CN106011126A - Compound immobilization method for acetylcholine esterase - Google Patents
Compound immobilization method for acetylcholine esterase Download PDFInfo
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- CN106011126A CN106011126A CN201610460093.5A CN201610460093A CN106011126A CN 106011126 A CN106011126 A CN 106011126A CN 201610460093 A CN201610460093 A CN 201610460093A CN 106011126 A CN106011126 A CN 106011126A
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- 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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- 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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- 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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- 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)
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Abstract
The invention belongs to the technical field of enzyme immobilization and particularly relates to a compound immobilization method for acetylcholine esterase. The method comprises the following steps: (1) carrier activation: the surface of an immobilization carrier is modified and activated, so that the immobilization carrier has the capacity in connection with spacer arm molecules; (2) preparation of an acetylcholine esterase immobilized carrier: the immobilization carrier is sequentially connected with the spacer arm molecules and key ligand molecules, and the acetylcholine esterase immobilized carrier is prepared; (3) preparation of an immobilized enzyme: the acetylcholine esterase immobilized carrier and acetylcholine esterase molecules are incubated, and the compound immobilized acetylcholine esterase is obtained.
Description
Technical field
The invention belongs to technical field of enzyme immobilization, particularly to the compound process for fixation of a kind of acetylcholinesterase.
Background technology
Enzyme is a kind of main biocatalyzer with albumen as architecture basics, compared with chemical catalyst, have reaction condition gentle,
Catalytic efficiency is high, specificity is strong, pollute the advantages such as low, is widely used in every field such as industry, agricultural, medicine and food.
Acetylcholinesterase (being called for short AChE) is a big class of acetylcholine esterase, is widely present in people, animal, insecticide etc. internal,
Can be choline and acetic acid by acetylcholine hydrolyzation in specific manner.Acetylcholinesterase can be by organophosphor, carbamate chemicals for agriculture spy
Opposite sex suppression, therefore acetylcholinesterase is widely used in multiple field such as Pesticides Testing, environment measuring.
Fixation techniques for enzyme (Enzyme Immobilization) refers to enzyme constraint or is limited in certain area, but still protects
Stay its catalysis characteristics a recyclable and reusable class technology.Immobilized enzyme, compared with resolvase, mainly has the advantage that (1)
Product is difficult to be polluted by enzyme, simplifies follow-up process for separating and purifying；(2) in most of the cases, enzyme is in immobilization rear stability meeting
It is improved and reusable, consequently facilitating continuous prodution, reduces production cost；(3) immobilized enzyme has certain machinery
Intensity, can use, it is simple to production process realizes pipeline, serialization and automatization in the way of using stirring or dress post.Due to
These characteristics, immobilized enzyme is widely adopted in every field such as industry, agricultural, medicine and food.
At present, acetylcholine ester enzyme immobilization many employings absorption method and cross-linking method.Absorption method is to utilize the Van der Waals force of molecule, hydrogen
Key effect or electrostatic interaction by enzyme Molecular Adsorption in the surface or space of specific porous material.Although the method can be the most fixing
Enzyme, but its substrate is by the hindering of mass transfer performances of porous material, causes the real reaction speed of adsorptive enzyme and activity to be decreased obviously,
Simultaneously because adsorption is affected by environment relatively big, once external environment condition change, the stability of immobilized enzyme can be largely influenced.
Cross-linking method carries out the crosslinking between pheron by (dual or multi) functional reagent, forms covalency between enzyme molecule and poly functional reagent
Key, coagulation is netted, generates water-fast two dimension cross-linked aggregates.Although cross-linking method can be greatly improved stablizing of immobilized enzyme
Property, but the covalent bond formed in cross-linking process is for randomly generating, it is difficult to controls, in addition to crosslinking between enzyme molecule, also
There is certain enzyme intramolecular crosslinking, this kind of enzyme intramolecular crosslinking has a strong impact on original structure and the conformation of enzyme, makes immobilized enzyme live
Impatient acute decline.
Owing to above-mentioned enzyme immobilization method exists notable defect, at present, existing Immobilization Method of Acetylcholinesterase can not meet
The performance requirement of actual application, ensures the high efficiency of immobilization acetylcholinesteraseelectrochemistry, high activity and high stability the most simultaneously.This asks
Topic has become the bottleneck of the actual application of immobilization acetylcholinesteraseelectrochemistry.Therefore, it is badly in need of a kind of knot being specifically designed for acetylcholinesterase
Structure and the enzyme immobilization method of functional characteristics design, improve the performance of existing immobilization acetylcholinesteraseelectrochemistry.
Summary of the invention
The invention provides the compound process for fixation of a kind of acetylcholinesterase, concrete technical scheme is as follows:
The compound process for fixation of a kind of acetylcholinesterase, comprises the following steps:
(1) support-activated: to carry out fixation support surface modifying activation so that it is there is the ability being connected with spacer molecule；
(2) preparation of acetylcholine ester enzyme immobilization carrier: by described fixation support and spacer molecule, crucial ligand molecule
It is sequentially connected with, is prepared as acetylcholine ester enzyme immobilization carrier；
(3) preparation of immobilized enzyme: described acetylcholine ester enzyme immobilization carrier is hatched with acetylcholinesterase molecule, obtains
Compound immobilization acetylcholinesteraseelectrochemistry.
The source of described acetylcholinesterase includes people, animal, plant, microorganism.
Described in step (1), fixation support includes polymer, electrode, mesoporous material, nano material, inorganic material, composite wood
Material waits all can the medium of enzyme immobilization.Preferably, fixation support described in step (1) includes molecular sieve mesoporous material, glass carbon electricity
Surface, pole, gold electrode surfaces, glass fibre membrane, quartz fibre film, filter paper.
In the present invention, spacerarm refers to the molecule for connecting fixation support and crucial ligand molecule, and its purposes is to increase
Fixation support and the distance of crucial ligand molecule, strengthen the flexibility ratio of crucial ligand molecule；Spacerarm includes but not limited to chain
Molecule, polymer molecule, dendrimer.Preferably, described spacerarm includes single chain molecule, strand shaped polymer, many officials
Can reunite compound, dendrimer nano material.
The structure of described crucial ligand molecule includes structure I, structure II, structure III, structure IV 4 type and respective chirality
Isomer, respective tautomer；
Wherein, R1、R2、R3、R4、R5、R6And R7Each can be identical or different, can be all hydrogen, halogen, hydroxyl, carboxylic
Base, amino, nitro, cyano group, sulfydryl, sulfonic group, methoxyl group, ethyoxyl, phenyl, phenylol, C1-C6Alkyl, C1-C6
Haloalkyl, C1-C4Alkyl amino, C5-C6Any one group in glycosyl.
In step (1), the method that fixation support surface carries out modifying activation includes chemical method, physical method or complex method.
Fixation support includes all direct, indirect and compound being connected chemically and physical connection with the connected mode of spacerarm.
Crucial ligand molecule includes all direct, indirect and compound being connected chemically and physical connection with the connected mode of spacerarm.
The method have the advantages that
1, immobilization acetylcholinesteraseelectrochemistry can retain the high activity of resolvase and specificity；The immobilization acetyl that the inventive method obtains
The response rate of acetylcholine esterase > 75%；
2, the stability of immobilization acetylcholinesteraseelectrochemistry is greatly improved, and range is the most extensive；After preserving 10 days,
The immobilization acetylcholinesteraseelectrochemistry enzyme that the inventive method obtains is lived still can keep more than 90%；
3, immobilization acetylcholinesteraseelectrochemistry is reusable repeatedly, and enzyme is lived and kept good；The immobilization acetyl that the inventive method obtains
Acetylcholine esterase is reused more than 10 times, and enzyme is lived and remained to be maintained at more than 70%；
4, the inventive method is big to the supported quantity of acetylcholinesterase, can be widely applied to commercial production.
The compound process for fixation of acetylcholinesterase of the present invention can be effectively improved the activity of immobilization acetylcholinesteraseelectrochemistry, stability
With fixing usefulness.
Detailed description of the invention
The present invention is described in detail by following embodiment, but those skilled in the art are not it will be appreciated that following embodiment is to the present invention
The restriction of protection domain, any improvement made on the basis of the present invention and change, all within protection scope of the present invention.
The activity test method of compound immobilization acetylcholinesteraseelectrochemistry
Reaction system final volume 0.2ml, 100 μ L 0.1mol/L, the phosphate buffer solution of pH=8.0, at the bottom of 50 μ L 0.75mmol/L
Thing (acetylthiocholine iodide), 50 μ L enzyme sources (adjust protein content at 40～80 μ g/mL) or etc. the immobilized enzyme of enzyme amount,
At 37 DEG C, react 5min, add 1.8ml DTNB-phosphate-ethanol reagent, under 412nm wavelength, carry out colorimetric determination,
The transmittance blank tube to 100% is adjusted after adding developer, to add and measure the enzyme liquid of pipe equivalent, to eliminate enzyme itself to light absorption
Impact.
Embodiment 1 structure I type
Fixation support selects the glass-carbon electrode of diameter 3mm, polishes first by alumina powder foot couple glassy carbon electrode surface,
Clean with distilled water afterwards and carry out support-activated with 0.1mol/L dust technology, using the toluene solution of 3-aminopropyl triethoxy
Above-mentioned glassy carbon electrode surface is modified by the method for backflow.Crucial ligand molecule structure is structure I types of molecules, wherein R1、
R4And R3For hydroxyl, R2、R5And R7For hydrogen, R6For amino.Selection glutaraldehyde is as spacerarm, under conditions of 25 DEG C
Glass-carbon electrode is inserted in the diethyl ether solution of 5% glutaraldehyde, auxiliary stirring reaction 2 hours, use ether to clean glass carbon electricity after reaction
3 times, surface, pole.Afterwards, under conditions of 25 DEG C, glass-carbon electrode is placed in the diethyl ether solution containing 1% crucial ligand molecule,
Auxiliary stirring reaction 2 hours, uses ether to clean glassy carbon electrode surface 3 times after reaction.By above-mentioned steps, after modifying
Glass-carbon electrode, spacerarm, crucial ligand molecule are sequentially connected, and form acetylcholine ester fixation support.Under conditions of 4 DEG C,
The phosphate buffer solution (pH=7.4,0.05mol/L) of acetylcholine ester enzyme immobilization carrier Yu acetylcholinesterase is hatched 24
Hour, it is thus achieved that compound immobilization acetylcholinesteraseelectrochemistry.
According to the operating process of " activity test method of compound immobilization acetylcholinesteraseelectrochemistry " part, to compound immobilization acetyl
The activity of acetylcholine esterase is tested, and result is as follows:
The activity recovery of the compound immobilization acetylcholinesteraseelectrochemistry that the present embodiment obtains is 83.2%.It is repeated 10 times use, enzyme
The activity of the immobilized enzyme that activity uses first can keep more than 79.3%.10 are preserved at-22 DEG C of 0.05M phosphate buffer solutions
My god, the activity of the immobilized enzyme that enzymatic activity used compared with first day can keep more than 96.1%.
Embodiment 2 structure III type
Fixation support selects diameter 3mm gold electrode, polishes first by alumina powder foot couple gold electrode surfaces, uses afterwards
Distilled water is cleaned and carries out support-activated with 0.1mol/L dust technology, stand-by.Crucial ligand molecule structure is structure III types of molecules,
Wherein R1、R2And R4For hydroxyl, R3And R6For hydrogen, R5For methyl, R7For amino.Select PAMAM (G4) branch
Gold electrode electrode, as spacerarm, is inserted the second of 5%PAMAM (G4) dendrimer under conditions of 35 DEG C by shape molecule
In alcoholic solution, auxiliary stirring reaction 3 hours, use ethanol purge glassy carbon electrode surface 3 times after reaction.Afterwards, at 30 DEG C
Under the conditions of gold electrode is placed in the ethanol solution containing 1% crucial ligand molecule, auxiliary stirring reaction 24 hours, make after reaction
By ethanol purge glassy carbon electrode surface 3 times.By above-mentioned steps, by the gold electrode after activation, spacerarm, crucial ligand molecule
It is sequentially connected, forms acetylcholine ester fixation support.Under conditions of 4 DEG C, by acetylcholine ester enzyme immobilization carrier and second
The phosphate buffer solution (pH=7.4,0.05mol/L) of acetylcholinesterase hatches 24 hours, it is thus achieved that compound immobilization acetylcholine
Esterase.
According to the operating process of " activity test method of compound immobilization acetylcholinesteraseelectrochemistry " part, to compound immobilization acetyl
The activity of acetylcholine esterase is tested, and result is as follows:
The activity recovery of the compound immobilization acetylcholinesteraseelectrochemistry that the present embodiment obtains is 78.5%.It is repeated 10 times use, enzyme
The activity of the immobilized enzyme that activity uses first can keep more than 75.8%.10 are preserved at-22 DEG C of 0.05M phosphate buffer solutions
My god, the activity of the immobilized enzyme that enzymatic activity used compared with first day can keep more than 93.5%.
Embodiment 3 structure IV type
Fixation support select silica gel particle, first in 20g silica gel particle add 2mL 3-bromopropyl trimethoxy silane and
100mL toluene solution, refluxes 48 hours, and toluene is cleaned and vacuum rotary steam removal solvent, makes the modified silica-gel of activation, stand-by.
Crucial ligand molecule structure is structure III types of molecules, wherein R1、R3And R4For hydroxyl, R2、R5And R6For hydrogen, R7For
Bromine.Select Polyethylene Glycol (molecular weight: 200Da) as spacerarm, under conditions of 25 DEG C, modified silica-gel is placed in 1%
In Polyethylene Glycol (molecular weight: 200Da) diethyl ether solution, and add 0.5%NaOH as catalyst, auxiliary stirring reaction 12
Hour, use ether to clean silica gel 3 times after reaction.Afterwards, the crucial aglucon that silica gel is placed under conditions of 25 DEG C 1% divides
In the diethyl ether solution of son, and add 0.5%NaOH as catalyst, auxiliary stirring reaction 24 hours, use ether after reaction
Clean glassy carbon electrode surface 3 times.By above-mentioned steps, modified silica-gel, spacerarm, crucial ligand molecule are sequentially connected composition
Acetylcholine ester fixation support.Under conditions of 4 DEG C, by the phosphorus of acetylcholine ester enzyme immobilization carrier Yu acetylcholinesterase
Acid buffering solution (pH=7.4,0.05mol/L) hatches 24 hours, it is thus achieved that compound immobilization acetylcholinesteraseelectrochemistry.
According to the operating process of " activity test method of compound immobilization acetylcholinesteraseelectrochemistry " part, to compound immobilization acetyl
The activity of acetylcholine esterase is tested, and result is as follows:
The activity recovery of the compound immobilization acetylcholinesteraseelectrochemistry that the present embodiment obtains is 75.9%.It is repeated 10 times use, enzyme
The activity of the immobilized enzyme that activity uses first can keep more than 72.3%.10 are preserved at-22 DEG C of 0.05M phosphate buffer solutions
My god, the activity of the immobilized enzyme that enzymatic activity used compared with first day can keep more than 92.6%.
Claims (10)
1. the compound process for fixation of an acetylcholinesterase, it is characterised in that comprise the following steps:
(1) support-activated: to carry out fixation support surface modifying activation so that it is there is the ability being connected with spacer molecule；
(2) preparation of acetylcholine ester enzyme immobilization carrier: by described fixation support and spacer molecule, crucial ligand molecule
It is sequentially connected with, is prepared as acetylcholine ester enzyme immobilization carrier；
(3) preparation of immobilized enzyme: described acetylcholine ester enzyme immobilization carrier is hatched with acetylcholinesterase molecule, obtains
Compound immobilization acetylcholinesteraseelectrochemistry.
Compound process for fixation the most according to claim 1, it is characterised in that the source of described acetylcholinesterase includes
People, animal, plant, microorganism.
Compound process for fixation the most according to claim 1, it is characterised in that fixation support bag described in step (1)
Include polymer, electrode, mesoporous material, nano material, inorganic material, composite.
Compound process for fixation the most according to claim 3, it is characterised in that fixation support bag described in step (1)
Include molecular sieve mesoporous material, glassy carbon electrode surface, gold electrode surfaces, glass fibre membrane, quartz fibre film, filter paper.
Compound process for fixation the most according to claim 1, it is characterised in that described spacerarm includes chain molecule, gathers
Adduct molecule, dendrimer.
Compound process for fixation the most according to claim 5, it is characterised in that described spacerarm includes single chain molecule, list
Chain polymer, polyfunctional group polymer, dendrimer nano material.
Compound process for fixation the most according to claim 1, it is characterised in that the structure of described crucial ligand molecule includes
Structure I, structure II, structure III, structure IV 4 type and respective chiral isomer, respective tautomer；
Wherein, R1、R2、R3、R4、R5、R6And R7For hydrogen, halogen, hydroxyl, carboxyl, amino, nitro, cyano group, mercapto
Base, sulfonic group, methoxyl group, ethyoxyl, phenyl, phenylol, C1-C6Alkyl, C1-C6Haloalkyl, C1-C4Alkyl amino,
C5-C6Any one group in glycosyl.
Compound process for fixation the most according to claim 1, it is characterised in that in step (1), to fixation support table
Face carries out modifying the method for activation and includes chemical method, physical method or complex method.
Compound process for fixation the most according to claim 1, it is characterised in that fixation support and the connection side of spacerarm
Formula includes all direct, indirect and compound being connected chemically and physical connection.
Compound process for fixation the most according to claim 1, it is characterised in that crucial ligand molecule and the company of spacerarm
The mode of connecing includes all direct, indirect and compound being connected chemically and physical connection.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760501A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | Co-crosslinking immobilization method of acetylcholinesterase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110760501A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | Co-crosslinking immobilization method of acetylcholinesterase |
| CN110760501B (en) * | 2019-05-07 | 2023-03-17 | 宁波大学 | Co-crosslinking immobilization method of acetylcholinesterase |
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