CN105441420B - A kind of orientation common immobilization method of multi-enzyme system - Google Patents
A kind of orientation common immobilization method of multi-enzyme system Download PDFInfo
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Abstract
The invention discloses a kind of orientation common immobilization method of multi-enzyme system.Multi-enzyme system is oriented co-immobilization on a carrier by the present invention using the biological affinity interaction between prothetic group and pheron.Advantage for present invention essentially consists in：The high orientation co-immobilization of multi-enzyme system is realized, the synergy being more beneficial between multi-enzyme system enzyme, drastically increases catalytic efficiency；Orientation affinity interaction immobilised enzymes is significantly improved than common absorption method activity of the immobilized enzyme, and a kind of feasible method is provided for further structure high efficiency, the living things catalysis system of low cost.
Description
Technical field
The present invention relates to a kind of orientation common immobilization method of multi-enzyme system, belong to protein immobilization field.
Background technology
Many enzymes in cell are frequently in a continuous reaction chain and worked, and continuous chain reaction refers to previous enzyme
The product of reaction is the substrate of the latter enzyme reaction.In complete intracellular a certain metabolic process, formed by several enzymes
React Chain System and be referred to as multi-enzyme system.Multi-enzyme system is also known as multienzyme complex, multi-enzyme system, multienzyme system, multienzyme cluster.It is continuous anti-
The product for answering previous reaction in system is the substrate of latter reaction, and reaction is sequentially connected, and forms a metabolic pathway or metabolism way
The part in footpath.Because this serial response is carried out in the multienzyme complex of a high-sequential, so as to improve urging for enzyme
Change efficiency, while beneficial to the regulation and control to enzyme.Many multi-enzyme systems in intact cell all have capacity of self-regulation.
By the flavo-enzyme with bioactivity and peroxidase orientation co-immobilization, flavo-enzyme and mistake can be made full use of
The cooperative effect of oxide enzyme, there is important application value in fields such as biology sensors.But traditional enzyme immobilization side
The immobilised enzymes structure that method is obtained, enzyme are to be connected in any site with carrier, and this method is mainly disadvantageous in that often
Significantly declining occurs in the activity for often resulting in immobilised enzymes.And flavo-enzyme and peroxidase orientation co-immobilization, it is to utilize enzyme
The affinity interaction of prothetic group and enzyme, flavo-enzyme is connected with peroxidase with orderly direction in the specific site of carrier,
Native conformation is held essentially constant, and this contributes to the biological function for efficiently realizing this multi-enzyme system, has prior research
With application value.
The content of the invention
The problem of the present invention is the provision of a kind of orientation common immobilization method of multi-enzyme system, it is therefore an objective to overcomes traditional single
A variety of deficiencies of enzyme immobilization method, realize the condition optimizing of the oriented immobilization of multi-enzyme system.
In order to solve above-mentioned problem, the present invention uses following technology：
The orientation common immobilization method of a kind of multi-enzyme system, it is characterised in that comprise the following steps：
Step 1, using the functional group on the carrier material with epoxy bond or carboxyl, with 3,5- diaminobenzoic acids (3,
Amino 5-DABA) is reacted, and covalent by 3,5- diaminobenzoic acids (3,5-DABA) is keyed on carrier material,
Acquisition is connected to the carrier material of 3,5- diaminobenzoic acids (3,5-DABA), is represented with S- (3,5-DABA)；Wherein 3,5- diaminos
The mass ratio of yl benzoic acid (3,5-DABA) and carrier material is 1-5:2；
Step 2, the S- that step 1 is obtained (3,5-DABA) react with polyaniline (PANI), and reaction passes through S- (3,5-
DABA the amino of aminobenzoic acid reacts with polyaniline on), formation covalent bond, and polyaniline and S- (3,5-DABA) mass ratio are
1-5:10, the carrier material for being connected to polyaniline is obtained, is represented with S- (3,5-DABA)-PANI；
Step 3, by the amino on the prothetic group flavin adenine dinucleotide (FAD) (FAD) of flavo-enzyme, with step 2 obtained by S-
(3,5-DABA)-PANI is reacted, and passes through the carboxyl and flavin adenine dinucleotide of the upper benzoic acid of S- (3,5-DABA)-PANI
The mass ratio of the amino reaction of acid, formation covalent bond, carrier material and flavin adenine dinucleotide (FAD) is 1-5:2, through amido link
Connection, the carrier for being connected to flavin adenine dinucleotide (FAD) is obtained, represented with S- (3,5-DABA)-PANI-FAD；
Step 4, the carboxyl on prothetic group (activated centre)-hemin (Hemin) of oxide enzyme, with step 3 institute
Obtain S- (3,5-DABA)-PANI-FAD to be reacted, pass through the ammonia of the polyaniline (PANI) on S- (3,5-DABA)-PANI-FAD
Base is reacted, and forms covalent bond, and products therefrom is represented with S- (3,5-DABA)-PANI-FAD-Hemin；With reducing agent to production
The mass ratio of thing progress reduction treatment, reducing agent and product is 8000-500:5；Wherein, hemin and S- (3,5-
DABA)-PANI-FAD mass ratio is 1:1-5.
Step 5, by S- (3,5-DABA) in the flavo-enzyme of apoenzyme, the peroxidase of apoenzyme and step 4-
PANI-FAD carries out affine absorption, the mass ratio of the flavo-enzyme of the apoenzyme of apoenzyme, the peroxidase of apoenzyme and carrier
For 1:1：1-5, obtain the multi-enzyme system of orientation co-immobilization.In the present invention, the peroxidating of the flavo-enzyme, apoenzyme of apoenzyme
Thing enzyme can be as obtained by normal outsourcing behavior；
Preferably, the carrier material described in a kind of step one oriented in common immobilization method of above-mentioned multi-enzyme system
For at least one of graphene oxide, graphene, CNT or graphite.
Preferably, the flavo-enzyme described in step three in a kind of orientation common immobilization method of above-mentioned multi-enzyme system is
In glucose oxidase (GOx), cholesterol oxidase, succinate dehydrogenase, D-AAO or acyl CoA dehydrogenases
At least one.
Preferably, the peroxide described in a kind of step four oriented in common immobilization method of above-mentioned multi-enzyme system
Enzyme is at least one of cytochromes, catalase or peroxidase.
Preferably, the reducing agent described in step five in a kind of orientation common immobilization method of above-mentioned multi-enzyme system is
At least one of hydrazine hydrate, sodium borohydride or sodium citrate..
Beneficial effect：The present invention compared with the existing technology has advantages below and effect：Oriented altogether using by multi-enzyme system
Immobilization, it can efficiently realize the bioactivity of multi-enzyme system.
Brief description of the drawings
Enzyme system adds GOD to Fig. 1 altogether：HRP=1:1(1mg/ml)；Substrate is 50mM glucose solutions, cyclic voltammetry
(CV) detection range is 0~0.8V, PANI (1)：The result of Mw=5400, Mn=4000 detection
Enzyme system adds GOD to Fig. 2 altogether：HRP=1:1(1mg/ml)；Substrate is 50mM glucose solutions.Cyclic voltammetry
(CV) detection range is 0~0.8V, PANI (chloroform)：The result of Mw=6300, Mn=4100 detection
Fig. 3 difference enzyme fixations electro-chemical activity contrasts
2R GO-3,5-PANI (2)-FAD apo-GOD+HRP GOD are engaged, HRP absorption
The double enzyme absorption of 3R GO-3,5-PANI (2) GOD+HRP
4R GO-3,5-PANI (2)-FAD-Hemin apo-GOD+apo-HRP GOD, HRP are engaged
5R GO-3,5-PANI (2)-Hemin GOD+apo-HRP HRP are engaged, GOD absorption
Embodiment
With reference to embodiment, the present invention is described in further detail, following examples be explanation of the invention and
The invention is not limited in following examples.
Embodiment 1
Experimental procedure：
1. graphene oxide (GO) -3,5- diaminobenzoic acids (3,5-DABA)
Graphene oxide 1.0g is weighed, is placed in beaker, adds 200ml THF, ultrasonic 2h；Add 1.0g3,5- diaminos
Yl benzoic acid (~6.57 × 10-3Mol), ultrasonic 30min is continued；Solution after ultrasound is transferred in three-necked flask, closing is anti-
Bottle is answered, is vacuumized, inflated with nitrogen, is heated to 70 DEG C of condensing refluxes, magnetic agitation 2h；Reaction solution centrifuges, and is washed with absolute ethyl alcohol
Wash precipitation for several times；Sediment is placed in vacuum drying oven and dried.
2.GO-3,5-DABA- polyaniline (PANI)
GO-3,5- diaminobenzoic acid 0.3009g are weighed, adds 60ml NMP mixing, ultrasonic disperse；Add polyaniline
0.1509g, stir；By 0.0710g ammonium persulfates (~3.11 × 10-4Mol) dissolved, delayed with 10ml 1.0M hydrochloric acid solutions
Slowly it is added dropwise to reaction bulb；After being added dropwise to complete, continue stirring reaction 2h；Reaction solution is centrifuged into (5000rpm, 5min) washing number
Secondary, precipitation is placed in vacuum drying oven and dried.
3.GO-3,5-DABA-PANI- flavin adenine dinucleotide (FAD) (FAD)
GO-3,5-DABA-PANI 20mg are weighed, with 20ml pH=5.27PBS buffer solutions, adds 0.1156g
NHS (1.0mM) and 0.2867g EDC (1.5mM), stir 30min；Centrifuge (10000rpm, 5min), wash away unreacted EDC
And NHS；Gained activation products are diluted to 5ml, ultrasonic 5min with pH=7.4 PBS；10mg FAD additions are weighed, are stirred
Mix overnight；Centrifuge washing, vacuum drying.
4.GO-3,5-DABA-PANI-FAD-hemin (Hemin)
GO-3,5-DABA-PANI-FAD are taken into 10mg, 10ml is diluted to pH=5.27PBS buffer solutions；Add
0.0577g NHS (0.5mM) and 0.1435g EDC (0.75mM), stir 30min；5mg Hemin additions are weighed, are stirred overnight；
Centrifuge washing, and be dried in vacuo.
5. reduce GO-3,5-DABA-PANI-FAD-Hemin
5mg GO-3,5-DABA-PANI-FAD-Hemin is taken to measure 20ml with pH=7.4PBS buffer solutions to 5ml
80% hydrazine hydrate adds, stirring reaction 4h；Centrifuge washing removes hydrazine hydrate, and vacuum drying is stand-by.
6. structure-glucose oxidase (GOD) of pair enzyme reaction system+horseradish peroxidase (HRP)
Take and reduced GO-3,5-DABA-PANI-FAD-Hemin 1mg, be dissolved in 1ml pH=7.4PBS buffer solutions, point
1ml 1mg/ml are not taken to go the GOD (apo-GOD) and 1ml 1mg/ml of prothetic group to go the HRP (apo-HRP) of prothetic group to add, stirring is anti-
30min is answered, it is stand-by under the conditions of depositing in 4 DEG C.
Active testing：
1.GOD-HRP dual-enzyme systems contrast with GOD single enzymes activity
1.1PANI(1)：Mw=5400, Mn=4000
Enzyme system adds GOD altogether：HRP=1:1(1mg/ml)；Substrate is 50mM glucose solutions.Cyclic voltammetry (CV)
Detection range is 0~0.8V, and Preliminary Determination activity is optimal to improve 4.24 times, as shown in figure 1,
1.2PANI (chloroform)：Mw=6300, Mn=4100
Enzyme system adds GOD altogether：HRP=1:1(1mg/ml)；Substrate is 50mM glucose solutions.Cyclic voltammetry (CV)
Detection range is 0~0.8V, and Preliminary Determination activity is optimal to improve 3.62, as shown in Figure 2.
2. different enzyme fixations electro-chemical activity contrasts
Institute's test sample product are respectively：
(1) the double enzyme absorption of R GO-3,5-PANI (2) GOD+HRP
(2) R GO-3,5-PANI (2)-FAD apo-GOD+HRP GOD are engaged, HRP absorption
(3) R GO-3,5-PANI (2)-Hemin GOD+apo-HRP HRP are engaged, GOD absorption
(4) R GO-3,5-PANI (2)-FAD-Hemin apo-GOD+apo-HRP GOD, HRP are engaged
From data in Fig. 3, the detection activity for the method acquisition that double enzymes are fixed with affinity adsorption is optimal.
Embodiment 2
Such as the identical embodiment of embodiment 1, it is as follows to adjust each material：
1. CNT (CNT) -3,5- diaminobenzoic acids (3,5-DABA)
CNT 1.0g is weighed, is placed in beaker, adds 200ml THF, ultrasonic 1h；Add 1.0g3,5- diaminourea
Benzoic acid (~6.57 × 10-3Mol), ultrasonic 30min is continued；Solution after ultrasound is transferred in three-necked flask, capping
Bottle, vacuumizes, inflated with nitrogen, is heated to 70 DEG C of condensing refluxes, magnetic agitation 2h；Reaction solution centrifuges, and is washed with absolute ethyl alcohol
Precipitation is for several times；Sediment is placed in vacuum drying oven and dried.
2.CNT-3,5-DABA- polyanilines (PANI)
CNT-3,5- diaminobenzoic acid 0.2g are weighed, adds 60ml NMP mixing, ultrasonic disperse；Add polyaniline
0.15g, stir；By 0.07g ammonium persulfates (~3.11 × 10-4Mol) dissolved with 10ml 1.0M hydrochloric acid solutions, slowly drop
Add reaction bulb；After being added dropwise to complete, continue stirring reaction 2h；Reaction solution is centrifuged into (5000rpm, 5min) washing for several times, sunk
Shallow lake is placed in vacuum drying oven and dried.
3.CNT-3,5-DABA-PANI- flavin adenine dinucleotide (FAD)s (FAD)
CNT-3,5-DABA-PANI 20mg are weighed, with 20ml pH=5.2PBS buffer solutions, adds 0.12g NHS
With 0.29g EDC, 30min is stirred；Centrifuge (10000rpm, 5min), wash away unreacted EDC and NHS；Gained activation products are used
PH=7.4 PBS is diluted to 5ml, ultrasonic 5min；10mg FAD additions are weighed, are stirred overnight；Centrifuge washing, vacuum
Dry.
4.CNT-3,5-DABA-PANI-FAD-hemin (Hemin)
CNT-3,5-DABA-PANI-FAD are taken into 10mg, 10ml is diluted to pH=5.27PBS buffer solutions；Add
0.06g NHS and 0.15g EDC, stir 30min；5mg Hemin additions are weighed, are stirred overnight；Centrifuge washing, and vacuum is done
It is dry.
5. reduce CNT-3,5-DABA-PANI-FAD-Hemin
5mg CNT-3,5-DABA-PANI-FAD-Hemin is taken to measure 20ml with pH=7.4PBS buffer solutions to 5ml
80% hydrazine hydrate adds, stirring reaction 4h；Centrifuge washing removes hydrazine hydrate, and vacuum drying is stand-by.
6. structure-glucose oxidase (GOD) of pair enzyme reaction system+catalase (CAT)
Take and reduced CNT-3,5-DABA-PANI-FAD-Hemin 1mg, be dissolved in 1ml pH=7.4PBS buffer solutions, point
1ml 1mg/ml are not taken to go the GOD (apo-GOD) and 1ml 1mg/ml of prothetic group to go the CAT (apo-CAT) of prothetic group to add, stirring is anti-
30min is answered, obtains glucose oxidase (GOD) and catalase (CAT) orientation co-immobilization multi-enzyme system.
Through product test, the bioactivity of multi-enzyme system can be efficiently realized.
Embodiment 3
1. graphene oxide (GO) -3,5- diaminobenzoic acids (3,5-DABA)
Graphene oxide 1.0g is weighed, is placed in beaker, adds 200ml THF, ultrasonic 1h；Add 1.0g3,5- diaminos
Yl benzoic acid (~6.57 × 10-3Mol), ultrasonic 30min is continued；Solution after ultrasound is transferred in three-necked flask, closing is anti-
Bottle is answered, is vacuumized, inflated with nitrogen, is heated to 70 DEG C of condensing refluxes, magnetic agitation 2h；Reaction solution centrifuges, and is washed with absolute ethyl alcohol
Wash precipitation for several times；Sediment is placed in vacuum drying oven and dried.
2.GO-3,5-DABA- polyaniline (PANI)
GO-3,5- diaminobenzoic acid 0.2g are weighed, adds 60ml NMP mixing, ultrasonic disperse；Add polyaniline
0.15g, stir；By 0.07g ammonium persulfates (~3.11 × 10-4Mol) dissolved with 10ml 1.0M hydrochloric acid solutions, slowly drop
Add reaction bulb；After being added dropwise to complete, continue stirring reaction 2h；Reaction solution is centrifuged into (5000rpm, 5min) washing for several times, sunk
Shallow lake is placed in vacuum drying oven and dried.
3.GO-3,5-DABA-PANI- flavin adenine dinucleotide (FAD) (FAD)
Weigh GO-3,5-DABA-PANI 20mg, with 20ml pH=5.2PBS buffer solutions, add 0.12g NHS and
0.29g EDC, stir 30min；Centrifuge (10000rpm, 5min), wash away unreacted EDC and NHS；Gained activation products pH
=7.4 PBS is diluted to 5ml, ultrasonic 5min；10mg FAD additions are weighed, are stirred overnight；Centrifuge washing, vacuum are done
It is dry.
4.GO-3,5-DABA-PANI-FAD-hemin (Hemin)
GO-3,5-DABA-PANI-FAD are taken into 10mg, 10ml is diluted to pH=5.27PBS buffer solutions；Add
0.06g NHS and 0.15g EDC, stir 30min；5mg Hemin additions are weighed, are stirred overnight；Centrifuge washing, and vacuum is done
It is dry.
5. reduce GO-3,5-DABA-PANI-FAD-Hemin
5mg GO-3,5-DABA-PANI-FAD-Hemin is taken to measure 20ml with pH=7.4PBS buffer solutions to 5ml
80% hydrazine hydrate adds, stirring reaction 4h；Centrifuge washing removes hydrazine hydrate, and vacuum drying is stand-by.
6. structure-cholesterol oxidase+horseradish peroxidase of pair enzyme reaction system
Take and reduced CNT-3,5-DABA-PANI-FAD-Hemin 1mg, be dissolved in 1ml pH=7.4PBS buffer solutions, point
Do not take 1ml 1mg/ml to go the cholesterol oxidase of prothetic group and 1ml 1mg/ml to go the horseradish peroxidase of prothetic group to add, stir
30min is reacted, obtains cholesterol oxidase and horseradish peroxidase orientation co-immobilization multi-enzyme system.
The bioactivity of multi-enzyme system can efficiently be realized.
Claims (5)
1. the orientation common immobilization method of a kind of multi-enzyme system, it is characterised in that comprise the following steps：
Step 1, using the functional group on the carrier material with epoxy bond or carboxyl, one with 3,5- diaminobenzoic acids
Amino is reacted, and by being covalently keyed on carrier material for 3,5- diaminobenzoic acids, acquisition is connected to 3,5- diaminobenzene first
The carrier material of acid, represented with S- (3,5-DABA)；Wherein the mass ratio of 3,5- diaminobenzoic acids and carrier material is 1-5:
2；
Step 2, the S- that step 1 is obtained (3,5-DABA) react with polyaniline, and reaction passes through amino on S- (3,5-DABA)
The amino of benzoic acid reacts with polyaniline, formation covalent bond, and polyaniline and S- (3,5-DABA) mass ratio are 1-5:10, obtain
The carrier material of polyaniline is connected to, is represented with S- (3,5-DABA)-PANI；
Step 3, by the amino on the prothetic group flavin adenine dinucleotide (FAD) of flavo-enzyme, with step 2 obtained by S- (3,5-DABA)-
PANI is reacted, anti-by the carboxyl of the upper benzoic acid of S- (3,5-DABA)-PANI and the amino of flavin adenine dinucleotide (FAD)
Answer, formation covalent bond, the mass ratio of carrier material and flavin adenine dinucleotide (FAD) is 1-5:2, through acid amides key connection, connect
There is the carrier of flavin adenine dinucleotide (FAD), represented with S- (3,5-DABA)-PANI-FAD；
Step 4, the carboxyl on prothetic group-hemin of oxide enzyme, with step 3 obtained by S- (3,5-DABA)-PANI-
FAD is reacted, and is reacted by the amino of the polyaniline on S- (3,5-DABA)-PANI-FAD, forms covalent bond, gained
Product is represented with S- (3,5-DABA)-PANI-FAD-Hemin；Reduction treatment, reducing agent and product are carried out to product with reducing agent
Mass ratio be 8000-500:5；Wherein, hemin and S- (3,5-DABA)-PANI-FAD mass ratio are 1:1-5.
Step 5, by S- (3,5-DABA)-PANI-FAD in the flavo-enzyme of apoenzyme, the peroxidase of apoenzyme and step 4
Affine absorption is carried out, the mass ratio of the flavo-enzyme of the apoenzyme of apoenzyme, the peroxidase of apoenzyme and carrier is 1:1：1-
5, obtain the multi-enzyme system of orientation co-immobilization.
2. according to a kind of orientation common immobilization method of multi-enzyme system described in claim 1, it is characterised in that in step 1
Described carrier material is at least one of graphene oxide, graphene, CNT or graphite.
3. according to a kind of orientation common immobilization method of multi-enzyme system described in claim 1, it is characterised in that in step 3
Described flavo-enzyme is glucose oxidase (GOx), cholesterol oxidase, succinate dehydrogenase, D-AAO or fat
At least one of acyl CoA dehydrogenases.
4. according to a kind of orientation common immobilization method of multi-enzyme system described in claim 1, it is characterised in that in step 5
Described peroxidase is at least one of cytochromes, catalase or peroxidase.
5. according to a kind of orientation common immobilization method of multi-enzyme system described in claim 1, it is characterised in that in step 4
Described reducing agent is at least one of hydrazine hydrate, sodium borohydride or sodium citrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610013733.8A CN105441420B (en) | 2016-01-08 | 2016-01-08 | A kind of orientation common immobilization method of multi-enzyme system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610013733.8A CN105441420B (en) | 2016-01-08 | 2016-01-08 | A kind of orientation common immobilization method of multi-enzyme system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105441420A CN105441420A (en) | 2016-03-30 |
| CN105441420B true CN105441420B (en) | 2018-01-02 |
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| CN1307131A (en) * | 2000-01-26 | 2001-08-08 | 沈树宝 | Method for immobilizing biocatalyst using animal casing film and use thereof |
| CN101974510A (en) * | 2010-11-09 | 2011-02-16 | 厦门大学 | Method for carrying out coupling immobilization on coenzyme and coenzyme dependent enzyme |
| CN103033549A (en) * | 2012-12-17 | 2013-04-10 | 同济大学 | Preparation method of double-enzyme glucose sensor based on graphene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1307131A (en) * | 2000-01-26 | 2001-08-08 | 沈树宝 | Method for immobilizing biocatalyst using animal casing film and use thereof |
| CN101974510A (en) * | 2010-11-09 | 2011-02-16 | 厦门大学 | Method for carrying out coupling immobilization on coenzyme and coenzyme dependent enzyme |
| CN103033549A (en) * | 2012-12-17 | 2013-04-10 | 同济大学 | Preparation method of double-enzyme glucose sensor based on graphene |
Non-Patent Citations (2)
| Title |
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| Carrier free co-immobilization of alpha amylase,glucoamylase and pullulanase as combined cross-linked enzyme aggregates(combi-CLE-As):a tri-enzyme biocatalyst with one pot starch hydrolytic activity;Talekar S等;《Bioresource Technology》;20131231;第269-275页 * |
| 多酶共固定化反应体系的研究进展;汤玉兰等;《中国生物工程杂志》;20151231;第35卷(第1期);第82-87页 * |
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