CN106480011A - Preparation method of purified coupled immobilized adenylate cyclase - Google Patents
Preparation method of purified coupled immobilized adenylate cyclase Download PDFInfo
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- CN106480011A CN106480011A CN201611122284.7A CN201611122284A CN106480011A CN 106480011 A CN106480011 A CN 106480011A CN 201611122284 A CN201611122284 A CN 201611122284A CN 106480011 A CN106480011 A CN 106480011A
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- 102000030621 adenylate cyclase Human genes 0.000 title claims abstract description 35
- 108060000200 adenylate cyclase Proteins 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 66
- 108090000790 Enzymes Proteins 0.000 claims abstract description 66
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical class OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000002184 metal Substances 0.000 claims abstract description 11
- 229910052751 metal Inorganic materials 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 20
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
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- 238000000746 purification Methods 0.000 claims description 13
- 229920005989 resin Polymers 0.000 claims description 12
- 239000011347 resin Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000012986 modification Methods 0.000 claims description 10
- 230000004048 modification Effects 0.000 claims description 10
- 229920000936 Agarose Polymers 0.000 claims description 9
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 9
- 239000012071 phase Substances 0.000 claims description 7
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- 239000004593 Epoxy Substances 0.000 claims description 6
- 238000006555 catalytic reaction Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 6
- HRYZWHHZPQKTII-UHFFFAOYSA-N chloroethane Chemical compound CCCl HRYZWHHZPQKTII-UHFFFAOYSA-N 0.000 claims description 6
- 229960003750 ethyl chloride Drugs 0.000 claims description 6
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 4
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- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 claims description 3
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 claims 1
- 101710095468 Cyclase Proteins 0.000 claims 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 claims 1
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims 1
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- 238000000034 method Methods 0.000 abstract description 23
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
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- 229910052737 gold Inorganic materials 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
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- LVIYYTJTOKJJOC-UHFFFAOYSA-N nickel phthalocyanine Chemical compound [Ni+2].C12=CC=CC=C2C(N=C2[N-]C(C3=CC=CC=C32)=N2)=NC1=NC([C]1C=CC=CC1=1)=NC=1N=C1[C]3C=CC=CC3=C2[N-]1 LVIYYTJTOKJJOC-UHFFFAOYSA-N 0.000 description 2
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- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- 150000008065 acid anhydrides Chemical class 0.000 description 1
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- 125000000539 amino acid group Chemical group 0.000 description 1
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- 235000011089 carbon dioxide Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
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- 239000012050 conventional carrier Substances 0.000 description 1
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- 150000002338 glycosides Chemical class 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229940005654 nitrite ion Drugs 0.000 description 1
- 125000004433 nitrogen atom Chemical group N* 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 125000004430 oxygen atom Chemical group O* 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N phosphoric acid Substances OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
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- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y406/00—Phosphorus-oxygen lyases (4.6)
- C12Y406/01—Phosphorus-oxygen lyases (4.6.1)
- C12Y406/01001—Aodenylate cyclase (4.6.1.1)
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Abstract
The invention discloses a preparation method of purified coupled immobilized adenylate cyclase, (1) preparing affinity adsorption medium: adding 5-100 g of metal chelating resin carrier with surface modified iminodiacetic acid into 10-200 mL of 0.01-1 mol/L NiCl2.6H2Oscillating the solution in the O solution for 2 to 4 hours at the temperature of 25 to 30 ℃ and the rpm of 150 to 200, and performing suction filtration to obtain an affinity adsorption medium; (2) immobilization of the enzyme: and (2) taking 0.1-1 g of the affinity adsorption medium obtained in the step (1), adding 1.5-15 mL of adenylate cyclase solution with the concentration of 0.1-2 mg/mL, carrying out oscillation for 2-4 h at 25-30 ℃ and 150-200 rpm, and centrifuging to obtain the immobilized adenylate cyclase. The immobilized enzyme obtained by the method has the advantages of high enzyme activity, reusability, less enzyme activity loss and the like.
Description
Technical field
The invention belongs to technical field of bioengineering, a kind of system of specific design purification co-immobilization adenyl cyclase
Preparation Method.
Background technology
Enzyme is the biomolecule that a class has catalysiss, its production with the mankind and closely related, the institute of world of living
There is biology to leave enzyme all can not survive.It is gentle etc. that the feature of enzyme includes high efficiency, specificity, reaction condition.But resolvase is stable
The poor, easy in inactivation of property and can not reusing, causes it to be difficult to apply in large-scale industrial production.Difficult in order to solve this
Topic, enzyme immobilization technology is arisen at the historic moment.Immobilized enzyme has many good qualities compared with resolvase, such as stability is high, can carry out technique processing,
Reusable.Conventional enzyme immobilizatio method includes absorption method, cross-linking method, covalent method and investment.Wherein absorption method and
Investment belongs to physical method, and cross-linking method and covalent method belong to chemical method.
Enzyme activity is the linear module of enzyme activity, and international biochemical meeting EC regulation is (the most suitable in optimum reaction conditionses
Substrate, optimum pH, the ionic strength of the most suitable buffer and 25 DEG C) under, catalysis per minute one micromole substrate is converted into product institute
The enzyme amount needing is an international enzyme activity unit.Enzyme activity is an important indicator weighing immobilized enzyme.Impact immobilization
The factor of enzyme enzyme activity has a lot, in addition to common temperature, pH, inhibitor, activator etc., also have some special impacts because
Element is for example：
(1) selection of carrier：Carrier selects to be directly connected to the activity of immobilization success and immobilized enzyme.Big absolutely
Most enzymes is all protein, its function of the structures shape of protein, its function of the structures shape of enzyme in the same manner.In enzyme molecule simultaneously
Not every position all plays a role, and the amino acid residue only having sub-fraction region in enzyme molecule can with Binding Capacity simultaneously
Catalytic substrate produces product, and this special area is the active center of enzyme, and the active center of enzyme is directly related with enzyme activity
Region, the structure of zymophore is closely related with the vigor of enzyme.During immobilized, if the carrier selecting does not conform to
Suitable, the active center of enzyme and the higher structure of pheron may be directly influenced, thus causing immobilized enzyme enzymatic activity low
Even inactivate.Following several principle is typically followed in the selection of fixed enzyme vector：1. contain in carrier and can react with enzyme
Functional group can strengthen the binding ability of itself and enzyme, improves the stability of immobilized enzyme.2. carrier has larger specific surface area
It is easy to loose structure and enzyme crosslinking, improve fixing rate.3. carrier should be water insoluble, can prevent enzyme from inactivating.4. carrier should have
Have preferable mechanical rigid and stability, improve the repeat usage of immobilized enzyme 5. carrier should select as far as possible reusable
, can substantially reduce immobilization cost 6. carrier should nontoxic, degradable, environment is no affected.Conventional carrier include organic and
Inorganic material two big class.Organic material is mainly ion exchange resin, and spent ion exchange resin makees the mainly excellent of carrier adsorption enzyme
Point is easy to operate, mild condition, is obtained in that higher immobilized enzyme and carrier are reusable after eluting.
(2) fixing direction：Enzyme immobilizatio has arbitrarily and the dividing of orientation.Arbitrarily immobilization refers to that enzyme passes through in enzyme molecule
Lysine residue be arbitrarily fixed on carrier, this immobilization can hinder substrate to enter into the avtive spot of enzyme thus reducing enzyme
Activity, and be usually that many sites combine when enzyme is arbitrarily immobilized on carrier, its fixed amount can be reduced.The directional at-tachment of enzyme
Changing is then to couple together enzyme in the specific part of enzyme with carrier, so that enzyme is arranged by certain direction in carrier surface, this solid
Fixedization can make the avtive spot of enzyme be exposed to outside, facilitate substrate to enter active site, can improve the activity of immobilized enzyme.Enzyme
Oriented immobilization method is a lot, passes through glycosyl part immobilization, enzyme and metal ion including the affine connection of enzyme and antibody, enzyme
Connection and the method for molecular biology.The connection of wherein enzyme and metal ion belongs to a kind of conventional oriented immobilization method.
W metal2+Can be fixed on what the protein of histidine mark oriented on carrier, realize the isolation and purification of target protein.
(3) permeability of immobilized enzyme：After research shows that enzyme is fixing, because enzyme-to-substrate is in different phases, substrate
Could need to contact with enzyme with the diffusion layer of surrounding through water insoluble net block, so the kinetics of immobilized enzyme is to a great extent
On by immobilized enzyme permeability determine.Improve immobilized enzyme permeability and can improve its enzyme activity.Large pore material duct width, passes
Matter resistance is little, can significantly improve the permeability of immobilized enzyme.Because these advantages make it in terms of relevant immobilized enzyme
Research is increasingly taken seriously.
CAMP is ring gland glycosides -3 ', 5 '-phosphoric acid, and it is intracellular second message,second messenger, participates in intracellular multiple metabolic responses such as
Adjust blood glucose balance, promote fat splitting to produce ATP energy supply and make the latter activate carbonic acid by activating carbonic anhydride protein kinase
The acid-base balance of acid anhydride enzyme adjustment cell.Also clinical research is had to show cAMP with multiple disease relationships closely, such as hypertension, coronary disease
Disease, diabetes etc., thus develop these diseases of Drug therapy based on cAMP.In the synthesis of protein, gene turn
Record and translation are all affected by cAMP.Therefore, medical experiment and research work are required to substantial amounts of cAMP.Traditional synthesis
The method of cAMP is mainly chemical synthesiss, adopts pyridine solvent the method more, not only can be to environment, also seriously
Impact workman's is healthy.In prior art, produce cAMP and mainly include two methods, a kind of is in traditional chemical routes
On the basis of improve, reduce pollution.Another kind is then to produce cAMP using adenyl cyclase catalysis ATP.At present, have no profit
Produce the technology of cAMP with immobilization adenyl cyclase.Producing cAMP hence with immobilization adenyl cyclase will be cAMP's
Produce and a new direction is provided.
1975, Porath utilized immobilization metal chelating affinity chromatograph technology (IMAC) to separate protein first, starts
Metal ion is used as a kind of affinity ligand, starts the frontier of protein affine technolog.Fixing metal affine absorption skill
Art has low cost compared with traditional affine technolog, adsorbent preparation easily, many advantages, such as separation process is simple.Just because of
These advantages, this technology is increasingly widely used on enzyme immobilizatio.In IMAC, common adsorbent includes LX-
1000IDA etc..The carrier of LX-1000IDA is agarose gel, and iminodiacetic acid (IDA) is its chelate group, and it can pass through shape
Become five-membered ring to form stable chelate with metal, metal ion is strapped in certain area of space.IDA and the knot of nickelous
Close be actually nitrogen-atoms above it, two oxygen atoms and nickelous chela and.IDA has lot of advantages, such as volume
Little, have hydrophilic and metal ion chela and afterwards be in electric neutrality.These advantages make adsorbent can subtract when protein is combined
Few steric restriction and non-specific adsorption.Certainly, LX-1000IDA also has its shortcoming, and the price of IDA is conventional with respect to other
Resin is expensive, this adds increased the cost of industrialized production.Meanwhile, also there is one with IDA for the adsorbent of chela mediating recipe can not neglect
Depending on problem, if IDA is combined insecure with metal ion, pollution or even the degeneration of protein can be caused.Control separating some
During the albumen treated, metal ion such as Ni2+、Cu2+Reveal and can produce serious harm to human body.
Content of the invention
The technical problem to be solved in the present invention is to provide extracellular polysaccharide in a kind of efficient clostridium acetobutylicum biomembrane
Separating and extracting process.(a kind of efficient, method of safety in production cAMP is provided)
For solving above-mentioned technical problem, the present invention provides following technical scheme：
A kind of preparation method of purification co-immobilization adenyl cyclase, comprises the steps：
(1) prepare affine adsorbing medium：Metal chelation resin carrier by the surface modification iminodiacetic acid of 5～100g
It is added to the NiCl of 10～200mL, 0.01～1mol/L2.6H2In O solution, shake under the conditions of 25～30 DEG C, 150～200rpm
Swing 2～4h, sucking filtration obtains affine adsorbing medium；
(2) enzyme immobilizatio：Take the affine adsorbing medium that 0.1～1g step (1) obtains, addition 1.5～15mL concentration is
The adenyl cyclase solution of 0.1～2mg/mL, on described adenyl cyclase carry histidine-tagged, 25～30 DEG C, 150
2～4h, centrifugation, being fixed adenyl cyclase is vibrated under the conditions of～200rpm.
Wherein, the metal chelation resin carrier of described surface modification iminodiacetic acid be prepared as follows as
Under：
(1a) by agarose by 6%～12% soluble in water make aqueous phase, be slowly poured into while hot in oil phase, stirring is all
Even, described oil phase composition is as follows：Toluene, chloroform, the volume ratio of Span-80 are 72～80：28～40：1～2, will mix
Compound is incubated 3～5min under the conditions of 50～60 DEG C, filters out the agarose microbeads that particle diameter is 60 mesh, cleans；
(2a) weigh in the NaOH aqueous solution that 15～20g agarose microbeads are added to 30～40mL, 2mol/L, add 100
～110mg sodium borohydride, 3～4mL epoxy ethyl chloride, 30～35 DEG C of vibrations, Deca 15～20mL successively in 10～12h
NaOH and 9～12mL epoxy ethyl chloride, are further continued for concussion reaction 10～12h, wash carrier；
(3a) carrier obtaining in step (2a) is added to the Na of 40～50mL, 2mol/L2CO3In solution, add 4～
The IDA of 5g vibrates 20～24h in 30～35 DEG C of shaking tables, is rinsed well with water, obtains the gold of surface modification iminodiacetic acid
Belong to chelating resin carrier.
Wherein, described adenyl cyclase solution is prepared as follows：
(1) structure of recombinant bacterium：Adenyl cyclase from arthrobacterium CGMCC 3584 is cloned on plasmid, will
Recombinant plasmid transformed E. coli, obtains recombinant bacterium.
(2) abduction delivering：Recombinant bacterium is connected in the LB fluid medium of 5mL, 30 DEG C, 200r/min shaking table culture 12h
Seed liquor, switching 4mL seed liquor in the LB fluid medium of 100mL, 30 DEG C, 200r/min shaking table culture to OD 600
Reach 0.8, add the IPTG induction 6h of final concentration of 0.6mmol/L, after induction terminates, 5000r/min centrifugation 10min collects and sends out
Zymotic fluid, removes supernatant, thalline subzero 20 DEG C frozen standby.
(3) crush thalline：Take 6g thalline to be added to the Tris-HCl buffer of the 0.1mol/L of 1.5L, concentrate 4 times, with even
By its breaking cellular wall, about in 500～900kPa, it is molten that the supernatant collected is adenyl cyclase to pressure during breaking cellular wall to matter machine
Liquid.
The immobilization adenyl cyclase that the preparation method of above-mentioned purification co-immobilization adenyl cyclase prepares
Within protection scope of the present invention.
Application in catalyzing and synthesizing cyclic adenosine monophosphate for the above-mentioned immobilization adenyl cyclase.
Wherein, catalystic converter system is as follows：
Adenyl cyclase 9.85～10.23U/mg, ATP 29.8～30.2g/L, acetone acid 2.6～2.7g/L,
MgCl2.6H2O15～16g/L, ammonia adjusts PH8.0～8.5；
Catalytic reaction condition is as follows：25～30 DEG C, 150～200rpm reaction 8～12h.
Beneficial effect：
In prior art, produce cAMP and mainly include two methods, one kind is to carry out on the basis of traditional chemical routes
Improve although pollution can be reduced, but production process is complex and inreal to eliminate pollution, another kind is then to utilize
Adenyl cyclase catalysis ATP produces cAMP, although the method high-efficient simple is pollution-free, adenyl cyclase used can not
Re-use, enzyme activity is easily suffered a loss.In the present invention immobilization adenyl cyclase produce cAMP technology not only remain above-mentioned
The advantage of method, also makes up the deficiency in these methods.The outstanding advantage of the method includes enzyme activity height, energy Reusability, enzyme activity
Loss is few.
Brief description
Fig. 1：The electrophoretogram of enzyme liquid after enzyme liquid and absorption, swimming lane 1 is crude enzyme liquid before purification, and swimming lane 2,3 is after purification
Effluent, 4 is marker.
Fig. 2：The electrophoretogram of purifying protein, swimming lane 1 is marker, and swimming lane 2 is the crude enzyme liquid before fixing, and swimming lane 3 is to fix
Remaining enzyme liquid afterwards, swimming lane 4 does not wash eluent for after fixing, and swimming lane 5 washs eluent for after fixing.
Fig. 3：Modify IDA resin carrier microsphere shape appearance figure.
Fig. 4：Mounting medium surface texture.
Fig. 5：Carrier inside section of structure.
Fig. 6：Carrier modification Ni2+Internal structure profile afterwards.
Fig. 7：Immobilized enzyme batch response diagram.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, it is as it will be easily appreciated by one skilled in the art that real
Apply the content described by example and be merely to illustrate the present invention, and should not be also without limitation on basis described in detail in claims
Invention.
Embodiment 1：
The metal chelation resin carrier of surface modification iminodiacetic acid is prepared as follows as follows：
(1a) by agarose by 6%～12% soluble in water make aqueous phase, be slowly poured into while hot in oil phase, stirring is all
Even, described oil phase composition is as follows：Toluene, chloroform, the volume ratio of Span-80 are 72～80：28～40：1～2, will mix
Compound is incubated 3～5min under the conditions of 50～60 DEG C, filters out the agarose microbeads that particle diameter is 60 mesh, cleans；
(2a) weigh in the NaOH aqueous solution of 15～20g agarose microbeads 30～40mL, 2mol/L, add 100～110mg
Sodium borohydride, 3～4mL epoxy ethyl chloride, 30～35 DEG C vibration, in 10～12h successively Deca 15～20mL NaOH and 9～
12mL epoxy ethyl chloride, is further continued for concussion reaction 10～12h, washes carrier；
(3a) carrier obtaining in step (2a) is added to the Na of 40～50mL, 2mol/L2CO3In solution, add 4～
The IDA of 5g vibrates 20～24h in 30～35 DEG C of shaking tables, is rinsed well with water, obtains the gold of surface modification iminodiacetic acid
Belong to chelating resin carrier.
Embodiment 2：Ni2+The mensure of adsorbance
Weigh the NiCl of 2.38g2.6H2O is settled in the volumetric flask of 100mL, Ni in solution2+Concentration is 0.1mol/L.Take
5mL is settled in the volumetric flask of 500mL, then taking-up 10mL is settled in 200mL volumetric flask from 500mL solution, now solution
Middle Ni2+Concentration is 5 × 10-5Mol/L, finally takes out 5mL, 10mL, 20mL, 50mL from 200mL solution successively and is respectively settled to
In 100mL volumetric flask, corresponding Ni2+Concentration is 2.5 × 10-5mol/L、1×10-5mol/L、5×10-6Mol/L and 2.5 ×
10-6mol/L.With Ni in diacetyldioxime metric measurement solution2+Content, by the Ni of each concentration preparing2+Solution is respectively inhaled
Take 10mL in 25mL volumetric flask, blank draws 10mL pure water, plus 2mL ammonium citrate solution and 1mL iodine solution are in test portion
In, add water to 20mL and shake up, plus 2mL diacetyldioxime solution, shake up, plus the Na of 2mL2- EDTA solution, adds water to graticule, shakes up.
Use 10mm cuvette, with water as reference liquid, measure the absorbance of nitrite ion under 530nm wavelength and deduct the extinction of blank assay
Degree.With each concentration as abscissa, absorbance is mapped for vertical coordinate.
The Ni of 1g resin absorption can be calculated by standard curve2+Concentration is 4.68 × 10-5Mol/L, about 0.278mg.Real
Apply example 3：The mensure of pheron adsorbance
First prepare the standard protein solution of 1g/L, take 7 test tubes label respectively, add each reagent by following form, in
Absorbance is measured, with A at 595nm595For vertical coordinate, standard protein concentration is abscissa, in coordinate plot on X axis standard curve.
With the affine adsorbing medium absorption enzyme liquid having obtained, enzyme liquid consumption 15mL, in 25 DEG C, 150r/min 2 hours of vibration, terminate
Afterwards according to aforesaid operations, measure its A595nm, then obtain protein content using standard curve.The supernatant take original enzyme liquid, having adsorbed
Liquid and carry out gel electrophoresiss experiment with the eluent after 500mmol imidazoles eluting immobilized enzyme, result such as Fig. 1 and Fig. 2.By marking
It is about 2.623mg that directrix curve can calculate the protein content that 1g resin can adsorb；In Fig. 1, band at 45KD for the supernatant becomes
Secretly illustrate that the affine adsorbing medium prepared has effect for the purification of albumen and absorption；Afforded with imidazoles in Fig. 2
Single band further demonstrates that the affine adsorbing medium of preparation has good effect for protein purification and absorption.
Table 1 determination of protein concentration table
Embodiment 4：Carrier and Ni2+Modify the electron-microscope scanning after carrier
The resin microsphere carrier that the iminodiacetic acid of the present invention is modified carries out electron-microscope scanning, and absorption Ni2+Afterwards
Carrier carries out electron-microscope scanning, obtains Fig. 2.Fig. 2 is to modify IDA resin carrier microsphere shape appearance figure；Fig. 3 is mounting medium surface texture
Electron-microscope scanning picture；Fig. 4 is the electron-microscope scanning picture of carrier inside structural profile；Fig. 5 is carrier modification Ni2+Internal junction afterwards
The electron-microscope scanning picture of structure section.By picture as can be seen that all there are many holes and pit, this hole in this carrier surface and inside
The abundant structure of amount is more beneficial for enzyme immobilizatio.
Embodiment 5：The research of immobilized enzyme stability in use.
Weigh the immobilized enzyme that 10g has prepared, preserve at 4 DEG C, its enzyme activity of results of regular determination.The storage of immobilized enzyme is steady
Qualitative test result as shown in fig. 6, Fig. 6 shows the stability of immobilized enzyme very well, the reuse through 8 batches, immobilized enzyme
Relative enzyme activity still up to more than the 90% of initial value.Immobilized enzyme enzyme activity is 9.85～10.23U/mg, and resolvase enzyme activity is
3.04～5.10U/mg.
Claims (7)
1. a kind of preparation method of purification co-immobilization adenyl cyclase is it is characterised in that comprise the steps：
(1) prepare affine adsorbing medium：The metal chelation resin carrier of the surface modification iminodiacetic acid of 5～100g is added
NiCl to 10～200mL, 0.01～1mol/L2.6H2In O solution, under the conditions of 25～30 DEG C, 150～200rpm vibration 2～
4h, sucking filtration obtains affine adsorbing medium；
(2) purification of enzyme and immobilization：Take the affine adsorbing medium that 0.1～1g step (1) obtains, add 1.5～15mL concentration
Adenyl cyclase solution for 0.1～2mg/mL, on described adenyl cyclase carry histidine-tagged, 25～30 DEG C,
2～4h, centrifugation, being fixed adenyl cyclase is vibrated under the conditions of 150～200rpm.
2. the preparation method of purification co-immobilization adenyl cyclase according to claim 1 is it is characterised in that described
The metal chelation resin carrier of surface modification iminodiacetic acid be prepared as follows：
(1a) by agarose by 6%～12% soluble in water make aqueous phase, be slowly poured in oil phase while hot, stir, institute
The oil phase composition stated is as follows：Toluene, chloroform, the volume ratio of Span-80 are 72～80：28～40：1～2, by mixture in
It is incubated 3～5min under the conditions of 50～60 DEG C, filters out the agarose microbeads that particle diameter is 60 mesh, clean；
(2a) weigh in the NaOH aqueous solution that 15～20g agarose microbeads are added to 30～40mL, 2mol/L, add 100～
110mg sodium borohydride, 3～4mL epoxy ethyl chloride, 30～35 DEG C of vibrations, Deca 15～20mL NaOH successively in 10～12h
With 9～12mL epoxy ethyl chloride, it is further continued for concussion reaction 10～12h, washes carrier；
(3a) carrier obtaining in step (2a) is added to the Na of 40～50mL, 2mol/L2CO3In solution, add 4～5g's
IDA vibrates 20～24h in 30～35 DEG C of shaking tables, is rinsed well with water, obtains the metal chelating of surface modification iminodiacetic acid
Resin carrier.
3. the preparation method of purification co-immobilization adenyl cyclase according to claim 1 is it is characterised in that described
Adenyl cyclase solution be prepared as follows：
(1) structure of recombinant bacterium：Adenyl cyclase from arthrobacterium CGMCC 3584 is cloned on plasmid, will recombinate
Plasmid transformation escherichia coli E.coli, obtains recombinant bacterium.
(2) abduction delivering：Recombinant bacterium is connected in the LB fluid medium of 5mL, 30 DEG C, 200r/min shaking table culture 12h must plant
Sub- liquid, the seed liquor of switching 4mL in the LB fluid medium of 100mL, 30 DEG C, 200r/min shaking table culture reaches to OD 600
0.8, add the IPTG induction 6h of final concentration of 0.6mmol/L, after induction terminates, 5000r/min centrifugation 10min collects fermentation
Liquid, removes supernatant, thalline subzero 20 DEG C frozen standby.
(3) crush thalline：Take 6g thalline to be added to the Tris-HCl buffer of the 0.1mol/L of 1.5L, concentrate 4 times, use homogenizer
By its breaking cellular wall, about in 500～900kPa, the supernatant collected is adenyl cyclase solution to pressure during breaking cellular wall.
4. the immobilization adenylic acid that the preparation method of purification co-immobilization adenyl cyclase described in claim 1 prepares
Cyclase.
5. application in catalyzing and synthesizing cyclic adenosine monophosphate for the immobilization adenyl cyclase described in claim 4.
6. application according to claim 3 is it is characterised in that catalystic converter system is as follows：
Adenyl cyclase 9.85～10.23U/mg, ATP 29.8～30.2g/L, acetone acid 2.6～2.7g/L,
MgCl2.6H2O15～16g/L, ammonia adjusts PH8.0～8.5.
7. application according to claim 6 is it is characterised in that catalytic reaction condition is as follows：25～30 DEG C, 150～
200rpm reacts 8～12h.
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| CN114107275A (en) * | 2021-11-19 | 2022-03-01 | 辽宁凯莱英医药化学有限公司 | Enzyme immobilization carrier and preparation method thereof, immobilized enzyme and preparation method thereof |
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