CN106582810A - Preparation method of graphene immobilized enzyme catalyst - Google Patents
Preparation method of graphene immobilized enzyme catalyst Download PDFInfo
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- CN106582810A CN106582810A CN201611062861.8A CN201611062861A CN106582810A CN 106582810 A CN106582810 A CN 106582810A CN 201611062861 A CN201611062861 A CN 201611062861A CN 106582810 A CN106582810 A CN 106582810A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J37/00—Processes, in general, for preparing catalysts; Processes, in general, for activation of catalysts
- B01J37/02—Impregnation, coating or precipitation
- B01J37/0215—Coating
- B01J37/0217—Pretreatment of the substrate before coating
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J37/00—Processes, in general, for preparing catalysts; Processes, in general, for activation of catalysts
- B01J37/32—Freeze drying, i.e. lyophilisation
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention provides a preparation method of a graphene immobilized enzyme catalyst. The preparation method utilizes a covalent bonding-based lipase immobilizing technology and comprises modifying the surface of graphene oxide through high specific surface area graphene oxide as a carrier, introducing a functional group undergoing a specific covalent reaction with an enzyme to the surface of the carrier, and carrying out bonding immobilization with the enzyme under mild conditions. Through synergistic catalysis effects, enzyme activity and a reaction conversion rate are improved and stability is improved. The preparation method can be applied to the enzyme catalyzed synthesis of esters, is environmentally friendly, has simple post-treatment processes, a high conversion rate, recyclability and a low production cost and is especially suitable for environmentally friendly synthesis of fatty acid esters.
Description
Technical field
A kind of Graphene fixes the preparation method of enzyme catalyst, belongs to living things catalysis organic synthesis field.
Background technology
Currently, organic esterses pass through based on the such as sulfuric acid catalytic esterification synthesis of highly acid material mostly, exist
Side reaction is more, high energy consumption, equipment corrosion are serious, environmental pollution the problems such as, seeking green synthesis techniques becomes the task of top priority.It is biological
Enzyme law catalysis esterification techniques have the advantages that catalytic selectivity height, reaction condition are gentle, environmentally friendly, with good industry
Change application prospect.However, biology enzyme there is also price height, easy in inactivation, be not easily recycled, the low shortcoming of repeat usage.It is current many
Biology enzyme is fixed on certain carrier using various technological means, to maintain higher enzymatic activity, improve enzyme stability and
Repeat usage.At present conventional process for fixation mainly has absorption method, investment, covalent coupling method and chemical crosslink technique.Its
Middle employing covalent coupling method, forms covalent bond and firmly connects using the active group of carrier surface with the functional activity group of enzyme molecule
Connect, enzyme stationarity is good, it has also become current main approaches.In recent years, the loaded catalyst of chemical modification has been obtained extensively
General attention, it not only can be recycled, even more because it includes multi-functional catalytic surface.Graphene has huge ratio table
Area, unique two-dimensional structure and chemical stability, have excellent electricity, mechanics, calorifics and engineering properties, it has also become currently open
Send out the frontier of novel catalyst carrier.The present invention as enzyme immobilization carrier, acts on Graphene by concerted catalysis, can be lifted
Enzymatic efficiency, it is significant to expanding lipase-catalyzed esterification technology application and development.Graphene derivative-graphene oxide
(GO), comprising multiple groups, such as hydroxyl, carboxyl, epoxide group and carbonyl, be easily modified by sulphation it is modified, and can in a solvent very
Disperse well.There are the functional groups such as many free hydroxyls, phenolic hydroxyl group, carboxyl, amino, sulfydryl on lipase surface, can with carboxyl,
There is the reaction such as etherificate, alkylation, acylated in aldehyde, ketone etc..Based on this, it is contemplated that by the graphite oxide using chemical modification
Alkene is cooperateed with and lifts enzymatic efficiency as the immobilization carrier of biology enzyme, improves esterification conversion ratio, is answered with expanding enzymatic industrialization
Use prospect.
The content of the invention
The present invention uses covalent coupling method fixed fat zymotechnic, by being used as load using high-specific surface area graphene oxide
Body, modifies surface of graphene oxide, and introducing in carrier surface can occur the function base of specific covalent reaction with enzyme
Group, and carry out with enzyme being bonded fixation in a mild condition；By synergic catalytic effect, the activity and reaction conversion ratio of enzyme are improved,
And improve its stability.This technology of preparing can apply to the synthesis of enzymatic esters, environment friendly and pollution-free, and post processing is simple, turn
Rate is high, repeats and utilizes, low production cost.Instant invention is especially suited for the green syt of fatty acid ester.
Technical scheme：A kind of Graphene fixes the preparation method of enzyme catalyst, concretely comprises the following steps：
1) preparation of functional graphene oxide
Step：A certain amount of graphene oxide GO (concentration 2mg/mL) aqueous dispersions are taken, appropriate NaOH and chloroethene is added
Sour sodium, water bath sonicator reaction certain hour；Neutralized with dilute HCl, then be washed with deionized to neutrality, freeze-drying, obtain table
The graphene oxide powder of face activated carboxylic, closed preservation is standby.
Above-mentioned gained acid activation graphene oxide powder is added in certain volume 0.2-0.3mol/L HCl solutions, in 45
DEG C stirring certain hour, then ultrasonic disperse 20-30min；Again above-mentioned dispersion liquid is added to into certain volume 5%2- (3,4- dihydroxy
Phenyl) in ethylamine solution, appropriate interfacial reaction agent FG-6 (interfacial reaction agent, laboratory system) is added, 0.5-1h is stirred vigorously,
Make fully chelating；By test solution centrifugation, gained solid deionized water cyclic washing, until cleaning solution pH is in neutrality；Exist again
15-24h is vacuum dried at 90 DEG C, the functional graphene oxide (being designated as A) of surface active groups modification is obtained.
2) Graphene fixes the preparation of enzyme catalyst
Step：A certain amount of A is added in the Tris-HCl buffer solutions (50mmol/L, pH7.0) of certain volume, ultrasound point
After scattered certain hour, the glutaraldehyde solution of certain volume 50% is added in above-mentioned solution, stirred under room temperature, activate certain hour
Afterwards, centrifugation, the washing of gained solid matter with deionized water；Again above-mentioned product is added to into the Tris- containing a certain amount of lipase
In HCl buffer solutions (0.05mol/L, pH7.0), after reacting certain hour at 4 DEG C, centrifugation, washing obtains Graphene and consolidates
Determine enzyme catalyst particle (being designated as B), it is standby at -20 DEG C are stored in after freeze-drying.
Beneficial effects of the present invention：Functionalization Activated Graphite alkene as obtained by prepared by the application present invention fixes enzyme catalyst
Can be used for catalyzing and synthesizing for fatty acid ester, with high conversion rate, reaction condition is gentle, product is easily separated, it is environmentally friendly the features such as；The party
Method is suitable to various enzyme immobilizatios, obtained fixed lipase catalyzed dose not easy in inactivation, difficult for drop-off, reaction conversion ratio
Height, repeat usage is high, reduces production cost, especially has broad application prospects in the green syt field of fatty acid ester.
Specific embodiment
Embodiment 1：
1) preparation of functional graphene oxide
Graphene oxide GO (concentration 2mg/mL) aqueous dispersions of 200mL are taken, 8g NaOH and 7g sodium chloroacetates, water is added
Bath ultrasound 30min；Neutralized with 0.2mol/L HCl, then be washed with deionized to neutrality, freeze-drying, obtain surface carboxyl groups work
The graphene oxide powder of change.Above-mentioned gained acid activation graphene oxide powder is added in 50mL 0.2mol/L HCl solutions,
30min, ultrasonic disperse 30min are stirred in 45 DEG C；Above-mentioned dispersion liquid is added to again the 2- (3,4- dihydroxy phenyls) of 60mL 5%
In ethylamine solution, 0.6g FG-6 (interfacial reaction agent, laboratory system) are added, be stirred vigorously 1h, make fully chelating；By test solution
Centrifugation, gained solid deionized water cyclic washing, until cleaning solution pH is in neutrality；Again 20h is vacuum dried at 90 DEG C,
Obtain the functional graphene oxide (being designated as A) of surface active groups modification.
2) Graphene fixes the preparation of enzyme catalyst
Step：Above-mentioned 100mg A are added in 150mL Tris-HCl buffer solutions (50mmol/L, pH 7.0), ultrasound
After dispersion 30min, the glutaraldehyde solution of 20mL 50% is added in above-mentioned solution, stirred under room temperature, after activation 4h, centrifugation point
From the washing of gained solid matter with deionized water；Again above-mentioned product is added to into the c1. lipase of Pseudomonas containing 200mg
In 300mL Tris-HCl buffer solutions (0.05mol/L, pH 7.0) of (laboratory system), after reacting 18h at 4 DEG C, centrifugation point
From washing, being fixed enzyme catalyst particle (being designated as B) is standby at -20 DEG C are stored in after freeze-drying.
This catalyst can be used for the synthesis of fatty acid ester, high conversion rate, and catalyst repeats reuse, keep high catalysis to live
Property.
Claims (1)
1. a kind of Graphene fixes the preparation method of enzyme catalyst, it is characterised in that comprise the steps：
1) preparation of functional graphene oxide
Step：A certain amount of graphene oxide GO (concentration 2mg/mL) aqueous dispersions are taken, appropriate NaOH and sodium chloroacetate is added,
Water bath sonicator reacts certain hour；Neutralized with dilute HCl, then be washed with deionized to neutrality, freeze-drying, obtain surface carboxyl groups
The graphene oxide powder of activation, closed preservation is standby.
Above-mentioned gained acid activation graphene oxide powder is added in certain volume 0.2-0.3mol/L HCl solutions, is stirred in 45 DEG C
Mix certain hour, then ultrasonic disperse 20-30min；Again above-mentioned dispersion liquid is added to into certain volume 5%2- (3,4- dihydroxy phenyls)
In ethylamine solution, appropriate interfacial reaction agent FG-6 (interfacial reaction agent, laboratory system) is added, be stirred vigorously 0.5-1h, make to fill
Divide chelating；By test solution centrifugation, gained solid deionized water cyclic washing, until cleaning solution pH is in neutrality；Again at 90 DEG C
Lower vacuum drying 15-24h, obtains the functional graphene oxide (being designated as A) of surface active groups modification.
2) Graphene fixes the preparation of enzyme catalyst
Step：A certain amount of A is added in the Tris-HCl buffer solutions (50mmol/L, pH7.0) of certain volume, ultrasonic disperse one
After fixing time, the glutaraldehyde solution of certain volume 50% is added in above-mentioned solution, stirred under room temperature, after activation certain hour,
Centrifugation, the washing of gained solid matter with deionized water；Again above-mentioned product is added to into the Tris-HCl containing a certain amount of lipase
In buffer solution (0.05mol/L, pH7.0), after reacting certain hour at 4 DEG C, centrifugation, washing obtains Graphene and fixes
Enzyme catalyst particle (is designated as B), standby at -20 DEG C are stored in after freeze-drying.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108579210A (en) * | 2018-05-21 | 2018-09-28 | 广州康滤净化科技有限公司 | The filtering material and preparation method of the compound carbonic anhydrase absorption carbon dioxide of graphene |
| ES2711838A1 (en) * | 2017-11-03 | 2019-05-07 | Univ Estadual Paulista Julio De Mesquita Filho Unesp | ENZYMATIC DERIVATIVE BASED ON IMMOBILIZED ENZYME ON GRAPHENE OXIDE (Machine-translation by Google Translate, not legally binding) |
| CN111172148A (en) * | 2019-12-20 | 2020-05-19 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Immobilized biological enzyme and preparation method thereof |
| CN112877321A (en) * | 2021-02-05 | 2021-06-01 | 南京工业大学 | Method for preparing graphene immobilized enzyme and application thereof |
| CN113862251A (en) * | 2021-09-09 | 2021-12-31 | 合肥工业大学 | Two-dimensional graphene-like carbon GMCs immobilized enzyme and preparation method and application thereof |
| WO2023196919A1 (en) * | 2022-04-07 | 2023-10-12 | Akron Polymer Solutions, Inc. | Pristine and ultra-reduced graphene oxide as a carrier for enzymes and catalysts |
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| CN102660533A (en) * | 2012-05-02 | 2012-09-12 | 东华大学 | Preparation method of graphene oxide immobilized alkali protease |
| CN104531665A (en) * | 2014-12-08 | 2015-04-22 | 江南大学 | Functionalized active attapulgite enzyme immobilization catalyst preparation method |
| WO2016057816A1 (en) * | 2014-10-08 | 2016-04-14 | University Of South Alabama | Modification of fibers with nanostructures using reactive dye chemistry |
| CN105521821A (en) * | 2015-12-16 | 2016-04-27 | 江南大学 | Preparation method of suspension type active immobilized enzyme catalyst |
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2016
- 2016-11-28 CN CN201611062861.8A patent/CN106582810A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102660533A (en) * | 2012-05-02 | 2012-09-12 | 东华大学 | Preparation method of graphene oxide immobilized alkali protease |
| WO2016057816A1 (en) * | 2014-10-08 | 2016-04-14 | University Of South Alabama | Modification of fibers with nanostructures using reactive dye chemistry |
| CN104531665A (en) * | 2014-12-08 | 2015-04-22 | 江南大学 | Functionalized active attapulgite enzyme immobilization catalyst preparation method |
| CN105521821A (en) * | 2015-12-16 | 2016-04-27 | 江南大学 | Preparation method of suspension type active immobilized enzyme catalyst |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2711838A1 (en) * | 2017-11-03 | 2019-05-07 | Univ Estadual Paulista Julio De Mesquita Filho Unesp | ENZYMATIC DERIVATIVE BASED ON IMMOBILIZED ENZYME ON GRAPHENE OXIDE (Machine-translation by Google Translate, not legally binding) |
| CN108579210A (en) * | 2018-05-21 | 2018-09-28 | 广州康滤净化科技有限公司 | The filtering material and preparation method of the compound carbonic anhydrase absorption carbon dioxide of graphene |
| CN111172148A (en) * | 2019-12-20 | 2020-05-19 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Immobilized biological enzyme and preparation method thereof |
| CN112877321A (en) * | 2021-02-05 | 2021-06-01 | 南京工业大学 | Method for preparing graphene immobilized enzyme and application thereof |
| CN113862251A (en) * | 2021-09-09 | 2021-12-31 | 合肥工业大学 | Two-dimensional graphene-like carbon GMCs immobilized enzyme and preparation method and application thereof |
| CN113862251B (en) * | 2021-09-09 | 2024-05-17 | 合肥工业大学 | Two-dimensional graphene-like carbon GMCs immobilized enzyme and preparation method and application thereof |
| WO2023196919A1 (en) * | 2022-04-07 | 2023-10-12 | Akron Polymer Solutions, Inc. | Pristine and ultra-reduced graphene oxide as a carrier for enzymes and catalysts |
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