CN106011126B - A kind of compound process for fixation of acetylcholinesterase - Google Patents
A kind of compound process for fixation of acetylcholinesterase Download PDFInfo
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- CN106011126B CN106011126B CN201610460093.5A CN201610460093A CN106011126B CN 106011126 B CN106011126 B CN 106011126B CN 201610460093 A CN201610460093 A CN 201610460093A CN 106011126 B CN106011126 B CN 106011126B
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- fixation
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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
- 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
- C12N11/06—Enzymes or microbial cells immobilised on or in an organic carrier attached to the carrier via a bridging agent
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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)
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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)
- C12Y301/01007—Acetylcholinesterase (3.1.1.7)
Abstract
The invention belongs to technical field of enzyme immobilization, in particular to the compound process for fixation of a kind of acetylcholinesterase.It the described method comprises the following steps: (1) support-activated: modification activation being carried out to fixation support surface, makes it have the ability connecting with spacer molecule；(2) preparation of acetylcholine ester enzyme immobilization carrier: the fixation support and spacer molecule, crucial ligand molecule are sequentially connected, acetylcholine ester enzyme immobilization carrier is prepared into；(3) preparation of immobilised enzymes: the acetylcholine ester enzyme immobilization carrier and acetylcholine ester enzyme molecule are incubated for, compound immobilization acetylcholinesteraseelectrochemistry is obtained.
Description
Technical field
The invention belongs to technical field of enzyme immobilization, in particular to the compound process for fixation of a kind of acetylcholinesterase.
Background technique
Enzyme is one kind mainly using albumen as the biocatalyst of structure basis, compared with chemical catalyst, has reaction item
Part is mild, high catalytic efficiency, specificity are strong, the low advantage of pollution, in every field applications such as industry, agricultural, medicine and food
Extensively.
Acetylcholinesterase (abbreviation AChE) is a major class of cholinesterase, is widely present in the bodies such as people, animal, insect
It is interior, it can be in specific manner choline and acetic acid by acetylcholine hydrolyzation.Acetylcholinesterase can be by organic phosphorus, carbamates agriculture
Medicine specificity inhibits, therefore acetylcholinesterase is widely used in the multiple fields such as Pesticides Testing, environment measuring.
Fixation techniques for enzyme (Enzyme Immobilization), which refers to, to be fettered enzyme or is limited in certain area,
But still retain its catalysis characteristics and a kind of technology that is recyclable and reusing.Immobilised enzymes is compared with resolvase, under mainly having
Column advantage: (1) product is not easy to be polluted by enzyme, simplifies subsequent process for separating and purifying；(2) in most cases, enzyme is in fixation
Changing rear stability can be improved and reusable, consequently facilitating continuous production, reduces production cost；(3) immobilised enzymes
With certain mechanical strength, can be used by the way of stirring or filling column, process easy to produce realizes pipeline, serialization
And automation.Due to these characteristics, immobilised enzymes is widely adopted in every field such as industry, agricultural, medicine and food.
Currently, acetylcholine ester enzyme immobilization mostly uses absorption method and cross-linking method.Absorption method is the Van der Waals using molecule
Enzyme molecule is adsorbed in the surface or gap of specific porous material by power, hydrogen bond action or electrostatic interaction.Although the method can
Effective immobilized enzyme, but obstruction of its substrate by the mass transfer performances of porous material lead to the real reaction rate and activity of adsorptive enzyme
It is decreased obviously, simultaneously because suction-operated is affected by environment larger, once external environment changes, the stability of immobilized enzyme be will receive
Tremendous influence.Cross-linking method carries out being crosslinked between zymoprotein by (dual or multi) functional reagent, enzyme molecule and poly functional reagent it
Between form covalent bond, agglutination generates two-dimentional cross-linked aggregates not soluble in water in netted.Although cross-linking method can greatly improve solid
The covalent bond determining the stability of enzyme, but being formed in cross-linking process is to be randomly generated, it is difficult to be controlled, in addition to occurring between enzyme molecule
Crosslinking is outer, and there is also being crosslinked in certain enzyme molecule, crosslinking seriously affects the original structure and conformation of enzyme in such enzyme molecule,
Decline fixed enzymatic activity sharply.
Since there are notable defects for above-mentioned enzyme immobilization method, currently, existing Immobilization Method of Acetylcholinesterase is not
It is able to satisfy the performance requirement of practical application, i.e., guarantees the high efficiency, high activity and high stable of immobilization acetylcholinesteraseelectrochemistry simultaneously
Property.This problem has become the bottleneck of immobilization acetylcholinesteraseelectrochemistry practical application.Therefore, it is badly in need of one kind specifically for acetylcholine
The enzyme immobilization method of the structure and function feature design of esterase, improves the performance of existing immobilization acetylcholinesteraseelectrochemistry.
Summary of the invention
The present invention provides a kind of compound process for fixation of acetylcholinesterase, specific technical solution is as follows:
A kind of compound process for fixation of acetylcholinesterase, comprising the following steps:
(1) support-activated: modification activation being carried out to fixation support surface, makes it have the energy connecting with spacer molecule
Power；
(2) preparation of acetylcholine ester enzyme immobilization carrier: by the fixation support and spacer molecule, crucial aglucon
Molecule is sequentially connected, and is prepared into acetylcholine ester enzyme immobilization carrier；
(3) preparation of immobilised enzymes: the acetylcholine ester enzyme immobilization carrier and acetylcholine ester enzyme molecule are incubated for,
Obtain compound immobilization acetylcholinesteraseelectrochemistry.
The source of the acetylcholinesterase includes people, animal, plant, microorganism.
Fixation support described in step (1) includes polymer, electrode, mesoporous material, nano material, inorganic material, answers
Condensation material etc. all can enzyme immobilization medium.Preferably, fixation support described in step (1) include molecular sieve mesoporous material,
Glassy carbon electrode surface, gold electrode surfaces, glass fibre membrane, quartz fibre film, filter paper.
In the present invention, spacerarm refers to the molecule for being connected and fixed carrier and crucial ligand molecule, and purposes exists
In increasing fixation support at a distance from crucial ligand molecule, enhance the flexibility ratio of crucial ligand molecule；Spacerarm includes but not
It is limited to chain molecule, polymer molecule, dendrimer.Preferably, the spacerarm includes single chain molecule, the polymerization of single-stranded shape
Object, polyfunctional group polymer, dendrimer nano material.
The structure of the key ligand molecule includes structure I, structure II, structure III, IV 4 seed type of structure and respective
Chiral isomer, respective tautomer；
Wherein, R1、R2、R3、R4、R5、R6And R7Respectively can be identical or different, it can be hydrogen, halogen, hydroxyl, carboxyl, ammonia
Base, nitro, cyano, sulfydryl, sulfonic group, methoxyl group, ethyoxyl, phenyl, phenylol, C1-C6Alkyl, C1-C6Halogenated alkyl, C1-
C4Alkyl amino, C5-C6Any one group in glycosyl.
It include chemical method, physical method or multiple to the method that fixation support surface carries out modification activation in step (1)
Conjunction method.
The connection type of fixation support and spacerarm includes all direct, indirect and compound is connected chemically and physics
Connection.
The connection type of crucial ligand molecule and spacerarm includes all direct, indirect and compound is connected chemically and object
Reason connection.
The invention has the following advantages:
1, immobilization acetylcholinesteraseelectrochemistry can retain the high activity and specificity of resolvase；The fixation that the method for the present invention obtains
Change the rate of recovery > 75% of acetylcholinesterase；
2, the stability of immobilization acetylcholinesteraseelectrochemistry is greatly improved, and use scope is more extensive；Saving 10
After it, the immobilization acetylcholinesteraseelectrochemistry enzyme activity that the method for the present invention obtains can still keep 90% or more；
3, immobilization acetylcholinesteraseelectrochemistry is reusable repeatedly, and enzyme activity keeps good；The fixation that the method for the present invention obtains
Change acetylcholinesterase reuse 10 times or more, enzyme activity remains to be maintained at 70% or more；
4, the method for the present invention is big to the supported quantity of acetylcholinesterase, can be widely applied to industrial production.
The compound process for fixation of acetylcholinesterase of the present invention can effectively improve immobilization acetylcholinesteraseelectrochemistry activity,
Stability and fixed efficiency.
Specific embodiment
The present invention is described in detail by following embodiments, but those skilled in the art are not it will be appreciated that following embodiments are pair
The limitation of the scope of the present invention, any improvements and changes made on the basis of the present invention, all in protection scope of the present invention
Within.
The activity test method of compound immobilization acetylcholinesteraseelectrochemistry
Reaction system final volume 0.2ml, the phosphate buffer solution of 100 μ L 0.1mol/L, pH=8.0,50 μ L
0.75mmol/L substrate (acetylthiocholine iodide) or waits enzyme amount at 50 μ L enzyme sources (adjustment protein content is in 40~80 μ g/mL)
Immobilised enzymes, react 5min at 37 DEG C, 1.8ml DTNB- phosphate-ethanol reagent be added, is carried out under 412nm wavelength
Colorimetric estimation adjusts light transmittance to 100% blank tube that the enzyme solution added with measurement pipe equivalent is added after color developing agent, to eliminate enzyme
Influence to light absorption in itself.
I type of embodiment 1 --- structure
Fixation support selects the glass-carbon electrode of diameter 3mm, is carried out first using alumina powder foot couple glassy carbon electrode surface
It polishes, cleaned later with distilled water and is carried out with 0.1mol/L dust technology and is support-activated, using 3- aminopropyl triethoxy
The method of toluene solution reflux modifies above-mentioned glassy carbon electrode surface.Crucial ligand molecule structure is I types of molecules of structure,
Wherein R1、R4And R3For hydroxyl, R2、R5And R7For hydrogen, R6For amino.Select glutaraldehyde as spacerarm, under conditions of 25 DEG C
Glass-carbon electrode is inserted into the diethyl ether solution of 5% glutaraldehyde, auxiliary is stirred to react 2 hours, cleans glass carbon using ether after reaction
Electrode surface 3 times.Later, glass-carbon electrode is placed under conditions of 25 DEG C in the diethyl ether solution containing 1% crucial ligand molecule,
Auxiliary is stirred to react 2 hours, is cleaned glassy carbon electrode surface 3 times after reaction using ether.Through the above steps, by the glass after modification
Carbon electrode, spacerarm, crucial ligand molecule are sequentially connected, and form acetylcholine ester fixation support.It, will under conditions of 4 DEG C
It is small that the phosphate buffer solution (pH=7.4,0.05mol/L) of acetylcholine ester enzyme immobilization carrier and acetylcholinesterase is incubated for 24
When, obtain compound immobilization acetylcholinesteraseelectrochemistry.
According to the operating process of " activity test method of compound immobilization acetylcholinesteraseelectrochemistry " part, to compound immobilization
The activity of acetylcholinesterase is tested, as a result as follows:
The activity recovery for the compound immobilization acetylcholinesteraseelectrochemistry that the present embodiment obtains is 83.2%.Being repeated 10 times makes
With the activity of the immobilized enzyme that enzymatic activity uses more for the first time can keep 79.3% or more.In -22 DEG C of 0.05M phosphate buffer solutions
It saves 10 days, the activity of the immobilized enzyme that enzymatic activity was used compared with first day can keep 96.1% or more.
III type of embodiment 2 --- structure
Fixation support selects diameter 3mm gold electrode, is polished first using alumina powder foot couple gold electrode surfaces, it
Cleaned afterwards with distilled water and carried out with 0.1mol/L dust technology it is support-activated, for use.Crucial ligand molecule structure is III class of structure
Type molecule, wherein R1、R2And R4For hydroxyl, R3And R6For hydrogen, R5For methyl, R7For amino.Select PAMAM (G4) dendrimer
As spacerarm, gold electrode electrode is inserted into the ethanol solution of 5%PAMAM (G4) dendrimer under conditions of 35 DEG C,
Auxiliary is stirred to react 3 hours, is cleaned glassy carbon electrode surface 3 times after reaction using ethyl alcohol.Later, by golden electricity under conditions of 30 DEG C
Pole is placed in the ethanol solution containing 1% crucial ligand molecule, and auxiliary is stirred to react 24 hours, cleans glass using ethyl alcohol after reaction
Carbon electrodes 3 times.Through the above steps, gold electrode, spacerarm, the crucial ligand molecule after activation are sequentially connected, are formed
Acetylcholine ester fixation support.Under conditions of 4 DEG C, by the phosphorus of acetylcholine ester enzyme immobilization carrier and acetylcholinesterase
Acid buffering solution (pH=7.4,0.05mol/L) is incubated for 24 hours, obtains compound immobilization acetylcholinesteraseelectrochemistry.
According to the operating process of " activity test method of compound immobilization acetylcholinesteraseelectrochemistry " part, to compound immobilization
The activity of acetylcholinesterase is tested, as a result as follows:
The activity recovery for the compound immobilization acetylcholinesteraseelectrochemistry that the present embodiment obtains is 78.5%.Being repeated 10 times makes
With the activity of the immobilized enzyme that enzymatic activity uses more for the first time can keep 75.8% or more.In -22 DEG C of 0.05M phosphate buffer solutions
It saves 10 days, the activity of the immobilized enzyme that enzymatic activity was used compared with first day can keep 93.5% or more.
IV type of embodiment 3 --- structure
Fixation support selects silica gel particle, and 2mL 3- bromopropyl trimethoxy silicon is added first into 20g silica gel particle
Alkane and 100mL toluene solution flow back 48 hours, and toluene is cleaned and vacuum rotary steam removes solvent, and the modified silica-gel of activation is made, to
With.Crucial ligand molecule structure is III types of molecules of structure, wherein R1、R3And R4For hydroxyl, R2、R5And R6For hydrogen, R7For bromine.Choosing
It uses polyethylene glycol (molecular weight: 200Da) as spacerarm, modified silica-gel is placed in 1% polyethylene glycol under conditions of 25 DEG C
In (molecular weight: 200Da) diethyl ether solution, and 0.5%NaOH is added as catalyst, auxiliary is stirred to react 12 hours, after reaction
It is cleaned silica gel 3 times using ether.Later, silica gel is placed in the diethyl ether solution of 1% crucial ligand molecule under conditions of 25 DEG C
In, and 0.5%NaOH is added as catalyst, auxiliary is stirred to react 24 hours, cleans glass-carbon electrode table using ether after reaction
Face 3 times.Through the above steps, modified silica-gel, spacerarm, crucial ligand molecule are sequentially connected composition acetylcholine ester immobilization
Carrier.Under conditions of 4 DEG C, by the phosphate buffer solution (pH=of acetylcholine ester enzyme immobilization carrier and acetylcholinesterase
7.4,0.05mol/L) it is incubated for 24 hours, obtains compound immobilization acetylcholinesteraseelectrochemistry.
According to the operating process of " activity test method of compound immobilization acetylcholinesteraseelectrochemistry " part, to compound immobilization
The activity of acetylcholinesterase is tested, as a result as follows:
The activity recovery for the compound immobilization acetylcholinesteraseelectrochemistry that the present embodiment obtains is 75.9%.Being repeated 10 times makes
With the activity of the immobilized enzyme that enzymatic activity uses more for the first time can keep 72.3% or more.In -22 DEG C of 0.05M phosphate buffer solutions
It saves 10 days, the activity of the immobilized enzyme that enzymatic activity was used compared with first day can keep 92.6% or more.
Claims (8)
1. a kind of compound process for fixation of acetylcholinesterase, which comprises the following steps:
(1) support-activated: modification activation being carried out to fixation support surface, makes it have the ability connecting with spacer molecule；
(2) preparation of acetylcholine ester enzyme immobilization carrier: by the fixation support and spacer molecule, crucial aglucon point
Son is sequentially connected, and is prepared into acetylcholine ester enzyme immobilization carrier；The spacerarm is chain molecule, polymer molecule or branch
Shape molecule；
It is described key ligand molecule structure be structure III, IV two types of structure and respective chiral isomer, it is respective mutually
Tautomeric；
Wherein, R1、R2、R3、R4、R5、R6And R7For hydrogen, halogen, hydroxyl, carboxyl, amino, nitro, cyano, sulfydryl, sulfonic group, first
Oxygroup, ethyoxyl, phenyl, phenylol, C1-C6Alkyl, C1-C6Halogenated alkyl, C1-C4Alkyl amino, C5-C6It is any in glycosyl
A kind of group；
(3) preparation of immobilised enzymes: the acetylcholine ester enzyme immobilization carrier and acetylcholine ester enzyme molecule are incubated for, obtained
To compound immobilization acetylcholinesteraseelectrochemistry.
2. compound process for fixation according to claim 1, which is characterized in that the source of the acetylcholinesterase includes
People, animal, plant, microorganism.
3. compound process for fixation according to claim 1, which is characterized in that fixation support packet described in step (1)
Include polymer, electrode, mesoporous material, nano material, inorganic material, composite material.
4. compound process for fixation according to claim 3, which is characterized in that fixation support packet described in step (1)
Include molecular sieve mesoporous material, glassy carbon electrode surface, gold electrode surfaces, glass fibre membrane, quartz fibre film, filter paper.
5. compound process for fixation according to claim 1, which is characterized in that the spacerarm includes single chain molecule, list
Chain polymer, polyfunctional group polymer, dendrimer nano material.
6. compound process for fixation according to claim 1, which is characterized in that in step (1), to fixation support surface
The method for carrying out modification activation includes chemical method, physical method or complex method.
7. compound process for fixation according to claim 1, which is characterized in that the connection side of fixation support and spacerarm
Formula includes all direct, indirect and compound is connected chemically and physical connection.
8. compound process for fixation according to claim 1, which is characterized in that the connection of crucial ligand molecule and spacerarm
Mode includes all direct, indirect and compound is connected chemically and physical connection.
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Citations (3)
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|---|---|---|---|---|
| CN103421878A (en) * | 2013-07-26 | 2013-12-04 | 江苏大学 | Preparation of polyphenol oxidase biosensor and detection of polyphenol oxidase biosensor to pesticide residues |
| CN104046610A (en) * | 2014-05-27 | 2014-09-17 | 中国农业大学 | Enzyme directed immobilization method based on protein surface screening |
| CN105044172A (en) * | 2015-07-06 | 2015-11-11 | 中国农业大学 | AChE enzyme oriented immobilization-based biosensor making method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421878A (en) * | 2013-07-26 | 2013-12-04 | 江苏大学 | Preparation of polyphenol oxidase biosensor and detection of polyphenol oxidase biosensor to pesticide residues |
| CN104046610A (en) * | 2014-05-27 | 2014-09-17 | 中国农业大学 | Enzyme directed immobilization method based on protein surface screening |
| CN105044172A (en) * | 2015-07-06 | 2015-11-11 | 中国农业大学 | AChE enzyme oriented immobilization-based biosensor making method |
Non-Patent Citations (2)
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| 《乙酰胆碱酯酶和香豆素460生物缀合物的传感性与分子对接》;蒋娟娟;《中国优秀硕士学位论文全文数据库基础科学辑》;20110315(第3期);全文 |
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