CN101092614A - Method for preparing immune magnetic microsphere of separating and immobilizing enzyme, and application - Google Patents
Method for preparing immune magnetic microsphere of separating and immobilizing enzyme, and application Download PDFInfo
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Abstract
This invention relates to a methodfor preparing magnetic immuno-microspheres for separating, purifying and immobilizing enzymes, and their application. The method immobilizes monoclonal or multiclonal antibody onto magnetic microspheres by bio-specific coupling to obtain magnetic immuno-microspheres for separating, purifying and immobilizing enzymes. The magnetic immuno-microspheres can be used for separating, purifying and immobilizing target enzymes from fermented liquid and cell lysate. The immobilized enzyme can be used in continuous catalysis of relative substrate. The method is novel, simple and rapid.
Description
[technical field] the invention belongs to biological technical field, relate to and utilize immune magnetic microsphere to carry out the separation and purification and the immobilized method of enzyme, particularly can be used for the preparation and the application in synthetic L-halfcystine thereof of the immune magnetic microsphere of separation and purification L-cysteine desulfhydrase, to improve the transformation efficiency of L-halfcystine.
On [background technology] traditional method, the separation and purification process of enzyme is very complicated comparatively, needs through cytoclasis, centrifugation, saltouts, processes such as hydrophobic chromatography, zwitterion displacement chromatography and gel permeation chromatography, causes the yield of enzyme lower.
The immobilization technology of enzyme has become in the biotechnology very one of active research field; mainly be to adopt methods such as absorption, embedding, crosslinked and covalent attachment traditionally; all have limitation separately, development of new, enzyme immobilization technology has become the development trend in this field efficiently.
(Magnetic Microspheres, MMS), or (Magnetic Beads is that developed recently gets up and is widely used in a kind of new multifunctional reagent in fields such as magneticsubstance, biotechnology and biological medicine MB) to magnetic microsphere to be magnetic micro-beads.Magnetic microsphere is meant the polymer microsphere that contains magneticmetal or metal oxide (as Fe, Co, Ni and oxide compound thereof) ultrafine powder and have magnetic responsiveness.Because its superparamagnetism has brought revolutionary development for the target organism product separation; And because it can fast enriching in magnetic environment, therefore for realize that immune analysis separates and target administration provide may.Because it is with low cost that magnetic microsphere possesses, plurality of advantages such as the good and biocompatibility height of material, its range of application is day by day extensive.
Immunomagnetic isolation (Immunomagnetic separation, IMS), be based on the new technology that magnetic microsphere grows up, by antibody being fixed on the magnetic microsphere carrier, realization is to a step separation and purification of target antigen, when having magnetic response performance efficiently, has the high degree of specificity of affinity chromatography again.The method that is used for antibody immobilization in early days mainly adopts physisorphtion and chemical coupling connection method, low and the problem that exists antibody to leak of the former joint efficiency, the latter is by means of chemical functional group's coupling len antibody, there is randomness, and because of sterically hindered existence reduces its antigenic combination activity, this has influenced the research of immunomagnetic isolation The Application of Technology undoubtedly.
[summary of the invention] the objective of the invention is to solve the above-mentioned problems in the prior art, opens up a new approach for the separation and purification and the immobilization technology of enzyme, relates to utilizing immune magnetic microsphere that the purpose enzyme is carried out a step separation and purification and an immobilized technology.
The present invention adopts staphylococcus aureus protein A (ProteinA) as bridging agent IgG antibody to be fixed on the magnetic microsphere carrier, traditional physisorphtion and chemical bonding have been replaced with biologic specificity coupling connection method, prepared immune magnetic microsphere can be applicable to a step separation and purification purpose enzyme, and further immobilization purpose enzyme, and then its substrate carried out the successive catalyzed reaction.
Particular content of the present invention comprises:
One. the preparation of immune magnetic microsphere:
Preparation is used for the immune magnetic microsphere of separation and purification and immobilized enzyme, is to spend the night in 4 ℃ of reactions according to magnetic microsphere and ProteinA solution after the described method activation of Chinese patent ZL03144274.9, to adopt PBS damping fluid thorough washing 6～8 times; The magnetic microsphere that again coupling is associated with Protein A places NaHCO
3-NaCO
3Adsorb with polyvalent antibody or odd contradictive hydroperitoneum in the damping fluid and be connected; After PBS fully cleans, adopt dimethyl pimelate (DMP) to carry out the crosslinked fixing of ProteinA and IgG and magnetic microsphere surface, add TBS damping fluid termination reaction at last, promptly obtain can be used for the immune magnetic microsphere of the separation and purification of purpose enzyme after fully cleaning, its antibody coupling connection amount is 4～10mg/mL.
Two. the separation and purification of purpose enzyme:
The immune magnetic microsphere that coupling is associated with anti-purpose enzyme antibody mixed with the cell pyrolysis liquid that contains the purpose enzyme, in 2～8 ℃ of oscillatory reactions 2～12 hours; Be placed on then and be used to adsorb the fixed magnetic microballoon on the magnetic bead collector, discard reaction solution, after PBS fully cleans, adopt glycine-hydrochloride buffer (pH 1.5～3.5) in 2～8 ℃ of vibrations purpose enzyme of absorption that dissociates; The magnetic resolution microballoon, and adopt in the PB damping fluid and dissociation solution, promptly obtain the purpose enzyme of purifying.Collected immune magnetic microsphere is after PBS cleans, and is reusable.
Three. the immobilization of purpose enzyme:
Coupling is associated with the immune magnetic microsphere of anti-purpose enzyme antibody, mixes with cell pyrolysis liquid, and in 2～8 ℃ of oscillatory reactions 2～12 hours; Be placed on then and be used to adsorb the fixed magnetic microballoon on the magnetic bead collector, remove reaction solution, after PBS fully cleaned, the immune magnetic microsphere that is combined with the purpose enzyme can be used for the specific substrate of catalysis and carries out the successive enzymatic reaction.
Compare with the method for separation and purification enzyme traditionally, adopt the immune magnetic microsphere of biologic specificity coupling connection method preparation to be used for the separation and purification and the immobilization of purpose enzyme, advantage such as have conveniently, novel, efficient.
Beneficial effect of the present invention: the method that the present invention adopts biologic specificity coupling connection, mono-clonal or polyclonal antibody are fixed on the magnetic microsphere carrier, obtain can be used for the immune magnetic microsphere of separation and purification and immobilized enzyme; By immune magnetic microsphere, need not carry out complicated pre-treatment to sample, can be directly from cell pyrolysis liquid, a step separation and purification and an immobilization purpose enzyme, step greatly simplifies the operation.This immune magnetic microsphere can be repeatedly used, and has improved efficient and has reduced cost.Difference of the present invention and traditional method are a kind of easy, enzyme purification and immobilized new technology fast and efficiently.
[description of drawings]
Fig. 1 .SDS-PAGE, Westem blot and L-cysteine desulfhydrase active coloring are analyzed
A:SDS-PAGE; B:Western blot; C:L-cysteine desulfhydrase active coloring.Each swimming lane is among the figure: swimming lane 1: e. coli bl21 (DE3) the recombinant bacterial strain cellular lysate liquid that comprises pETcd; Swimming lane 2: the L-cysteine desulfhydrase recombinant protein of purifying; Swimming lane 3: pseudomonas putida TS1138 cellular lysate liquid; Swimming lane 4: through the pseudomonas putida TS1138 cellular lysate liquid of immune magnetic microsphere processing; Swimming lane 5: the L-cysteine desulfhydrase that elutes from magnetic microsphere.
Fig. 2. immune magnetic microsphere is handled TS1138 cellular lysate liquid and is transformed production L-halfcystine
A: L-semicystinol concentration/time curve in the solution; B: pyruvic acid concentration/time curve in the solution.Symbol among the figure " ■ " and " " represent primary TS1138 cellular lysate liquid; TS1138 cellular lysate liquid after " ▲ " and " △ " representative is handled through immune magnetic microsphere.
[embodiment]
Embodiment one
The contriver separates acquisition one strain pseudomonas putida TS1138 bacterial strain (Pseudomona sputida TS1138) voluntarily, and its cell pyrolysis liquid can catalytic substrate DL-2-amino-△
2-thiazoline-4-carboxylic acid (DL-2-amino-△
2-thiazoline-4-carboxylic acid, DL-ATC) synthetic L-halfcystine.
Described pseudomonas putida TS1138 (Pseudomonas putida TS1138), be preserved on January 17th, 2007 that " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (in No. 13 Institute of Microorganism, Academia Sinica of ZhongGuanCun north, Haidian District, BeiJing City-bar), preserving number is CGMCC No.1920.
Yet, the L-halfcystine that generates can be decomposed into pyruvic acid, H owing to have the L-cysteine desulfhydrase simultaneously in the lysate
2S and NH
3, greatly influenced the accumulation of purpose product L-halfcystine.This patent utilizes the immune magnetic microsphere of L-cysteine desulfhydrase Antibody Preparation, can remove the L-cysteine desulfhydrase in the lysate step, and the lysate after employing is handled is the synthetic L-halfcystine of catalytic substrate again, can improve its transformation efficiency greatly.
1.L-the clone of cysteine desulfhydrase gene
According to desulfhydrase gene (cd) sequence of Pseudomonas putida KT2440, design L-cysteine desulfhydrase upstream region of gene primer 5 ' CCG voluntarily
GGA TCCATG AAG TTG CCG ATC TAC CTT G-3 ' and downstream primer 5 ' CCC
AAG CTTTTA GTG GGC GGC CCA CTC-3 ', with Pseudomonas putida TS1138 genomic dna is template, pcr amplification obtains the desulfhydrase gene fragment of about 1.2kb size, the long 1215bp of being of its open reading frame is determined in the order-checking back, 404 amino acid of encoding, application also obtain Genbank sequence number: AY675347.
2.L-the recombinant expressed and purifying of cysteine desulfhydrase
With L-cysteine desulfhydrase gene subclone to expression vector pET-21a (+), construction recombination plasmid pETcd, through after the sequence verification, recombinant plasmid transformed is gone into e. coli bl21 (DE3), obtain to comprise e. coli bl21 (DE3) recombinant bacterial strain of recombinant plasmid pETcd.Recombinant bacterial strain is inoculated in 4mL contains in the LB substratum of penbritin 100 μ g/mL, behind 37 ℃ of shaking culture 12～16h, be forwarded to the 50mL fresh culture, continue to be cultured to OD with 1% inoculum size
600When reaching 0.6-0.8, adopting final concentration is that the IPTG of 1mmol/L was in 20 ℃ of abduction deliverings 24 hours, the thalline of collecting adopts the PBS damping fluid resuspended, ultrasonic disruption was handled 5 minutes, make the abundant cracking of thalline, utilize the L-cysteine desulfhydrase that comprises in the Ni-NTA affinity chromatography column separating purification lysate.Prove all that through SDS-PAGE, Western blot and the experiment of L-cysteine desulfhydrase active coloring purified L-cysteine desulfhydrase has high purity and good the enzyme activity.(Figure 1A, B, C. swimming lane 1,2)
3. the preparation of anti-L-cysteine desulfhydrase antibody:
Get the reorganization L-cysteine desulfhydrase albumen 0.5mL of 0.4mg/mL, with after the emulsification of isopyknic Freund's complete adjuvant thorough mixing L-cysteine desulfhydrase emulsification antigen.Get 0.1mL emulsification antigen and respectively 4 BALB/c mouse (female, 8 weeks) are carried out abdominal injection.Append injection and adopt Freund's incomplete adjuvant emulsification to prepare antigen, every two all booster shots once, totally three times.Last immunity is after 6 days, and the eyeball blood sampling is left standstill whole blood 2 hours, and centrifugal 20 minutes of 3000rpm collects serum deactivation 30 minutes in 55 ℃ of water-baths, after the packing in-20 ℃ of freezing preservations.
4. the preparation of Mierocrystalline cellulose immune magnetic microsphere
To spend the night in 4 ℃ of reactions according to cellulose magnetic microsphere and the ProteinA solution after the described method activation of Chinese patent ZL03144274.9, adopt PBS damping fluid thorough washing 6～8 times; The cellulose magnetic microsphere that coupling is associated with Protein A places NaHCO
3-NaCO
3In the damping fluid, adsorb with above-mentioned antiserum(antisera) and to be connected; After PBS fully cleans, adopt dimethyl pimelate (DMP) to carry out the crosslinked fixing of Protein A and IgG and cellulose magnetic microsphere surface, add TBS damping fluid termination reaction at last, promptly obtain can be used for the immune magnetic microsphere of separation and purification L-cysteine desulfhydrase after fully cleaning, adopt sandwich method ELISA to determine that its antibody coupling connection amount is 10mg/mL.
5. the preparation of pseudomonas putida TS1138 cellular lysate liquid
Picking one ring pseudomonas putida TS1138 inserts (glucose 20g, ATC3g, corn steep liquor 5g, urea 3g, NaCl 1.5g, MnSO the 4mL seed culture medium from flat board
4H
2O 0.1g, K
2HPO
43g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g is settled to 1L, and pH 7.5), 28 ℃ of 120r/min cultivated after 16 hours, were transferred to 100mL with 1% inoculum size and produced (glucose 30g, ATC 4g, corn steep liquor 1g, urea 3g, NaCl 1.5g, MnSO in the enzyme substratum
4H
2O 0.1g, K
2HPO
43g, MgSO
47H
2O 0.5g, FeSO
47H
2O 0.01g is settled to 1L, and pH 7.5), 28 ℃ of 120r/min cultivated after 16 hours, centrifugal, collect thalline, wash thalline 1 to 2 time with glycine-sodium hydrate buffer solution (pH8.0), again with the resuspended thalline of damping fluid, behind the ultrasonic disruption cell, centrifugal, collect supernatant, be cellular lysate liquid.
6.L-the immunomagnetic isolation purifying of cysteine desulfhydrase
Get the Mierocrystalline cellulose immune magnetic microsphere 1mL of preparation, place the Glass Containers of magnetic resolution device; Get 20mL pseudomonas putida TS1138 cellular lysate liquid (total protein content is 40mg), join in the Glass Containers, in 4 ℃ of slow oscillatory reaction 3h.The magnetic resolution microballoon is collected reaction solution and is used for follow-up synthetic L-halfcystine reaction.Clean microballoon 6 times with PBS, add 4mL 0.1M glycine-hydrochloride buffer (pH2.5) again,, dissociate by L-cysteine desulfhydrase adsorbed on the microballoon in 4 ℃ of oscillatory reactions 2 hours.The magnetic resolution microballoon is collected dissociation solution, uses a small amount of 1M PB damping fluid (pH7.0) neutralization immediately, obtains the L-cysteine desulfhydrase of purifying.Immune magnetic microsphere is after PBS cleans, in 4 ℃ of preservations, in order to reusing.SDS-PAGE, Western blot and the experiment of L-cysteine desulfhydrase active coloring be proof all, isolating L-cysteine desulfhydrase have very high purity and good the enzyme activity, and the cellular lysate liquid after handling through immune magnetic microsphere no longer contains L-cysteine desulfhydrase activity.(Figure 1A, B, C. swimming lane 3,4,5)
7.L-the conversion of halfcystine is synthetic
Respectively with mycelidium lysate and the cellular lysate liquid 20mL and 40mL substrate solution (0.2%DL-ATC, the 1.5%K that pass through after immune magnetic microsphere separates the L-cysteine desulfhydrase
2HPO
4, pH8.0) mix, and react at 37 ℃, measure the transformation efficiency of the synthetic L-halfcystine of its catalytic substrate DL-ATC in the differential responses time.
Record when reaction proceeds to 10h, the cellular lysate liquid after handling through immune magnetic microsphere can reach peak rate of conversion 96.4%, and after 14h all keep stable; And undressed original lysate reaches its peak rate of conversion 62.7% in reaction behind the 4h owing to there is the Decomposition of L-cysteine desulfhydrase, transformation efficiency after reaction times in decline rapidly.
8. the recycling effect of immune magnetic microsphere
Utilize this immune magnetic microsphere, repeat above-mentioned separation and purification L-cysteine desulfhydrase and transform synthetic L-halfcystine process 5 times.The data of table 1 show the L-cysteine desulfhydrase that each reaction is all contained in the separation and purification cellular lysate liquid fully, the average conversion of the synthetic L-halfcystine of cellular lysate liquid catalytic substrate DL-ATC after treatment reaches 96.2%, increase significantly than the mycelidium lysate, shown that this immune magnetic microsphere has good reuse.
The recycling effect of table 1 immune magnetic microsphere
| Number of times | L-cysteine desulfhydrase activity | Transformation efficiency | ||
| Original lysate (U) | Handle back lysate (U) | Original lysate (%) | Handle back lysate (%) | |
| 1 2 3 4 5 | 5.63±0.20 5.85±0.13 5.44±0.21 6.15±0.17 5.76±0.16 | Do not have | 62.7±1.7 61.5±2.0 63.0±2.3 60.8±1.9 61.8±1.8 | 96.4±1.8 95.6±2.0 97.0±1.4 96.2±2.1 96.0±1.7 |
| On average | 5.77±0.38 | Do not have | 61.9±1.1 | 96.2±0.8 |
Embodiment two,
1.L-the clone of cysteine desulfhydrase gene, Recombinant Protein Expression purifying, and the preparation method of anti-L-cysteine desulfhydrase antibody is with example one.
2. the preparation of agarose immune magnetic microsphere
To spend the night in 4 ℃ of reactions according to agarose magnetic microsphere and the Protein A solution after the described method activation of Chinese patent ZL03144274.9, adopt PBS damping fluid thorough washing 6～8 times; The agarose magnetic microsphere that coupling is associated with Protein A places NaHCO
3-NaCO
3Adsorb with L-cysteine desulfhydrase polyvalent antibody in the damping fluid and be connected; After PBS fully cleans, adopt glutaraldehyde with Protein A and the crosslinked agarose magnetic microsphere surface of being fixed in of IgG, add the glycine termination reaction at last, promptly obtain can be used for the immune magnetic microsphere of immobilization L-cysteine desulfhydrase after fully cleaning, adopt sandwich method ELISA to determine that its antibody coupling connection amount is 4mg/mL.
3.L-the immobilization of cysteine desulfhydrase
Get prepared immune magnetic microsphere 1mL, with 20mL pseudomonas putida TS1138 cellular lysate liquid (total protein content is 40mg), in 4 ℃ of slow oscillatory reaction 3h; Magnetic resolution discards reaction solution, after PBS fully cleans, promptly is fixed the immune magnetic microsphere of L-cysteine desulfhydrase, in 4 ℃ of preservations.
4. the activity of microsphere immobilized L-cysteine desulfhydrase is investigated
The 0.05%L-halfcystine solution of 5 parts of 4mL of configuration, get the immune magnetic microsphere that the 1mL immobilization has the L-cysteine desulfhydrase, under 37 ℃ of conditions successively with each 2h of each part L-halfcystine solution reaction, the content that comprises the degradation production pyruvic acid after the detection reaction in the solution generates the required enzyme amount of 1 μ mol product pyruvic acid as a L-cysteine desulfhydrase unit of activity (U) with per minute.
Table 2 result shows: immobilization has the microballoon of L-cysteine desulfhydrase all can keep comparatively stable activity in 5 secondary responses, has shown that this immune magnetic microsphere has immobilization effect preferably to the L-cysteine desulfhydrase.
The activity of the L-cysteine desulfhydrase that table 2. is microsphere immobilized is investigated
| Reaction | L-cysteine desulfhydrase activity (U) |
| 1 2 3 4 5 | 4.84±0.19 4.75±0.13 4.68±0.21 4.70±0.17 4.59±0.16 |
Claims (6)
1, the preparation method of the immune magnetic microsphere of a kind of separation and purification and immobilization purpose enzyme is characterized in that: magnetic microsphere after will activating and staphylococcus aureus protein A solution reaction, adopt PBS damping fluid thorough washing; The magnetic microsphere that again coupling is associated with staphylococcus aureus protein A adsorbs with the polyvalent antibody of the IgG antibody of anti-purpose enzyme or odd contradictive hydroperitoneum and is connected; After PBS fully cleans, adopt glutaraldehyde or dimethyl pimelate with staphylococcus aureus protein A and the crosslinked magnetic microsphere surface that is fixed on of IgG, add TBS damping fluid termination reaction at last, promptly obtain can be used for the separation and purification and the immobilized magnetic microsphere of purpose enzyme after fully cleaning, the coupling connection amount of its enzyme is 1～10mg/mL.
2, method according to claim 1 is characterized in that the magnetic microsphere that is adopted is the magnetic microsphere that contains the hydroxyl activity group, i.e. agarose magnetic microsphere, cellulose magnetic microsphere or polyvinyl alcohol magnetic microsphere.
3, method according to claim 1 and 2 is characterized in that described purpose enzyme is the L-cysteine desulfhydrase, and the IgG antibody of anti-purpose enzyme is anti-L-cysteine desulfhydrase antibody.
4, the application of the immune magnetic microsphere of the described method preparation of a kind of claim 1 is characterized in that immune magnetic microsphere is used for going on foot separation and purification purpose enzyme from the cell pyrolysis liquid one of complexity; Its separation and purification process is the immune magnetic microsphere that coupling is associated with anti-purpose enzyme antibody, with cell pyrolysis liquid in 2～8 ℃ of oscillatory reactions 2～12 hours; Be placed on then and be used to adsorb the fixed magnetic microballoon on the magnetic bead collector, remove reaction solution, after PBS fully cleaned, the glycine-hydrochloride buffer that adopts pH1.5～3.5 was in 2～8 ℃ of vibrations purpose enzyme of absorption that dissociates; The magnetic resolution microballoon is collected dissociation solution, immediately with the PB damping fluid neutralization of pH6.5～10.0, can obtain the purpose enzyme of purifying; After this microballoon is cleaned with PBS, reusable in 2～8 ℃ of preservations.
5, application according to claim 4, it is characterized in that described immune magnetic microsphere is the immune magnetic microsphere that is fixed with the described anti-L-cysteine desulfhydrase antibody of claim 3, and be used for the synthetic of L-halfcystine, detailed process is as follows: above-mentioned immune magnetic microsphere is joined in the pseudomonas putida TS1138 cellular lysate liquid, separate absorption L-cysteine desulfhydrase, collect immune magnetic microsphere then, the reaction solution of removing desulfhydrase can be used for follow-up microbial enzyme method and transforms the L-halfcystine, improves the transformation efficiency of synthetic L-halfcystine.
6, the application of the immune magnetic microsphere of the described method preparation of a kind of claim 1, it is characterized in that immune magnetic microsphere is used for the immobilization of purpose enzyme, its process is: the immune magnetic microsphere that coupling is associated with anti-purpose enzyme antibody, mix with cell pyrolysis liquid, in 2～8 ℃ of oscillatory reactions 2～12 hours; Be placed on then and be used to adsorb the fixed magnetic microballoon on the magnetic bead collector, remove reaction solution, after PBS fully cleaned, the immune magnetic microsphere that is fixed with the purpose enzyme promptly can be used for specific substrates is carried out continuous catalytic reaction.
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| CN108314701A (en) * | 2018-04-09 | 2018-07-24 | 青岛汉德森生物科技有限公司 | A method of utilizing magnetic bead extraction and antibody purification |
| CN109781986A (en) * | 2019-03-11 | 2019-05-21 | 复旦大学附属妇产科医院 | A kind of kit and preparation method thereof of the surface magnetic microparticle chemiluminescence immune detection CA125 Tn antigen |
| CN111308066A (en) * | 2020-03-10 | 2020-06-19 | 北京国科融智生物技术有限公司 | Novel method for enriching and detecting biomedical and environmental samples by magnetic particles |
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