CN102409037A - Immobilization method of acetylcholine esterase and application - Google Patents
Immobilization method of acetylcholine esterase and application Download PDFInfo
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- CN102409037A CN102409037A CN2011103125024A CN201110312502A CN102409037A CN 102409037 A CN102409037 A CN 102409037A CN 2011103125024 A CN2011103125024 A CN 2011103125024A CN 201110312502 A CN201110312502 A CN 201110312502A CN 102409037 A CN102409037 A CN 102409037A
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Abstract
The invention relates to a method for preparing immobilized recombinant acetylcholine esterase with Ni-NTA (nickel-nitrilotriacetic acid) resins. The method comprises the steps of mixing a crude enzyme liquid of recombinant acetylcholine esterase prepared by a gene engineering technique with the Ni-NTA resins treated by a buffer solution, and centrifuging to prepare the immobilized acetylcholine esterase, wherein in a process for preparing the recombinant acetylcholine esterase by using the gene engineering technique, reasonable selection and matching of a restriction enzyme, a transfer plasmid vector, a competent cell and a host can further influence the preparation of the immobilized acetylcholine esterase; and the use amount of the Ni-NTA resins and the enzyme liquid as well as centrifugation speed and time further influence the preparation effect of the immobilized acetylcholine esterase. The immobilized recombinant acetylcholine esterase prepared by the method has high activity ad good stability, is easy to store, and can be directly and effectively applied to substrate detection, inhibitor screening and pesticide residue detection. The method has short flow, is simple to operate, saves large amount of labors and materials, and is suitable for industrial application.
Description
Technical field
The present invention relates to a kind of process for fixation and application of enzyme, specifically, is that the reorganization E.C. 3.1.1.7 is fixed to the method and the application thereof of processing immobilization acetylcholinesteraseelectrochemistry on the Ni-NTA resin.
Background technology
E.C. 3.1.1.7 (AChE) is a kind of important lytic enzyme.It is the catalytic substrate acetylcholine hydrolyzation optionally, and its catalytic activity can be suppressed by organophosphorus or carbamate chemicals for agriculture.Utilize this characteristic E.C. 3.1.1.7 to can be used for detecting organophosphorus and carbamate chemicals for agriculture.Thereby AChE has important use in fields such as environmental protection, medical science, agricultural, military affairs.But resolvase character is unstable, and very easily inactivation can not be reused.Compare with resolvase, it is strong that immobilized enzyme has stability, and favorable reproducibility such as can reuse at advantage.Therefore, the research of the immobilization technology of enzyme has become the important component part of enzyme engineering.
At present, the process for fixation of E.C. 3.1.1.7 has absorption method, entrapping method and crosslinking.Absorption method is adsorbed in E.C. 3.1.1.7 on the carrier according to the physical adsorption principle, and the advantage of this method is that method is simple, and enzyme is lived intact.But because a little less than the physisorption power, so adopting absorption method fixed E.C. 3.1.1.7 to be prone to run off, enzyme is lived and is descended soon.Entrapping method is wrapped in E.C. 3.1.1.7 in the polymkeric substance grid, though solved the enzyme losing issue,, harsh polymerizing condition can make the E.C. 3.1.1.7 inactivation, and the E.C. 3.1.1.7 that is embedded in the polymkeric substance depths is difficult to play a role.Crosslinking adopts bifunctional reagent that the form of enzyme with covalent linkage is connected on the carrier securely, but violent chemical reaction still can make the E.C. 3.1.1.7 inactivation.Therefore search out the suitable carriers material, the immobilization reorganization E.C. 3.1.1.7 that develop enzymic activity height, good stability, is easy to preserve is that related industries department is extremely expected.
Ni-NTA resin (His label purifying resin) is a kind of purification media that is used for purifying His label recombinant protein, and it is to be coupled a kind of four tooth sequestrant NTA and to be got by 4% crosslinked gel, has Ni
2+At present, the Ni-NTA resin has been used to purifying and has contained histidine-tagged reorganization E.C. 3.1.1.7, be about to recombinant protein and treated Ni-NTA mixed with resin after, utilize that affinity chromatography combines, washing, wash-out be with the method for purification of recombinant proteins.
Summary of the invention
The object of the present invention is to provide a kind of method with Ni-NTA resins immobilization reorganization E.C. 3.1.1.7.
The contriver is in the process of studying for a long period of time; Unexpected find not wash, elution step can make that to contain histidine-tagged reorganization E.C. 3.1.1.7 immobilized effectively to the Ni-NTA resin; Directly be used for enzyme activity determination, inhibitor screening and Detecting Pesticide, and have acetylcholine esterase active height, good stability, be easy to advantages such as preservation.
The objective of the invention is to realize through following technical measures:
A kind of method for preparing immobilization reorganization E.C. 3.1.1.7 is characterized in that:
A. raw material is prepared
Adopt genetic engineering technique to obtain to have histidine-tagged reorganization E.C. 3.1.1.7 crude enzyme liquid;
With damping fluid washing Ni-NTA resin, centrifugal treating;
B. with the Ni-NTA mixed with resin after reorganization E.C. 3.1.1.7 crude enzyme liquid that makes in the steps A and the processing, centrifugal treating.
Above-mentioned genetic engineering technique is meant some special genes or dna segment, sends into recipient cell through carrier or other means, makes a kind of genetics operation that they breed in recipient cell and express.Step comprises:
(1) obtaining of dna fragmentation, the i.e. separation of goal gene and preparation
(2) being connected of dna fragmentation and carrier, make recombinant DNA
(3) the external source recombinant dna fragment imports host cell
(4) select
(5) destination gene expression
The contriver finds in actual mechanical process; In using genetic engineering technique preparation reorganization E.C. 3.1.1.7 process; Restriction enzyme, transferring plasmid vector, competent cell, host's selection is also very important, the choose reasonable of these factors with cooperate the preparation that can further influence immobilization acetylcholinesteraseelectrochemistry.
Preferably, above-mentioned host is pichia spp Pichia pastoris KM71.
More preferably, competent cell is the e. coli jm109 bacterial strain in the above-mentioned recombinant DNA preparation.
Most preferably, the restriction endonuclease sites that goal gene is introduced in above-mentioned purpose gene isolation and the preparation is EcoR I, and transferring plasmid vector is pPIC9K, and making the linearizing restriction enzyme of recombinant expression plasmid carrier is Sal I.
The contriver finds utilizing this legal system to be equipped with in the immobilization reorganization E.C. 3.1.1.7 process simultaneously, and the consumption of Ni-NTA resin and enzyme liquid, has further influence to the preparation effect of immobilization acetylcholinesteraseelectrochemistry at centrifugal speed and time.
Preferably, the total protein content of above-mentioned reorganization E.C. 3.1.1.7 crude enzyme liquid is 500 μ gml
-1And more than, crude enzyme liquid with the processing after the Ni-NTA resin mix by 3: 1 volume ratio.
More preferably, the prescription of above-mentioned damping fluid is 300mM NaCl, 50mM Na3PO4, and the 10mM imidazoles, pH 7.4.
Further preferably, above-mentioned damping fluid consumption is 5 times of Ni-NTA resin volume.Most preferably, the rotating speed of above-mentioned centrifugally operated is 3000rpm, and the time was 30 seconds.
Specifically, the operation steps of genetic engineering technique is following:
1. be designed for the upstream and downstream primer of amplification acetylcholinesterasegene gene sequence, all introduce restriction enzyme digestion sites at primer 5 '-end, histidine-tagged six of downstream primer introducings, so that be attached on the Ni-NTA resin;
2. pcr amplification acetylcholinesterasegene gene cDNA;
3. the acetylcholinesterasegene gene cDNA fragment that the digestion with restriction enzyme amplification that is 1. provided with step obtains produces sticky end;
4. with step digestion with restriction enzyme transferring plasmid vector 1., carry out dephosphorylation after cutting fully and handle;
5. with step 3. gained acetylcholinesterasegene gene cDNA fragment and step 4. the transferring plasmid vector of gained be connected the transformed competence colibacillus cell;
6. perhaps carry out PCR with transferring plasmid vector upstream primer and acetylcholinesterasegene gene downstream primer, the correct transformant of screening closure, order-checking with acetylcholinesterasegene gene upstream primer and transfer vector downstream primer;
7. extract the recombinant expression plasmid after sequence verification, make the expression plasmid linearizing with being different from step digestion with restriction enzyme 1.;
8. the method that adopts electric shock to transform changes the linearizing expression plasmid over to as heterologous gene expression system host's competent cell;
9. the picking positive transformant places the bottle that shakes that 25mL BMGY substratum is housed, and in 30 ℃, 250rpm is cultured to OD
600=2.The centrifugal 5min of 3000rpm under the room temperature collects thalline, with the resuspended thalline of 50mL BMMY inducing culture, is positioned over 30 ℃, and on the shaking table of 250rpm continued growth 7-9 days, every 24h adds 100% methyl alcohol to final concentration in substratum be 2.0%;
10. the centrifugal 5min of 3000rpm under the room temperature collects the inducing culture supernatant, promptly gets to have histidine-tagged reorganization E.C. 3.1.1.7 crude enzyme liquid.If the content of reorganization E.C. 3.1.1.7 is lower, can adopt the method for ultrafiltration to concentrate.
The present invention has following beneficial effect:
1. the inventive method flow process is short, simple to operate, has saved great amount of manpower and material resources; Realize quick preparation immobilization reorganization E.C. 3.1.1.7, be suitable for industry and laboratory applications.
Active height, good stability of the immobilization acetylcholinesteraseelectrochemistry of this law preparation, be easy to preservation.
3. the immobilization acetylcholinesteraseelectrochemistry of this law preparation directly and effectively is used for substrate detection, inhibitor screening and Detecting Pesticide.
Embodiment
Through embodiment the present invention is carried out concrete description below; Be necessary to be pointed out that at this following examples only are used for the present invention is further specified; Can not be interpreted as the restriction to protection domain of the present invention, the technician in this field can make some nonessential improvement and adjustment to the present invention according to the invention described above content.
Embodiment 1
A. raw material is prepared
Adopt genetic engineering technique to obtain to have histidine-tagged reorganization E.C. 3.1.1.7 crude enzyme liquid;
1. be designed for the upstream and downstream primer of amplification Asiatic migrotory locust acetylcholinesterasegene gene sequence, all introduce EcoR I restriction enzyme site at primer 5 '-end, histidine-tagged six of downstream primer introducings, so that be attached on the Ni-NTA;
2. pcr amplification Asiatic migrotory locust acetylcholinesterasegene gene cDNA;
3. cut the Asiatic migrotory locust acetylcholinesterasegene gene cDNA fragment that amplification obtains with EcoR I enzyme, produce EcoR I sticky end;
4. cut carrier pPIC9K with EcoR I enzyme, carry out dephosphorylation after cutting fully and handle;
5. the Asiatic migrotory locust acetylcholinesterasegene gene cDNA fragment after EcoR I enzyme being cut is connected transformed into escherichia coli JM109 with the pPIC9K carrier that passes through after enzyme of the same race is cut;
6. use pPIC9K carrier upstream primer and Asiatic migrotory locust acetylcholinesterasegene gene downstream primer, the correct transformant of screening closure, order-checking;
7. extract the recombinant expression plasmid pPIC9K after sequence verification, cut with Sal I enzyme and carry out the expression plasmid linearizing;
8. the method that adopts electric shock to transform changes the linearizing expression plasmid over to pichia spp (Pichia pastoris KM71) competent cell;
9. the picking positive transformant places the bottle that shakes that 25mL BMGY substratum is housed, and in 30 ℃, 250rpm is cultured to OD
600=2.The centrifugal 5min of 3000rpm under the room temperature collects thalline, with the resuspended thalline of 50mL BMMY inducing culture, is positioned over 30 ℃, and on the shaking table of 250rpm continued growth 7-9 days, every 24h adds 100% methyl alcohol to final concentration in substratum be 2.0%;
10. the centrifugal 5min of 3000rpm under the room temperature collects the inducing culture supernatant, promptly gets to have histidine-tagged reorganization E.C. 3.1.1.7 crude enzyme liquid.
In the 1.5mL centrifuge tube, pack into the Ni-NTA resin (Novagen company, 100mL dress) of 60 μ L under the normal temperature with the 3000rpm rotating speed centrifugal 30 seconds, is abandoned supernatant; With the binding buffer liquid of 5 times of volumes (the 10mM imidazoles, pH 7.4 for 300mMNaCl, 50mM Na3PO4) washing resin three times, centrifugal, abandon supernatant; B. in centrifuge tube, add crude enzyme liquid (90 μ L), resuspended resin, slight jolting is centrifugal after 30 minutes, abandons supernatant.
Detect the activity of immobilized E.C. 3.1.1.7 according to following steps:
1. with the resuspended resin of 250 μ L test buffered soln, add 25 μ L 5,5 '-dithio two (2-dinitrobenzoic acid) and acetylthiocholine solution respectively, make its final concentration be respectively 0.40mmolL
-1And 0.25mmolL
-1
2. mixing was hatched 5 minutes for 30 ℃;
3. centrifugal, get supernatant;
4. supernatant was hatched 30 seconds at 50 ℃;
5. do not receive the sample article and be blank to add, measure to add and receive the absorbancy of sample article gained supernatant at 412 nano wave length places through the supernatant of same treatment.
6. after half a year, 1.～5. measure immobilized acetylcholine esterase active 4 ℃ of refrigerator storage according to above-mentioned steps.
Above-mentioned ordinary method is with after recombinant protein and the treated Ni-NTA mixed with resin, utilizes the resulting recombinant proteins of purification step such as affinity chromatography combines, washing, wash-out, detects the activity of E.C. 3.1.1.7 then by above-mentioned steps.
Can find out high, the anti-storage of the reorganization Asiatic migrotory locust acetylcholine esterase active of Ni-NTA after immobilized from The above results.
The step of carrying out the substrate detection with immobilized E.C. 3.1.1.7 is following:
1. detect the resuspended resin of damping fluid with 250 μ L;
2. add 25 μ L 5,5 '-dithio two (2-dinitrobenzoic acid), making its final concentration is 0.40mmolL
-1
3. add 25 μ L material to be checked, making its final concentration is 0.25mmolL
-1
4. mixing was hatched 5 minutes for 30 ℃;
5. centrifugal, get supernatant;
6. supernatant was hatched 30 seconds at 50 ℃;
7. do not add the absorbancy of material gained supernatant to be checked to add material to be checked and to be blank, to measure at 412 nano wave length places through the supernatant of same treatment;
8. confirm according to absorbance whether material to be checked is the effect substrate of E.C. 3.1.1.7.
The step of carrying out inhibitor screening with immobilized E.C. 3.1.1.7 is following:
1. detect the resuspended resin of damping fluid with 240 μ L;
2. add 10 μ L suppressor factor to be screened, making its final concentration is 1.0mmolL
-1
3. mixing was hatched 3 minutes for 30 ℃;
4. add 25 μ L 5,5 '-dithio two (2-dinitrobenzoic acid) and acetylthiocholines respectively, make its final concentration be respectively 0.40mmolL
-1And 0.25mmolL
-1
5. mixing was hatched 5 minutes for 30 ℃;
6. centrifugal, get supernatant;
7. supernatant was hatched 30 seconds at 50 ℃;
8. do not wait to screen material and be blank to add, measure to add and waits to screen the absorbancy of material gained supernatant at 412 nano wave length places through the supernatant of same treatment;
9. confirm according to absorbance whether wait to screen material is the suppressor factor of E.C. 3.1.1.7.
The step of carrying out Detecting Pesticide with immobilized E.C. 3.1.1.7 is following:
1. detect the resuspended resin of damping fluid with 240 μ L;
2. add 10 μ L sample to be checked;
3. mixing was hatched 3 minutes for 30 ℃;
4. add 25 μ L 5,5 '-dithio two (2-dinitrobenzoic acid) and acetylthiocholines respectively, make its final concentration be respectively 0.40mmolL
-1And 0.25mmolL
-1
5. mixing was hatched 5 minutes for 30 ℃;
6. centrifugal, get supernatant;
7. supernatant was hatched 30 seconds at 50 ℃;
8. do not receive the sample article and be blank to add, measure to add and receive the absorbancy of sample article gained supernatant at 412 nano wave length places through the supernatant of same treatment;
9. qualitatively judging according to absorbance receives the sample article whether pesticide residue are arranged.
According to above-mentioned use immobilized E.C. 3.1.1.7 to carry out step that substrate detects whether detect the sulfo-propionylcholine respectively as shown in table 1 as the effect substrate mensuration result of E.C. 3.1.1.7 with the sulfo-BuCh:
Table 1 E.C. 3.1.1.7 effect substrate detected result
| Examined object matter | The sulfo-propionylcholine | The sulfo-BuCh | Blank |
| Absorbance | 0.847 | 0.094 | 0.089 |
The gained result can find out from table 2, and the sulfo-propionylcholine is the effect substrate of E.C. 3.1.1.7, and the sulfo-BuCh then is not the effect substrate of E.C. 3.1.1.7.
The step of carrying out inhibitor screening according to the immobilized E.C. 3.1.1.7 of above-mentioned usefulness filters out the stronger chemical insecticide of restraining effect has Malathion, thiophos, Chlorpyrifos 94.
Table 21.0mmolL
-1Malathion, thiophos, Chlorpyrifos 94 are to the restraining effect of E.C. 3.1.1.7
| Suppressor factor | The Malathion | Thiophos | Chlorpyrifos 94 | Unrestraint agent contrast |
| Absorbance | 0.091 | 0.073 | 0.100 | 0.840 |
The step of carrying out Detecting Pesticide according to the immobilized E.C. 3.1.1.7 of above-mentioned usefulness can detect organophosphorus or carbamate insecticides is residual, and its detectability can reach 10
-6MolL
-1
Table 3 immobilization acetylcholinesteraseelectrochemistry is to the detection case (concentration unit: 10 of Malathion and Chlorpyrifos 94
-6MolL
-1)
Claims (9)
1. a method for preparing reorganization E.C. 3.1.1.7 fixation support is characterised in that, adopts the following steps preparation:
A. raw material is prepared
Adopt genetic engineering technique to obtain to have histidine-tagged reorganization E.C. 3.1.1.7 crude enzyme liquid;
With damping fluid washing Ni-NTA resin, centrifugal treating;
B. with the Ni-NTA mixed with resin after reorganization E.C. 3.1.1.7 crude enzyme liquid that makes in the steps A and the processing, centrifugal treating.
2. the method for claim 1 is characterized in that: described genetic engineering technique comprises that the separation of goal gene and preparation, the preparation of recombinant DNA, external source recombinant dna fragment import steps such as host cell;
Said host is pichia spp Pichia pastoris KM71.
3. want 2 described methods like right, it is characterized in that: in the preparation of said recombinant DNA, the competent cell of employing is the e. coli jm109 bacterial strain.
4. like claim 2 or 3 described methods; It is characterized in that: the restriction endonuclease sites that goal gene was introduced during said goal gene separated and prepares is EcoR I; Transferring plasmid vector is pPIC9K, and making the linearizing restriction enzyme of recombinant expression plasmid carrier is the Sal I.
5. according to claim 1 or claim 2 method, it is characterized in that: the total protein content of said reorganization E.C. 3.1.1.7 crude enzyme liquid is 500 mgml
-1More than, crude enzyme liquid with handle after the Ni-NTA resin mix by the volume ratio of 3:1.
6. according to claim 1 or claim 2 method, it is characterized in that: the prescription of damping fluid is 300 mM NaCl in the said steps A, 50 mM Na3PO4,10 mM imidazoles, pH 7.4.
7. like claim 1,2 or 5 described methods, it is characterized in that: said damping fluid consumption is 5 times of Ni-NTA resin volume.
8. like claim 1,2,5 or 6 described methods, it is characterized in that: said centrifugal rotation speed is 3000rpm, and the time is 30 seconds.
9. the method for claim 1 is characterized in that:
A. raw material is prepared
Adopt genetic engineering technique to obtain to have histidine-tagged reorganization E.C. 3.1.1.7 crude enzyme liquid, described genetic engineering technique comprises that the separation of goal gene and preparation, the preparation of recombinant DNA, external source recombinant dna fragment import steps such as host cell; Said host is pichia spp Pichia pastoris KM71; In the preparation of said recombinant DNA, the competent cell of employing is the e. coli jm109 bacterial strain; The restriction endonuclease sites that goal gene was introduced during said goal gene separated and prepares is EcoR I, and transferring plasmid vector is pPIC9K, and making the linearizing restriction enzyme of recombinant expression plasmid carrier is the Sal I; Damping fluid (300 mM NaCl with 5 times of volumes; 50 mM Na3PO4,10 mM imidazoles, pH 7.4) washing resin three times; With the 3000rpm rotating speed centrifugal 30 seconds, abandon supernatant;
B. the Ni-NTA resin after reorganization E.C. 3.1.1.7 crude enzyme liquid that makes in the steps A and the processing is mixed by the volume ratio of 3:1, centrifugal treating, the total protein content of reorganization E.C. 3.1.1.7 crude enzyme liquid is 500 mgml-1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882000A (en) * | 2014-03-17 | 2014-06-25 | 中国科学院过程工程研究所 | Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof |
| CN110760501A (en) * | 2019-05-07 | 2020-02-07 | 宁波大学 | Co-crosslinking immobilization method of acetylcholinesterase |
-
2011
- 2011-10-14 CN CN 201110312502 patent/CN102409037B/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
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| 《Romote Sensing，Environment and Transportation Engineering (RSETE),2011 international conference on》 20110626 Fei Zhu, et al. Integrated Purification and Immobilization of Glutamate Decarboxylase 第6903-6906页 1-9 , * |
| 《湖北农业科学》 20100831 吕延成 等 乙酰胆碱酯酶固定化条件的研究 第1887-1889页 1-9 第49卷, 第8期 * |
| FEI ZHU, ET AL.: "Integrated Purification and Immobilization of Glutamate Decarboxylase", 《ROMOTE SENSING，ENVIRONMENT AND TRANSPORTATION ENGINEERING (RSETE),2011 INTERNATIONAL CONFERENCE ON》 * |
| 吕延成 等: "乙酰胆碱酯酶固定化条件的研究", 《湖北农业科学》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103882000A (en) * | 2014-03-17 | 2014-06-25 | 中国科学院过程工程研究所 | Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof |
| 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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