed, the Coulomb repulsion increase between enzyme molecule.Therefore in general, in order to improve urging for enzyme
Change performance, immobilised enzymes need to have it is following some：1) activated centre of enzyme can be fully contacted substrate；2) enzyme and carrier material
It is completely embedded；3) carrier material has good biocompatibility；4) carrier material has enough specific surface areas.
In order to solve the problems, such as resolvase, there has been proposed many solutions：Publication No. CN104569099A
Chinese patent application disclose a kind of quick detection phenol electrode and preparation method thereof, using 1- amino pyrene modify oxygen reduction fossil
Black alkene is carrier material, fixes tyrosinase using glutaraldehyde cross-linking, and the modification of 1- amino pyrenes causes redox graphene table
Face has amino, and the crosslinking for being more beneficial for enzyme is fixed and the high-specific surface area of Graphene provides place to react, and its electric conductivity has
Beneficial to the detection sensitivity of electrode；The Chinese patent application of Publication No. CN105154428A discloses a kind of carboxylated three-dimensional to be had
Sequence mesoporous carbon-lysozyme composite and its preparation method and application, lysozyme is by covalent cross-linking in carboxylated three-dimensional order
In mesoporous carbon, which fixes higher enzymatic activity, good stability and repeatable utilization.The patent with carboxylated three-dimensional ordered mesoporous carbon is
Carrier, three-dimensional ordered mesoporous carbon have that aperture is homogeneous, the neat and orderly unique knot for having wicket connection and hole between in duct
Structure, this is more beneficial for transmission of the enzyme molecule in duct and makes which more accessible to enzymatic activity point.Authorization Notice No. is
The Chinese patent of CN100359324C discloses a kind of biologic sensor enzyme functional susceptivity film containing stripped MnO 2 and its system
Preparation Method, using stripped MnO 2 specific surface area it is big, Active sites are more, negatively charged and have the spy such as satisfactory electrical conductivity
Point immobilized enzyme.
Although above-mentioned patent application a certain degree of can solve the problems, such as resolvase, still there is resolvase and fix
Property it is poor, be unfavorable for improve enzyme activity and operational stability.
The content of the invention
It is an object of the invention to overcome above-mentioned technical deficiency, a kind of immobilised enzymes, fixed enzyme vector and its system are proposed
Preparation Method, in solution prior art, fixed enzyme vector is poor to the stationarity of resolvase, be unfavorable for the activity of raising enzyme and operation
The technical problem of stability.
To reach above-mentioned technical purpose, technical scheme provides a kind of fixed enzyme vector, including graphite oxide
Alkene and modify inorganic nanoparticles containing polyelectron group in the surface of graphene oxide.
Preferably, the inorganic nanoparticles are including but not limited in zinc oxide, manganese dioxide, hydrotalcite, titanium dioxide
One or more.
Preferably, the graphene oxide and the mass ratio of inorganic nanoparticles are 7:3.
Meanwhile, the present invention also provides a kind of preparation method of fixed enzyme vector, comprises the steps：
(1) graphene oxide is added in solvent, be subsequently adding dressing agent or surfactant, ultrasonically treated 1 hour；
(2) surfactant is added in the mixed liquor that dressing agent is added in step (1) or surface is added in step (1)
Dressing agent is added in the mixed liquor of activating agent, stirring makes surfactant fully react with dressing agent and formed to modify in oxidation stone
The inorganic nanoparticles on black alkene surface；
(3) wash, be dried, obtain final product.
Preferably, in the step (1) solvent be deionized water, the dressing agent that adds be zinc acetate, the step (2) is
The mixed solution and sodium citrate of NaOH and ethylene glycol are sequentially added under stirring, it is after stirring 1h, anti-at 100 DEG C
Answer 12 hours.
Preferably, in the step (1) solvent be isopropanol, add dressing agent be MnCl2·4H2O, the step (2)
To add ammonia spirit, ultrasonic agitation to react 12 hours at 120 DEG C after 30 minutes.
Preferably, in the step (1) solvent be deionized water, add surfactant be DBSA
Sodium, the step (2) are to add the Mg (NO for stirring3)2·6H2O、Al(NO3)3·9H2O, urea and hydrogen peroxide solution
Mixed liquor, persistently stirs and is heated to 150 DEG C under nitrogen protection, and at 150 DEG C, condensing reflux reacts 8 hours.
And, the present invention also provides a kind of immobilised enzymes, including fixed enzyme vector and is fixed on the immobilised enzymes and carries
Lipase on body.
Preferably, the lipase is pig pancreas enzyme, the fold Candida enzyme of fermentable, antarctic candida enzyme
Or rhizomucor miehei lipase.
Compared with prior art, the present invention modifies surface of graphene oxide using oxide nano particles, on the one hand increases
Surface of graphene oxide oxygen-containing functional group and increased the load capacity of enzyme, on the other hand remain graphene oxide high-ratio surface
Product and layer structure, alleviate graphene oxide reunion in the solution in addition.Enzyme is fixed on into GO/ oxide nano particles load
Body material surface, is conducive to enzyme to be fully contacted substrate, and high surface and provides place and fast mass transmission for reaction.Fixing
During enzyme recycling, as the reunion of graphene oxide is suppressed, so that the enzyme for being fixed on surface can not be hidden
Cover and give full expression to enzymatic activity, therefore with good repeat performance.
Description of the drawings
Heat endurance curve comparison figures of the Fig. 1 for the immobilised enzymes of embodiments of the invention 1；
Reusability curve comparison figures of the Fig. 2 for the immobilised enzymes of embodiments of the invention 1；
Scanning electron microscope (SEM) photographs of the Fig. 3 for the GO/ZnO carriers fixation CRL enzymes of embodiments of the invention 1.
Specific embodiment
In order that the objects, technical solutions and advantages of the present invention become more apparent, it is below in conjunction with drawings and Examples, right
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is only to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1
Embodiments of the invention 1 provide a kind of fixed enzyme vector, and its concrete preparation process is as follows：
Take a certain amount of graphene oxide (substituting with GO below) to be scattered in deionized water, add proper amount of acetic acid zinc, wherein
Add the amount of zinc acetate to ensure the mass percent of zinc oxide in the fixed enzyme vector to be formed as 30%, ultrasonically treated 1 is little
When；In the case of stirring, the mixed solution of NaOH and ethylene glycol is added, and the wherein amount of NaOH is identical with zinc acetate mole
Amount, and the mass parts of NaOH are 1 with the parts by volume of ethylene glycol:10, specially when NaOH is 1g, ethylene glycol is 10ml；Then
Continue stirring, add the sodium citrate of 10 times of GO mass, stirring mixed liquor to be placed in reactor after 1 hour, and at 100 DEG C
Lower reaction 12 hours；After reaction terminates, filtered with ethanol and water washing, vacuum drying at 60 DEG C obtains GO/ZnO carrier materials.
Fixed enzyme vector manufactured in the present embodiment is GO/ZnO carriers, specially GO and is modified in the ZnO in the GO faces.
After preparing above-mentioned fixed enzyme vector, GO/ZnO immobilised enzymes can be prepared by following manner, for the ease of description,
The present embodiment is illustrated by taking the fold Candida enzyme of fermentable as an example, wherein the fold of the present embodiment fermentable
Candida enzyme is with CRL enzymes abbreviation.
Concrete preparation process is as follows：3g CRL enzymes are scattered in the PBS cushioning liquid (0.1M, PH=7.0) of 150ml,
Enzyme liquid is centrifuged after fully dispersed, takes supernatant, determined its enzyme content and preserve at 4 DEG C；
Take 0.6g GO/ZnO carrier materials to be scattered in 10ml PBS cushioning liquid, ultrasonic 20min；Take 90ml CRL enzymes
Supernatant be added in the mixed liquor after ultrasound, and mixed liquor is placed in 37 DEG C of shaking table, in the reaction 3 of 160rp/min
Hour；Then the immobilised enzymes of above-mentioned gained is washed with PBS after 3 times, after centrifugation lyophilization, is stored in 4 DEG C of environment
Under.
The superiority of fixed enzyme vector manufactured in the present embodiment for convenience of description, the present embodiment will using said method
CRL enzymes are separately fixed on GO carriers, GO/ZnO carriers, ZnO carriers and are contrasted with resolvase respectively, detect the catalysis of enzyme
Activity, heat endurance and repeat usage, as shown in Figures 1 to 3, as a result show：After immobilised enzymes repeatedly use, GO/
Though the enzymatic activity of ZnO@CRL enzymes has fluctuation but loses less, and GO@CRL enzymes and ZnO@CRL enzymes loss of activity after repeated
It is larger, this is because GO reunites than more serious during reuse so that the enzyme for being fixed on surface is covered by graphite linings, and
ZnO immobilized enzymes are unstable, and easily during reuse, enzyme departs from ZnO carriers and causes enzymatic activity to reduce；At 50 DEG C, with
The increase in reaction time, the relative activity of immobilised enzymes are stable compared with resolvase, and the relative activity of GO/ZnO@CRL enzymes is higher, this
Illustrate that enzyme is fixed on the GO/ZnO carrier materials of the present invention, its heat endurance is significantly improved.
Embodiment 2
Embodiments of the invention 2 provide a kind of fixed enzyme vector, and its concrete preparation process is as follows：
Weigh a certain amount of GO to be scattered in isopropanol, add a certain amount of MnCl2·4H2O, wherein adding MnCl2·
4H2The amount of O to ensure the mass percent of manganese dioxide in the fixed enzyme vector to be formed as 30%, ultrasonically treated 1 hour；Again
The ammonia spirit of 10ml 2mol/L is added in above-mentioned mixed liquor, continues ultrasonic agitation 30min；Then mixed liquor is poured into instead
In answering kettle, heat 12 hours at 120 DEG C；After reaction terminates, then deionized water cyclic washing to neutrality is done at 80 DEG C again
It is dry, calcination is carried out after grinding and obtains GO/MnO2Carrier material.
The method that immobilised enzymes is prepared in the present embodiment and embodiment 1 is essentially identical, therefore here is not described in detail.
Embodiment 3
Embodiments of the invention 2 provide a kind of fixed enzyme vector, and its concrete preparation process is as follows：
Take 10g surfactant sodium dodecyl base benzene sulfonic acid sodium salts to be dissolved in 100ml deionized waters, the GO for weighing 1g disperses which
In and be ultrasonically formed uniform GO suspension；Weigh 1.025g Mg (NO3)2·6H2O、0.375g Al(NO3)3·9H2O and
Urea is added to and is contained with the there-necked flask of 200ml hydrogen peroxide solutions, and wherein the mass concentration of hydrogen peroxide solution is 20%, will
It is added in GO suspension after solution movement stirring 15min in there-necked flask, persistently stirs, by the solution after stirring in oil bath
150 DEG C are heated in pot and under the protection of nitrogen, keep being stirred vigorously, and condensing reflux reaction 8 hours at 150 DEG C, instead
Product is filtered and used after milli-Q water 4 times, air drying 1～2 day at 25 DEG C after should terminating, that is, GO/ is obtained
LDH carrier materials.Wherein, the present embodiment LDH is hydrotalcite.
The method that immobilised enzymes is prepared in the present embodiment and embodiment 1 is essentially identical, therefore here is not described in detail.
The fixing means that CRL enzymes press the present embodiment 1 is fixed on carrier material prepared by the present embodiment 1～3 respectively, detection
The heat endurance and repeat usage of the immobilised enzymes of formation, shown in table specific as follows：
For resolvase, temperature be 50 DEG C, the reaction time be 180min when, enzyme relative activity be 30%.
It can be seen in table 1 that the heat endurance of the fixed enzyme vector immobilized enzyme of the preparation of the present embodiment 1～3 is relative to free
Enzyme has higher increase, and the repeat usage of immobilised enzymes is higher, and this aspect illustrates that enzyme is relatively more steady in carrier material
Fixed, on the other hand during immobilized enzyme is reused, reunions of the GO in reaction system have received suppression well for explanation, from
And enable the enzyme for being fixed on its surface sufficiently participate in reaction and it is not covered.The graphite linings of GO high-specific surface areas, are enzyme
Catalytic reaction provides good place and mass transfer place.
The specific embodiment of present invention described above, does not constitute limiting the scope of the present invention.Any basis
Various other corresponding change and deformation that the technology design of the present invention is made, should be included in the guarantor of the claims in the present invention
In the range of shield.
Claims (9)
1. a kind of fixed enzyme vector, it is characterised in that including graphene oxide and modify in the surface of graphene oxide
Inorganic nanoparticles containing polyelectron group.
2. fixed enzyme vector according to claim 1, it is characterised in that the inorganic nanoparticles including but not limited to
One or more in zinc oxide, manganese dioxide, hydrotalcite, titanium dioxide.
3. fixed enzyme vector according to claim 1 and 2, it is characterised in that the graphene oxide and inorganic nano
The mass ratio of particle is 7:3.
4. a kind of preparation method of fixed enzyme vector, it is characterised in that comprise the steps：
(1) graphene oxide is added in solvent, be subsequently adding dressing agent or surfactant, ultrasonically treated 1 hour；
(2) surfactant is added in the mixed liquor that dressing agent is added in step (1) or surface-active is added in step (1)
Add dressing agent in the mixed liquor of agent, stirring makes surfactant fully react and be formed with dressing agent to modify in graphene oxide
The inorganic nanoparticles on surface；
(3) wash, be dried, obtain final product.
5. preparation method according to claim 4, it is characterised in that solvent is deionized water, adds in the step (1)
Dressing agent be zinc acetate, the step (2) is the mixed solution that NaOH and ethylene glycol are sequentially added under stirring
And sodium citrate, after stirring 1h, react 12 hours at 100 DEG C.
6. preparation method according to claim 4, it is characterised in that solvent is isopropanol, adds in the step (1)
Dressing agent is MnCl2·4H2O, the step (2) 120 DEG C at react 12 after 30 minutes to add ammonia spirit, ultrasonic agitation
Hour.
7. preparation method according to claim 4, it is characterised in that solvent is deionized water, adds in the step (1)
Surfactant be neopelex, the step (2) is to add the Mg (NO that stir3)2·6H2O、Al
(NO3)3·9H2The mixed liquor of O, urea and hydrogen peroxide solution, persistently stirs and is heated to 150 DEG C under nitrogen protection, 150
At DEG C, condensing reflux reacts 8 hours.
8. a kind of immobilised enzymes, it is characterised in that including the arbitrary described fixed enzyme vector of claims 1 to 3 and be fixed on
Lipase on the fixed enzyme vector.
9. immobilised enzymes according to claim 8, it is characterised in that the lipase is pig pancreas enzyme, fermentable
Fold Candida enzyme, antarctic candida enzyme or rhizomucor miehei lipase.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107354146A (en) * | 2017-07-31 | 2017-11-17 | 苏州凯邦生物技术有限公司 | Preparation method using modified hydrotalcite as the immobilization biological enzyme of carrier |
| CN109266639A (en) * | 2018-08-31 | 2019-01-25 | 华南协同创新研究院 | A kind of dual immobilised enzymes and its preparation method and application |
| CN110228855A (en) * | 2019-07-19 | 2019-09-13 | 宁波石墨烯创新中心有限公司 | The preparation method and sewage water treatment method of graphene oxide composite material |
| CN112871993A (en) * | 2021-02-18 | 2021-06-01 | 杭州楠大环保科技有限公司 | Phase-change water production treatment process for perishable garbage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107354146A (en) * | 2017-07-31 | 2017-11-17 | 苏州凯邦生物技术有限公司 | Preparation method using modified hydrotalcite as the immobilization biological enzyme of carrier |
| CN109266639A (en) * | 2018-08-31 | 2019-01-25 | 华南协同创新研究院 | A kind of dual immobilised enzymes and its preparation method and application |
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| CN112871993A (en) * | 2021-02-18 | 2021-06-01 | 杭州楠大环保科技有限公司 | Phase-change water production treatment process for perishable garbage |
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