CN118028283A - Aminated chelate metal ion catalyst and preparation method and application thereof - Google Patents
Aminated chelate metal ion catalyst and preparation method and application thereof Download PDFInfo
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- 229910021645 metal ion Inorganic materials 0.000 title claims abstract description 27
- 239000013522 chelant Substances 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title abstract description 17
- 102000004190 Enzymes Human genes 0.000 claims abstract description 63
- 108090000790 Enzymes Proteins 0.000 claims abstract description 63
- 229910001425 magnesium ion Inorganic materials 0.000 claims abstract description 54
- 238000006243 chemical reaction Methods 0.000 claims abstract description 53
- 108091000080 Phosphotransferase Proteins 0.000 claims abstract description 30
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 30
- 102000020233 phosphotransferase Human genes 0.000 claims abstract description 30
- 229910001453 nickel ion Inorganic materials 0.000 claims abstract description 22
- VEQPNABPJHWNSG-UHFFFAOYSA-N Nickel(2+) Chemical compound [Ni+2] VEQPNABPJHWNSG-UHFFFAOYSA-N 0.000 claims abstract description 18
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 57
- 229920000936 Agarose Polymers 0.000 claims description 51
- 239000000243 solution Substances 0.000 claims description 40
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 29
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- 229910052759 nickel Inorganic materials 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 15
- 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 14
- 239000007853 buffer solution Substances 0.000 claims description 14
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- 239000000758 substrate Substances 0.000 claims description 12
- OOXNYFKPOPJIOT-UHFFFAOYSA-N 5-(3-bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d]pyrimidin-4-amine;dihydrochloride Chemical compound Cl.Cl.C=12C(N)=NC=NC2=NC(C=2C=NC(=CC=2)N2CCOCC2)=CC=1C1=CC=CC(Br)=C1 OOXNYFKPOPJIOT-UHFFFAOYSA-N 0.000 claims description 11
- 102100032534 Adenosine kinase Human genes 0.000 claims description 11
- 108010076278 Adenosine kinase Proteins 0.000 claims description 11
- 229920000642 polymer Polymers 0.000 claims description 11
- 239000000872 buffer Substances 0.000 claims description 10
- 239000012064 sodium phosphate buffer Substances 0.000 claims description 7
- 229920002873 Polyethylenimine Polymers 0.000 claims description 6
- QHMLGYJOGFXZOP-UHFFFAOYSA-N [Ni]N Chemical compound [Ni]N QHMLGYJOGFXZOP-UHFFFAOYSA-N 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 102000001253 Protein Kinase Human genes 0.000 claims description 5
- 108060006633 protein kinase Proteins 0.000 claims description 5
- 108010039918 Polylysine Proteins 0.000 claims description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 4
- 229920000656 polylysine Polymers 0.000 claims description 4
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- 102000005548 Hexokinase Human genes 0.000 claims description 2
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- 102000012435 Phosphofructokinase-1 Human genes 0.000 claims description 2
- 108010022684 Phosphofructokinase-1 Proteins 0.000 claims description 2
- 229920002401 polyacrylamide Polymers 0.000 claims description 2
- 108010011110 polyarginine Proteins 0.000 claims description 2
- FAGUFWYHJQFNRV-UHFFFAOYSA-N tetraethylenepentamine Chemical compound NCCNCCNCCNCCN FAGUFWYHJQFNRV-UHFFFAOYSA-N 0.000 claims description 2
- -1 polyetheramine Polymers 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 34
- 238000001179 sorption measurement Methods 0.000 abstract description 14
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 10
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 abstract description 10
- 238000005576 amination reaction Methods 0.000 abstract description 10
- 238000009776 industrial production Methods 0.000 abstract description 3
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- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 3
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 3
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 3
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- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 3
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- 229910019142 PO4 Inorganic materials 0.000 description 2
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- 239000011777 magnesium Substances 0.000 description 1
- ATTFYOXEMHAYAX-UHFFFAOYSA-N magnesium nickel Chemical compound [Mg].[Ni] ATTFYOXEMHAYAX-UHFFFAOYSA-N 0.000 description 1
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- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
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Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention belongs to the technical field of catalysis and application of enzymes, and particularly relates to an aminated chelate metal ion catalyst and a preparation method and application thereof. The preparation method of the catalyst comprises the steps of modifying magnesium ions on an affinity medium chelating nickel ions; then carrying out amination treatment; and finally, fixing the kinase on the surface of the catalyst to realize the recycling of the kinase. The catalyst obtained by the invention can catalyze the reaction, improve the utilization rate of ATP, obviously improve the enzyme activity, can be used as a carrier to immobilize the enzyme through affinity adsorption, has better adsorption effect and higher carrier rate, and realizes the repeated use of the enzyme, thus having wide application prospect in the large-scale industrial production of the immobilized enzyme.
Description
Technical Field
The invention belongs to the technical field of catalysis and application of enzymes, and particularly relates to an aminated chelate metal ion catalyst and a preparation method and application thereof.
Background
Enzymes are a class of biomolecules with catalytic functions that are closely related to human production and life, and all organisms in the world cannot survive from the enzymes. The characteristics of the enzyme include high efficiency, specificity, mild reaction conditions and the like. However, the free enzyme has poor stability, is easy to inactivate and cannot be reused, so that the free enzyme is difficult to apply to large-scale industrial production. To solve this problem, immobilized enzyme technology has been developed. The immobilized enzyme has many advantages over the free enzyme, such as high stability, process and repeated use. Common enzyme immobilization methods include adsorption, cross-linking, covalent and entrapment. Wherein the adsorption method and the embedding method belong to physical methods, and the crosslinking method and the covalent method belong to chemical methods.
However, these conventional methods have a number of disadvantages. Adsorption methods tend to cause desorption of enzymes; covalent bonding is easy to destroy the structure of the enzyme, so that the recovery rate of the enzyme activity is greatly lost; embedding is easy to cause mass transfer limitation of a substrate; the crosslinking reaction is more intense, and the loss of enzyme activity is larger. Affinity adsorption has now become a new trend in immobilized enzymes, and has become a widely used immobilization method in recent years. The affinity adsorption immobilized enzyme hardly affects the activity or folding of the protein by the adsorption of the enzyme orientation. In the affinity immobilization, enzyme molecules can form a relatively stable affinity covalent bond with metal ions on a carrier, and the binding force is more stable than the electrostatic acting force and the hydrophobic acting force, so that the enzyme molecules are not easy to fall off in the reaction process. Compared with the traditional immobilization method, the affinity immobilization has both activity and stability.
The invention uses immobilized metal ion affinity chromatography (Immobilized Metal Ion Affinity Chromatography, IMAC) which is based on the coordination of transition metal ions (such as Cu 2+、Co2+ and Ni 2+) with alpha-amino groups on amino acid residues and atoms (S, O, N) containing lone pair electrons in the side chains for enzyme purification and immobilization. The high selectivity of IMAC for recombinant proteins containing polyhistidine tags allows for the firm immobilization of the target protein on the carrier via metal ions, while the hybrid protein is not able to further sequester the metal ions due to the lack of histidine tags. IMAC has the advantages of low cost, convenient chelation, large capacity, high concentration, stability and easy regeneration compared with other affinity immobilization. When separating some therapeutic proteins, the leakage of metal ions such as Ni 2+、Cu2+ can cause serious harm to human bodies, and the affinity medium obtained by the method is easy to have the problems of low immobilized enzyme activity, low reaction speed and poor batch stability when the immobilized enzyme is used.
Phosphate transfer and hydrolysis reactions are the basis of kinase and phosphatase signaling pathways, critical to cell function and growth regulation, efficient catalysis of kinases requires the participation of cofactors such as NADH, ATP, etc., and the assistance of magnesium ions. ATP is expensive as a phosphate donor in the reaction. In general immobilization, there are often problems of low ATP utilization rate and low yield, so that it is necessary to invent a catalyst which can not only catalyze a reaction and increase ATP utilization rate, but also can be used as a carrier to immobilize an enzyme by affinity adsorption, thereby realizing the reuse of the enzyme.
Disclosure of Invention
The invention aims to solve the technical problem of providing an aminated chelate metal ion catalyst and a preparation method thereof aiming at the defects of the prior art.
The invention also solves the technical problem of providing the application of the aminated chelate metal ion catalyst in immobilized kinase catalytic reaction.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for preparing an aminated chelate metal ion catalyst, comprising the steps of:
(1) Placing the affinity medium for chelating nickel ions into PBS buffer solution, and then adding magnesium chloride solution for reaction to obtain the affinity medium for chelating nickel ions-magnesium ions;
(2) And (3) placing the nickel ion-magnesium ion chelating affinity medium obtained in the step (1) into a polyamino polymer solution for reaction to obtain an amino nickel ion-magnesium ion chelating affinity medium, namely the catalyst.
Wherein in the step (1), the affinity medium comprises any one or a combination of a plurality of agarose, resin and chitosan; preferably the affinity medium is agarose, resin or chitosan.
Wherein in the step (1), the PBS buffer solution is 30-70 mM sodium phosphate buffer solution with pH value of 8-8.5. Preferably, the PBS buffer is 50mM sodium phosphate buffer, pH 8.2.
Wherein, in the step (1), the concentration of the affinity medium for chelating nickel ions in the PBS buffer solution is 50-150 mg/mL; preferably, the concentration is 100mg/mL.
Wherein in the step (1), the concentration of the magnesium chloride solution is 30-70 mM, the solvent is water, and the volume ratio of the magnesium chloride solution to the PBS buffer solution is 1 (0.5-1.5). Preferably, the concentration of the magnesium chloride solution is 50mM, the solvent is water, and the volume ratio of the magnesium chloride solution to the PBS buffer solution is 1:1.
Wherein in step (1), the reaction is oscillation, and preferable reaction conditions are: the temperature is 25 ℃, the oscillation time is 1.5-2.5 hours, and the rotating speed is 180-200 rpm.
Wherein in the step (2), the polyamino polymer comprises any one or a combination of a plurality of polylysine, polyethyleneimine, polyacrylamide, polyarginine, polyether amine, glycosaminoglycan and tetraethylenepentamine; preferably, the polyamino polymer is polylysine or polyethyleneimine.
Wherein in the step (2), the concentration of the affinity medium chelating nickel ions-magnesium ions in the polyamino polymer solution is 4-10 mg/mL; preferably, the concentration is 5mg/mL.
Wherein in the step (2), the concentration of the polyamino polymer solution is 30-70 mM, and the solvent is pure water. Preferably, the concentration of the polyamino polymer solution is 50mM, and the solvent is pure water.
Wherein in step (2), the reaction is oscillation, and the preferable reaction conditions are: the temperature is 25 ℃, the shaking is carried out for 0.5 hour, and the rotating speed is 180-200 rpm.
In the step (1) and the step (2), after the reaction is finished, the corresponding affinity medium is obtained through washing and suction filtration. Specifically, the washing is pure water washing for 2-4 times, and the suction filtration is vacuum filtration, and the suction filtration is carried out until the water is dried.
The aminated chelate metal ion catalyst prepared by the preparation method is also in the scope of the invention.
Specifically, the aminated chelate metal ion catalyst is a catalyst for catalyzing reaction of interface activated kinase, namely, the catalyst can activate all kinases on the surface of an affinity medium to catalyze reaction.
Specifically, the aminated chelate metal ion catalyst can also be used as a carrier, and kinase can be reused by immobilized kinase through affinity adsorption.
The use of the above-described aminated chelate metal ion catalysts for catalytic reactions of immobilized kinases is also within the scope of the present invention.
Wherein the kinase is provided with a histidine tag, and the kinase comprises any one or a combination of a plurality of uridine-cytidine kinase, adenosine kinase, protein kinase, 6-phosphofructokinase and hexokinase; preferably, the kinase is a uridine-cytidine kinase or an adenosine kinase, more preferably a uridine-cytidine kinase.
The application is that an aminated chelate metal ion catalyst is added into Tris-HCL buffer solution and enzyme solution of kinase to carry out immobilization reaction to obtain immobilized kinase, and then the immobilized kinase is used for catalytic reaction.
Wherein the Tris-HCl buffer is 50mM Tris-HCl buffer with pH of 8.5.
Wherein the amount of the aminated chelate metal ion catalyst is not related to the enzyme activity of the enzyme solution of the kinase, and is mainly related to the protein concentration and the volume of the enzyme solution of the kinase. Specifically, the dosage ratio of the amination chelate metal ion catalyst to Tris-HCL buffer solution to enzyme solution of kinase is as follows: 0.3g (1-100 mL): (1.5-100 mL). Preferably, the ratio of the aminated chelate metal ion catalyst to Tris-HCL buffer solution to enzyme solution of kinase is as follows: 0.3g:1mL:1.5mL. Preferably, the kinase enzyme solution has a kinase protein concentration of 0.6g/L.
Wherein, the reaction conditions of the immobilization reaction are as follows: the reaction is carried out for 1 hour at the temperature of 4 to 10 ℃ and the rpm of 180 to 200 rpm. Preferred reaction conditions are: the mixture was shaken at 10℃and 200rpm for 1 hour.
Wherein, the catalytic reaction is carried out by adding immobilized kinase into Tris-HCL buffer solution, and then adding substrate reaction solution.
Wherein, the immobilized kinase is added into Tris-HCL buffer, specifically, 0.3-0.5 g immobilized kinase is added into Tris-HCL buffer until the total volume is 3-5 mL.
Wherein, the volume ratio of the substrate reaction solution to the Tris-HCL buffer solution containing immobilized kinase is as follows: 1: (1-1.5). The preferred volume ratio is 1:1.
Wherein the substrate comprises ATP and magnesium chloride; specifically, the concentration of ATP is 30-60 mM. The preferred ATP concentration is 45mM.
Wherein, the catalytic reaction has the following reaction conditions: reacting for 5-20 minutes at 30-50 ℃ and 180-200 rpm. Preferably, the reaction conditions are: the reaction was carried out at 45℃and 200rpm for 5 minutes.
The beneficial effects are that:
(1) The magnesium ions and beta and gamma phosphate groups of ATP form a complex (Mg.ATP 2-) and are connected with a substrate to form a transition state, and the magnesium ions on the catalyst enable the concentration of the nearby ATP and the substrate to be higher, thereby being beneficial to the combination of the substrate and the active center of enzyme, catalyzing the reaction, improving the utilization rate of ATP and obviously improving the enzyme activity.
(2) After being modified by polyamino polymer, the positive charge on the surface of the catalyst is higher, which is favorable for the adsorption of enzymes with negative charges on the surface, the adsorption effect is better, and the loading rate of the catalyst is higher.
(3) The invention can not only catalyze the reaction and improve the utilization rate of ATP, but also can be used as a carrier to fix the enzyme by affinity adsorption, has better adsorption effect, and realizes the repeated use of the enzyme, thereby having wide application prospect in the large-scale industrial production of the immobilized enzyme.
Drawings
The foregoing and/or other advantages of the invention will become more apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings and detailed description.
FIG. 1 is a graph showing the morphology of nickel ion-chelating agarose of 100 μm, 10 μm, and 2. Mu.m.
FIG. 2 is a 100 μm, 10 μm, and 2 μm profile of an aminated nickel ion chelating agarose.
FIG. 3 is a 100 μm, 10 μm, 2 μm morphology of an aminated nickel ion-magnesium ion chelating agarose catalyst.
FIG. 4 is a table of XPS element content analysis.
FIG. 5 is a graph comparing relative enzyme activities and protein loads of different modified agarose.
FIG. 6 is a graph comparing the relative enzyme activities of different modified agarose at different concentrations of ATP.
FIG. 7 is a graph of agarose catalyst batch stability for an amination chelate of nickel ion-magnesium ion.
Detailed Description
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
Example 1: amination sepharose catalyst immobilized uridine-cytidine kinase with chelate nickel ion-magnesium ion
1. Preparation of aminated nickel ion-magnesium ion chelating agarose catalyst
(1) 5G of nickel ion chelating agarose microsphere (namely the crosslinked agarose compatible medium described in patent CN113231049B, the specific preparation method of which is disclosed in patent CN 113231049B) is dispersed in 50mL of 50mM sodium phosphate buffer solution with pH of 8.2, 50mL of 50mM magnesium chloride solution is added for reaction, the reaction process is that the agarose microsphere is oscillated for 2 hours in a shaking table with 200rpm at 25 ℃, the agarose microsphere is washed 3 times by pure water after the reaction is finished, and the agarose microsphere chelating nickel ion-magnesium ion is obtained by suction filtration until the agarose microsphere is dried.
(2) And (2) dispersing 5g of the nickel ion-magnesium ion chelating agarose microspheres prepared in the step (1) in 50mL of 5g/L polyethylenimine solution, oscillating for 0.5 hour in a shaking table at 200rpm at 25 ℃, washing 3 times with pure water after the reaction is finished, and filtering to dryness to obtain the amino nickel ion-magnesium ion chelating agarose with more positive charges on the surface.
The aminated nickel ion-magnesium ion chelating agarose is a catalyst for interface activated kinase catalytic reaction, namely the aminated nickel ion-magnesium ion chelating agarose catalyst.
And (3) taking 5g of agarose microspheres chelating nickel ions, and repeating the preparation process of the step (2) to prepare the aminated agarose chelating nickel ions.
SEM examination was performed on agarose chelating nickel ions, aminated agarose chelating nickel ions, and aminated agarose chelating nickel ions-magnesium ions, respectively, and the results are shown in FIGS. 1, 2, and 3. 1-3, the gaps become smaller and the surface becomes smooth after the medium is modified, which indicates that the medium is filled and covered by the modification groups. Meanwhile, XPS element content analysis is carried out, the results are shown in table 1 and fig. 4, the increase of magnesium ion content is found, and the success of magnesium ion modification is determined; the N content was found to increase and the amination modification was successful.
TABLE 1XPS elemental content analysis results
2. Aminated nickel ion-magnesium ion chelating agarose immobilized uridine-cytidine kinase and catalytic reaction
The uridine-cytidine kinase has a histidine tag, and 1mL of 50mM Tris-HCL buffer solution at pH8.5 and 1.5mL of enzyme solution of uridine-cytidine kinase with a protein concentration of 0.6g/L are added to 0.3g of aminated nickel ion-magnesium ion chelating agarose catalyst. The system was shaken at 10℃and 200rpm for 1 hour, after the completion, centrifuged at 4℃and 4000rpm for 4 minutes, the first supernatant was retained, the protein load was measured as compared with the free enzyme, and the immobilized enzyme was washed with Tris-HCl buffer and repeated three times to obtain immobilized uridine-cytidine kinase.
Tris-HCl buffer was added to 0.3g of immobilized uridine-cytidine kinase to bring the total volume to 5mL. 5mL of a reaction solution containing 30mM cytidine, 45mM ATP and 0.5mL of 0.2M magnesium chloride as substrates was added. The catalytic reaction was carried out by shaking at 45℃and 200rpm for 5 minutes. And after the reaction is finished, taking supernatant for inactivation, carrying out liquid phase detection, calculating relative enzyme activity, measuring the concentration of free enzyme solution and the concentration of supernatant protein after the first washing and centrifugation, and calculating the load rate.
Wherein the enzyme activity of uridine-cytidine kinase is defined as: under standard enzyme activity assay conditions, the amount of enzyme required to catalyze the production of 1. Mu. Mol/L cytidine acid from the substrate cytidine for 1min is defined as one enzyme activity unit, i.e., 1U.
The relative enzyme activity and protein load were calculated as follows:
the immobilized and catalytic reaction processes are repeated by taking agarose of chelating nickel ions and agarose of aminated chelating nickel ions as a control, and the performance of the aminated agarose immobilized uridine-cytidine kinase of chelating nickel ions-magnesium ions is compared with that of the immobilized uridine-cytidine kinase of magnesium ions before the amination modification. As shown in FIG. 5, it is apparent from FIG. 5 that the relative enzyme activity and protein loading rate of the aminated nickel ion-magnesium ion chelating agarose are greatly improved after the magnesium ion and the amination modification.
3. Catalytic reactions at different ATP concentrations
The relative enzyme activities were calculated by catalytic reaction using agarose of aminated chelate nickel-magnesium ion and agarose of aminated chelate nickel ion as catalysts at ATP concentrations of 0.01M, 0.02M, 0.03M, 0.045M, 0.06M, and 0.1M, and the results are shown in FIG. 6. The result shows that the relative enzyme activity of the agarose which takes the aminated chelating nickel ion-magnesium ion as the catalyst is higher along with the increase of the ATP concentration, and the magnesium ion on the catalyst proves that the concentration of ATP and substrate near the carrier is higher, thereby being beneficial to the combination of the substrate and the active center of the enzyme, promoting the reaction and improving the utilization rate of ATP.
4. Reusable property test
The resulting aminated nickel ion-magnesium ion chelating agarose was centrifugally washed after the completion of the catalytic reaction, and the procedure of the catalytic reaction was repeated 10 times again, and the relative enzyme activities were calculated, and the results are shown in FIG. 7. As can be seen from the figure, after the agarose of the amination chelate nickel ion-magnesium ion is reused for 10 times, the relative enzyme activity of uridine-cytidine kinase can still reach more than 65 percent. Agarose, which illustrates that the amination chelated nickel ion-magnesium ion, can be reused as a catalyst immobilized enzyme.
Example 2: aminated nickel ion-magnesium ion chelating resin catalyst immobilized uridine-cytidine kinase
(1) 5G of nickel ion chelating resin (the preparation method of the nickel ion chelating resin refers to the preparation method of a cross-linked agarose affinity medium in patent CN 113231049B), dispersing in 50mL of 50mM sodium phosphate buffer with pH of 8.2, adding 50mL of 50mM magnesium chloride solution for reaction, oscillating for 2 hours in a shaking table at 25 ℃ and 200rpm, washing with pure water for 3 times after the reaction is finished, and filtering to dryness to obtain the nickel ion-magnesium ion chelating resin.
(2) Dispersing 5g of the nickel ion-magnesium ion chelating resin prepared in the step (1) in 50mL of 5g/L polyethyleneimine solution, oscillating for 0.5 hour in a shaking table at 200rpm at 25 ℃, washing 3 times with pure water after the reaction is finished, and carrying out suction filtration to dryness to obtain the amino nickel ion-magnesium ion chelating resin catalyst with more positive charges on the surface.
(3) And (3) immobilizing uridine-cytidine kinase by using the nickel ion-magnesium ion chelating resin catalyst prepared in the step (2) according to the immobilization method in the example 1, and then carrying out catalytic reaction by using the immobilized uridine-cytidine kinase to determine the relative enzyme activity and protein load rate of the immobilized uridine-cytidine kinase. The result shows that the relative enzyme activity of the immobilized uridine-cytidine kinase of the aminated nickel ion-magnesium ion chelating resin catalyst can reach about 90 percent, and the protein carrier rate can reach 96 percent.
(4) After the catalytic reaction, the immobilized uridine-cytidine kinase is centrifugally washed, the process of the catalytic reaction is repeated for 10 times, and the relative enzyme activity of the immobilized uridine-cytidine kinase is detected through liquid phase, so that the relative enzyme activity can still reach 50% after the catalytic reaction is repeatedly used for 10 times.
Example 3: aminated nickel ion-magnesium ion chelating chitosan catalyst immobilized uridine-cytidine kinase
(1) 5G of chitosan chelating nickel ions (the preparation method of chitosan chelating nickel ions refers to the preparation method of cross-linked agarose affinity medium in patent CN 113231049B), the specific preparation method refers to patent CN113231049A, the chitosan chelating nickel ions and the chitosan chelating nickel ions are dispersed in 50mL of 50mM sodium phosphate buffer solution with pH of 8.2, 50mL of 50mM magnesium chloride solution is added for reaction, the reaction process is oscillating for 2 hours in a shaking table with 200rpm at 25 ℃, washing is carried out for 3 times by pure water after the reaction is finished, and suction filtration is carried out until the chitosan chelating nickel ions and magnesium ions are dried.
(2) And (2) dispersing 5g of the nickel ion-magnesium ion chelating chitosan prepared in the step (1) in 50mL of 5g/L polyethyleneimine solution, oscillating for 0.5 hour in a shaking table at 200rpm at 25 ℃, washing 3 times with pure water after the reaction is finished, and carrying out suction filtration to dryness to obtain the amino nickel ion-magnesium ion chelating chitosan catalyst with more positive charges on the surface.
(3) The immobilized chelate nickel ion-magnesium ion chitosan catalyst prepared in the step (2) is immobilized with uridine-cytidine kinase according to the immobilization method in example 1. And then, carrying out a catalytic reaction by using immobilized uridine-cytidine kinase, and measuring the relative enzyme activity and protein load rate of the immobilized uridine-cytidine kinase. The result shows that the relative enzyme activity of the immobilized uridine-cytidine kinase of the aminated nickel ion-magnesium ion chelating chitosan catalyst can reach about 85 percent, and the protein carrier rate can reach 80 percent.
(4) After the catalytic reaction, the immobilized uridine-cytidine kinase is centrifugally washed, the process of the catalytic reaction is repeated for 10 times, and the relative enzyme activity of the immobilized uridine-cytidine kinase is detected through liquid phase, so that the relative enzyme activity can still reach 42% after the catalytic reaction is repeated for 10 times.
Example 4: amination sepharose catalyst immobilized adenosine kinase for chelating nickel ion-magnesium ion
(1) 5G of agarose for chelating nickel ions (agarose microsphere for chelating nickel ions is a cross-linked agarose affinity medium described in patent CN113231049B, the specific preparation method of the agarose is disclosed in patent CN 113231049B) is dispersed in 50mL of 50mM sodium phosphate buffer solution with pH of 8.2, 50mL of 50mM magnesium chloride solution is added for reaction, the reaction process is that the agarose is oscillated for 2 hours in a shaking table with 200rpm at 25 ℃, the agarose microsphere is washed by pure water for 3 times after the reaction is finished, and the agarose is suction-filtered to be dried, so that the agarose for chelating nickel ions-magnesium ions is obtained.
(2) And (2) dispersing 5g of the nickel ion-magnesium ion chelating agarose prepared in the step (1) in 50mL of 5g/L polylysine solution, oscillating for 0.5 hour in a shaking table at 200rpm at 25 ℃, washing with pure water for 3 times after the reaction is finished, and carrying out suction filtration to dryness to obtain the amino nickel ion-magnesium ion chelating agarose catalyst with more positive charges on the surface.
(3) The immobilized agarose catalyst of chelating nickel ion-magnesium ion prepared in the step (2) was used for immobilizing adenosine kinase according to the immobilization method in example 1. And then, carrying out catalytic reaction by using the immobilized adenosine kinase, and measuring the relative enzyme activity and protein load rate of the immobilized adenosine kinase. The result shows that the relative enzyme activity of the aminated nickel ion-magnesium ion chelating agarose catalyst immobilized adenosine kinase can reach about 89%, and the protein load rate can reach 92%.
(4) After the catalytic reaction, the immobilized adenosine kinase is centrifugally washed, the process of the catalytic reaction is repeated for 10 times, and the relative enzyme activity of the immobilized adenosine kinase is detected through a liquid phase, so that the relative enzyme activity can still reach 52% after the immobilized adenosine kinase is repeatedly used for 10 times.
The invention provides an aminated chelated metal ion catalyst, a preparation method and an application thought and method thereof, and particularly the method and the method for realizing the technical scheme are numerous, the above is only a preferred embodiment of the invention, and it should be pointed out that a plurality of improvements and modifications can be made by those skilled in the art without departing from the principle of the invention, and the improvements and modifications are also considered as the protection scope of the invention. The components not explicitly described in this embodiment can be implemented by using the prior art.
Claims (10)
1. A method for preparing an aminated chelate metal ion catalyst, comprising the steps of:
(1) Placing the affinity medium for chelating nickel ions into PBS buffer solution, and then adding magnesium chloride solution for reaction to obtain the affinity medium for chelating nickel ions-magnesium ions;
(2) And (3) placing the nickel ion-magnesium ion chelating affinity medium obtained in the step (1) into a polyamino polymer solution for reaction to obtain an amino nickel ion-magnesium ion chelating affinity medium, namely the catalyst.
2. The method according to claim 1, wherein in the step (1), the affinity medium comprises any one or a combination of agarose, resin and chitosan.
3. The method according to claim 1, wherein in the step (1), the PBS buffer is 30-70 mM sodium phosphate buffer having a pH of 8-8.5; the concentration of the affinity medium for chelating nickel ions in the PBS buffer solution is 50-150 mg/mL; the concentration of the magnesium chloride solution is 30-70 mM, the solvent is water, and the volume ratio of the magnesium chloride solution to the PBS buffer solution is 1 (0.5-1.5); the reaction conditions are as follows: 25 ℃, 1.5-2.5 hours, and the rotating speed is 180-200 rpm.
4. The method according to claim 1, wherein in the step (2), the polyamino polymer comprises any one or a combination of several of polylysine, polyethyleneimine, polyacrylamide, poly-arginine, polyetheramine, glycosaminoglycan, and tetraethylenepentamine.
5. The method according to claim 1, wherein in the step (2), the concentration of the affinity medium chelating nickel ions-magnesium ions in the polyamino polymer solution is 4 to 10mg/mL; the concentration of the polyamino polymer solution is 30-70 mM, and the solvent is pure water; the reaction conditions are as follows: 25 ℃,0.5 hour, and the rotating speed is 180-200 rpm.
6. An aminated chelate metal ion catalyst prepared by the method of any one of claims 1 to 5.
7. Use of an aminated chelate metal ion catalyst according to claim 6 for catalytic reactions of immobilized kinases.
8. The use according to claim 7, wherein the kinase is provided with a histidine tag, comprising any one or a combination of several of uridine-cytidine kinase, adenosine kinase, protein kinase, 6-phosphofructokinase, hexokinase.
9. The use according to claim 7, wherein the immobilized kinase is obtained by adding an aminated chelate metal ion catalyst to an enzyme solution of Tris-HCL buffer and kinase for immobilization reaction, and then carrying out catalytic reaction by using the immobilized kinase.
10. The use according to claim 9, wherein the ratio of the amount of the aminated chelate metal ion catalyst to the Tris-HCL buffer, the enzyme solution of kinase is: 0.3g (1-100 mL): (1.5-100 mL);
The immobilization reaction has the following reaction conditions: reacting for 1 hour at the temperature of 4-10 ℃ and the rpm of 180-200 rpm; the substrate of the catalytic reaction comprises ATP and magnesium chloride, and the reaction conditions are as follows: reacting for 5-20 minutes at 30-50 ℃ and 180-200 rpm.
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