CN115611813A - Preparation method and application of artificial enzyme with carboxylesterase and carbonic anhydrase double-enzyme activities - Google Patents
Preparation method and application of artificial enzyme with carboxylesterase and carbonic anhydrase double-enzyme activities Download PDFInfo
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 69
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 69
- 230000000694 effects Effects 0.000 title claims abstract description 66
- 108090000209 Carbonic anhydrases Proteins 0.000 title claims abstract description 33
- 102000003846 Carbonic anhydrases Human genes 0.000 title claims abstract description 32
- 102000013392 Carboxylesterase Human genes 0.000 title claims abstract description 20
- 108010051152 Carboxylesterase Proteins 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000011701 zinc Substances 0.000 claims abstract description 95
- 239000002244 precipitate Substances 0.000 claims abstract description 29
- 238000006243 chemical reaction Methods 0.000 claims abstract description 27
- 229910052723 transition metal Inorganic materials 0.000 claims abstract description 23
- 150000003624 transition metals Chemical class 0.000 claims abstract description 23
- 238000001338 self-assembly Methods 0.000 claims abstract description 19
- 239000006228 supernatant Substances 0.000 claims abstract description 17
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- 238000001291 vacuum drying Methods 0.000 claims abstract description 12
- 229910052725 zinc Inorganic materials 0.000 claims abstract description 12
- 239000007864 aqueous solution Substances 0.000 claims abstract description 11
- 239000007787 solid Substances 0.000 claims abstract description 10
- QDFQBILHZPUAAH-CQSZACIVSA-N C(=O)(OCC1=CC=CC=C1)[C@@](N)(CC1=CNC=N1)C(=O)O Chemical compound C(=O)(OCC1=CC=CC=C1)[C@@](N)(CC1=CNC=N1)C(=O)O QDFQBILHZPUAAH-CQSZACIVSA-N 0.000 claims abstract description 9
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- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 6
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- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 description 6
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- 238000002835 absorbance Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
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- MFLKDEMTKSVIBK-UHFFFAOYSA-N zinc;2-methylimidazol-3-ide Chemical compound [Zn+2].CC1=NC=C[N-]1.CC1=NC=C[N-]1 MFLKDEMTKSVIBK-UHFFFAOYSA-N 0.000 description 2
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- LXBGSDVWAMZHDD-UHFFFAOYSA-N 2-methyl-1h-imidazole Chemical compound CC1=NC=CN1 LXBGSDVWAMZHDD-UHFFFAOYSA-N 0.000 description 1
- SLAMLWHELXOEJZ-UHFFFAOYSA-N 2-nitrobenzoic acid Chemical class OC(=O)C1=CC=CC=C1[N+]([O-])=O SLAMLWHELXOEJZ-UHFFFAOYSA-N 0.000 description 1
- OTLNPYWUJOZPPA-UHFFFAOYSA-M 4-nitrobenzoate Chemical compound [O-]C(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-M 0.000 description 1
- MVPKIPGHRNIOPT-UHFFFAOYSA-N 5,6-dimethyl-2h-benzotriazole Chemical compound C1=C(C)C(C)=CC2=NNN=C21 MVPKIPGHRNIOPT-UHFFFAOYSA-N 0.000 description 1
- 102000001049 Amyloid Human genes 0.000 description 1
- 108010094108 Amyloid Proteins 0.000 description 1
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- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
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- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
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- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 102000057327 human CA2 Human genes 0.000 description 1
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- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
- 239000013110 organic ligand Substances 0.000 description 1
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- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
- C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
- C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/22—Organic complexes
- B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
- B01J31/2208—Oxygen, e.g. acetylacetonates
- B01J31/2217—At least one oxygen and one nitrogen atom present as complexing atoms in an at least bidentate or bridging ligand
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/10—Complexes comprising metals of Group I (IA or IB) as the central metal
- B01J2531/16—Copper
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/20—Complexes comprising metals of Group II (IIA or IIB) as the central metal
- B01J2531/26—Zinc
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a preparation method and utilization of artificial enzyme with carboxylesterase and carbonic anhydrase double-enzyme activities, wherein aqueous solution of chloride of transition metal zinc and copper is added into aqueous solution of N alpha-carbobenzoxy-D-histidine, and is stirred in weak alkaline buffer solution to be uniformly mixed so as to complete coordination chelation reaction, and the mixture is kept stand at normal temperature for 24-72h to complete self-assembly; centrifuging at 8000-10000rpm for 20-30min, discarding supernatant, collecting precipitate, and vacuum drying the precipitate to obtain BpA-Cu and BpA-Zn solid. The preparation method is simple, the reaction condition is mild, the operation is easy, the synthesis condition of the unnatural amino acid is mature, the price is low, and the source is wide. The activity can keep 80.1% of the maximum activity under alkaline conditions and 90.1% at high temperature of 80 ℃, and the hydration of carbon dioxide is 8.4 times of that of a blank control group.
Description
Technical Field
The invention relates to a preparation method and application of an artificial metalloenzyme with carboxylesterase and carbonic anhydrase dual-enzyme activities, which is formed by self-assembly of N alpha-benzyloxycarbonyl-D-histidine and transition metal such as zinc or copper ions through coordination, belongs to the technical field of artificial enzyme preparation and application, and particularly relates to an artificial enzyme with carboxylesterase and carbonic anhydrase dual-enzyme activities, a preparation method and application thereof.
Background
Enzymes are biocatalysts produced by organisms that convert substrates into products efficiently and specifically in mild biological environments. The enzyme has the advantages of high efficiency, specificity, mild reaction condition and the like, so the enzyme is widely applied to the fields of medical treatment and medicine, food sanitation, chemical engineering and the like. However, natural enzymes have limited sources, and intrinsic defects such as high purification cost due to expensive equipment and poor stability in the separation and purification process, which are difficult to satisfy the needs of modern industries, have been designed to develop a variety of novel artificial catalysts to replace the functions of natural enzymes (Chemical Society Reviews 2019,48, 1004-1076).
Esterases (esterases, EC 3.1.1.1) are carboxylesterases in the general sense, a generic term for enzymes that catalyze ester bond hydrolysis and synthesis, are widely found in plants, animals, and microorganisms, and have become an important industrial enzyme due to their wide range of action and high substrate specificity. When esters are hydrolyzed catalytically, the cleavage of ester bonds is catalyzed to produce alcohols and fatty acids (Journal of Agricultural and Food Chemistry,2011,59, 2019-2025). CA (EC 4.2.1.1) is a group represented by Zn 2+ One of the most catalytically efficient metalloenzymes as cofactors for the catalytic activity centers, which can catalyze CO 2 The catalytic rate can reach 10 in the natural state 7 Multiple (Biotechnology Progress,2009,25, 68-74). However, natural carboxylesterases and carbonic anhydrases, as natural proteins, still suffer from the significant disadvantage of being labile to inactivation. On the one hand, under the environment of high temperature or extreme acid, extreme alkali and the like, the higher structure of the protein is easy to unfold, so that the active structure is damaged, and the irreversible activity loss is caused. On the other hand, the natural enzymes are easily soluble in water, the enzymes free in water are not recoverable and after long-term storage, the activity is reduced or even completely lost, and the defects in the two aspects greatly limit the application of the enzymes in the industrial field. Therefore, it is necessary to develop new compoundsThe enzyme activity can also meet the stability requirement.
Currently, many mimetics for esterases and carbonic anhydrases are amyloid-based short peptides, and metalloenzymes having esterase and carbonic anhydrase activities are synthesized rarely using small organic chemical molecules, dipeptides, amino acids, non-protein amino acids, and the like as organic ligands. Since the self-assembling peptide building blocks derived from amyloid sequences with catalysis have been reported, a series of heptapeptide molecules based on amyloid fibrils were designed, and heptapeptide supramolecules of these alternating hydrophobic and hydrophilic residues possess esterase activity (Protein Science,2015,24, 188-188). After which the acyl ester hydrolysis can be catalyzed by screening in the presence of zinc ions. Synthesis of biomimetic based ZIF-8 Using 2-methylimidazole with Zinc ion, ZIF-8 Artificial enzyme showed similar affinity for 4-nitrobenzoate (pNPA) and promoted CO compared to human Carbonic anhydrase II 2 Hydration (Nanoscale, 2019,11, 5960-5966). Non-nuclear carbonic anhydrase mimetics were synthesized using the self-assembly of 5, 6-dimethyl-1, 2, 3-benzotriazole with zinc ions and showed higher carbonic anhydrase activity (inorganic chemistry,2019,58, 9916-9921). Nano-fiber (Nature Catalysis,2019,2, 977-985) with carbonic anhydrase activity, which is synthesized by using phenylalanine and zinc ions in a CO-assembly manner and can not only hydrolyze pNPA into pNP but also promote CO 2 And then using cyclic histidine and zinc ions to synthesize homogeneous dipeptide nano supramolecular materials with carboxylesterase and carbonic anhydrase activities by an ultrasound-assisted solid phase peptide method (angelwet chemistry. International Edition in English,2021,60, 17164-17170). However, the preparation process and method of the related materials are complex; the performances of reusability, catalytic activity, stability and the like are not good enough.
Disclosure of Invention
The invention aims to solve the problems of poor biocompatibility, complex preparation process and the like of materials used in the existing simulated esterase, and provides an artificial metalloenzyme which is formed by coordination chelation of N alpha-benzyloxycarbonyl-D-histidine and transition metal such as zinc or copper and has the activity of both carboxylesterase and carbonic anhydrase, and the enzyme has certain significance in the field of reducing carbon dioxide emission. The preparation method is simple, the reaction condition is mild, the operation is easy, and the catalytic nano material with the activity of the carboxylesterase and the carbonic anhydrase can be synthesized by the abundant, cheap and easily obtained unnatural amino acids and the transition metal ions, so that the cost for treating the carboxylic ester and reducing the emission of carbon dioxide can be saved.
The technical scheme of the invention is summarized as follows:
a preparation method and application of an artificial enzyme with carboxylesterase and carbonic anhydrase double-enzyme activities; the nanomaterial with double enzyme activity mimics, which is formed by self-assembly of N alpha-benzyloxycarbonyl-D-histidine and transition metal such as zinc or copper ions through coordination, is marked as N alpha-CDH-M, wherein N alpha-CDH is N alpha-benzyloxycarbonyl-D-histidine, and M represents transition metals such as zinc and copper.
The preparation method of the artificial enzyme with the dual-enzyme activity of carboxylesterase and carbonic anhydrase comprises the following steps:
1) Adding an aqueous solution of N alpha-benzyloxycarbonyl-D-histidine and an aqueous solution of a chloride salt of transition metal copper or zinc into an alkalescent buffer solution, and magnetically stirring to complete a coordination chelation reaction; standing at normal temperature for 24-72h to complete self-assembly;
2) Centrifuging at 8000-10000rpm for 10-30min, discarding supernatant, collecting precipitate, cleaning the precipitate with ultrapure water for 3-5 times, and vacuum drying the precipitate containing partial water after the last centrifugation to obtain Nalpha-CDH-Zn or Nalpha-CDH-Cu solid sample;
the water specification of the N alpha-CDH and the transition metal salt solution prepared in the step 1) is at least ultrapure water; the concentration of the N alpha-CDH solution is 20-50mM; the concentration of the transition metal salt solution is 20-100mM; the addition of the transition metal salt solution to the na-CDH solution needs to be performed at normal temperature.
The molar ratio of the transition metal salt solution in the step 1) to the N alpha-CDH aqueous solution is 1.
The reaction conditions of the step 1) after the transition metal salt solution is added are as follows: self-assembling for 24-72h at normal temperature.
The temperature of the vacuum drying oven in the step 2) is set to be 40-80 ℃, and the drying time is 24-72 hours until the precipitate containing a small amount of water is dried to powder Nalpha-CDH-Zn or Nalpha-CDH-Cu.
The invention relates to an application of an artificial enzyme with the activities of carboxylesterase-like enzyme and carbonic anhydrase in the fields of simulating the activities of carboxylesterase and carbonic anhydrase and reducing carbon dioxide in the environment.
The invention uses N alpha-carbobenzoxy-D-histidine and transition metal such as zinc or copper ions to self-assemble by coordination to form a supermolecular material with a double-enzyme activity simulant, and utilizes the self-assembled nano material prepared by the method to simulate the activity of carboxylesterase and carbonic anhydrase. The carboxylesterase activity and stability of the compound are measured by using 4-nitrobenzene butyrate pNPB as a substrate, because the pNPB generates a yellow substance p-nitrophenol pNP after being hydrolyzed, and the substance has a strong absorption peak at 405 nm; by using CO 2 Determination of CaCO as substrate 3 Thereby determining CO 2 The transformation ability of (a). The catalytic kinetic parameters of N alpha-CDH-Zn are respectively Km of 0.49mM and Vmax of 1.08 mu M.S -1 The affinity of the N alpha-CDH-Zn and the substrate pNPB is higher. And can still keep 80.1% of the maximum activity under the condition of pH9.0, the catalytic activity of N alpha-CDH-Zn can reach 90.1% of the maximum activity at 80 ℃, which indicates that the artificial enzyme N alpha-CDH-Zn has higher stability. In addition, when the concentration of the N alpha-CDH-Zn reaches 800 mu g/mL, the cell activity can be kept at 76.2%, which indicates that the protein has good biocompatibility. In addition, caCO formed by N alpha-CDH-Zn 3 The weight of the compound is 8.4 times that of the blank control, and the content of carbon dioxide in the environment can be reduced by the N alpha-CDH-Zn.
The invention has the advantages of simple preparation conditions, mature synthesis conditions of the unnatural amino acid, low price and wide source. The catalytic process of the mimic enzyme can be monitored by an ultraviolet-visible spectrophotometer and an enzyme-labeling instrument. Compared with the materials reported in the previous literature, the invention has the advantages that:
1) The prepared artificial enzyme has higher catalytic rate of 1.08 MuM.S -1 And lower Km value of 0.49mM;
2) The prepared mimic enzyme can keep 80.1 percent of the original activity under the alkaline condition and 90.1 percent of the activity under the high-temperature condition of 80 ℃, so that the mimic enzyme can be applied to the industry.
Drawings
FIG. 1 is an SEM image of the catalytic material N α -CDH-Zn formed by self-assembly of example 1 at different times.
FIG. 2 is a UV absorption spectrum of N α -CDH-Zn catalyzed carboxylic ester hydrolysis of example 7.
FIG. 3 is a graph of the ability of self-assembly of EXAMPLE 8N α -CDH-Zn to catalyze carboxylate hydrolysis at various times.
FIG. 4 is a graph showing the relative catalytic activity of the artificial enzyme N.alpha. -CDH-Zn for carboxylic acid esters in buffers of different pH values in example 9.
FIG. 5 is a graph showing the relative catalytic activity of the artificial enzyme N.alpha. -CDH-Zn of example 10 on carboxylic acid esters after incubation at different temperatures for 30 min.
FIG. 6 is a graph showing the relative catalytic activity of the artificial enzyme N.alpha. -CDH-Zn for carboxylic acid esters in buffers containing different salt concentrations in example 11.
FIG. 7 is a graph showing the relative catalytic activities of the artificial enzyme N α -CDH-Zn in example 12 in catalyzing the repeated use of carboxylic ester hydrolysis.
FIG. 8 shows CaCO formed by over-weighing the artificial enzyme Nalpha-CDH-Zn in example 13 3 To evaluate CO 2 The conversion of (1).
FIG. 9 is a graph showing cytotoxicity of the self-assembled artificial enzyme N.alpha. -CDH-Zn and the starting material in example 14.
Detailed Description
The method of the present invention is further illustrated by the following examples and figures, but the examples described herein are for the purpose of illustration only and are not intended to limit the invention in any way.
The preparation method of the artificial enzyme with the activity of carboxylesterase and carbonic anhydrase formed by self-assembly of the unnatural amino acid and the transition metal ion through coordination comprises the following steps:
1) Dissolving BpA with 10-50mM sodium hydroxide solution prepared with ultrapure water to obtain 20-50mM Nalpha-CDH aqueous solution, and preparing 20-100mM ZnCl with ultrapure water 2 、CuCl 2 Solution, adding the obtained aqueous solution of Nalpha-CDHAdding the transition metal salt solution into a weakly alkaline buffer solution, and then slowly adding the transition metal salt solution into the aqueous solution of the Nalpha-CDH, wherein magnetic stirring is needed in the process to fully and uniformly mix the transition metal salt solution until the transition metal chloride solution is completely added, and the ratio of the transition metal chloride solution to the aqueous solution of the Nalpha-CDH to be controlled in the process is 1-1. Fully mixing, and self-assembling at normal temperature for 24-72h to obtain the final self-assembled supermolecular aggregate suspension.
2) Centrifuging the suspension obtained after the reaction at high speed, namely centrifuging at 4-25 ℃ and 8000-10000rpm for 20-30min, discarding the supernatant, retaining the precipitate, and washing the precipitate with ultrapure water for 3-5 times; and (3) putting the washed precipitate in a vacuum drying oven at 40-80 ℃ for 24-72h to obtain the self-assembled artificial enzyme powder with the activity of the carboxylesterase-like enzyme and the carbonic anhydrase. The activity, stability and biocompatibility of the artificial enzyme powder were determined.
Example 1ZnCl 2 And N alpha-CDH, the molar ratio of the N alpha-CDH to the carboxylesterase-like enzyme and carbonic anhydrase double-enzyme activity is 1.
2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was weighed and dissolved in 800mL of deionized water to obtain 10mM HEPES buffer, and then adjusted to pH 8.0 with NaOH to a volume of 1L, which was then sterilized by filtration through a 0.45 μm filter. 578.584mg of Nalpha-CDH was dissolved in ultrapure water to obtain 20mM Nalpha-CDH solution, and 5.456g of ZnCl was weighed 2 Dissolving in ultrapure water to obtain ZnCl with the concentration of 40mM respectively 2 And (3) solution. The N.alpha. -CDH solution and 10mM HEPES buffer pH 8.0 were mixed, followed by dropwise addition of 40mM ZnCl 2 And stirring the solution by using a magnetic stirrer during the period to ensure that the solution and the solution are fully contacted, and then placing the system at the normal temperature for 24 hours to enable the system to self-assemble into the active nano material N alpha-CDH-Zn.
The mixture formed by self-assembly after standing for different time is centrifuged at 8000rpm for 30min to remove supernatant, and the collected precipitate is washed with deionized water for 5 times to remove unreacted reactants. And (3) putting the precipitate obtained after the last centrifugation in a constant-temperature vacuum drying oven, and drying for 48 hours at 60 ℃ to obtain solid powder.
In FIG. 1, (a), (c), (e), (g) are 24h, 36h, 48h, 60h and 72h magnified by 2.5 ten thousand times, where the scale length represents 1 μm, and (b), (d), (f), (h) are 24h, 36h, 48h, 60h and 72h magnified by 5 ten thousand times, where the scale represents 500nm. As can be seen from fig. 1: when the self-assembly time is 24h, a regular structure is formed, the structure is a special regular structure formed by stacking multiple layers of N alpha-CDH-Zn, and the artificial enzyme N alpha-CDH-Zn with double enzyme activity is successfully constructed, the difference of the morphologies of the N alpha-CDH-Zn is not obvious after the self-assembly time is prolonged, but the thickness of the monolayer is gradually increased along with the self-assembly time from an enlarged graph.
Example 2ZnCl 2 And N alpha-CDH, wherein the molar ratio of the carboxylesterase-like enzyme to carbonic anhydrase double-enzyme activity is 1.
2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was weighed and dissolved in 800mL of deionized water to obtain 10mM HEPES buffer, and then adjusted to pH 8.0 with NaOH to a volume of 1L, which was then sterilized by filtration through a 0.45 μm filter. 723.23mg of Nalpha-CDH was dissolved in ultrapure water to obtain a 25mM Nalpha-CDH solution, and 3.41g of ZnCl was weighed 2 Dissolving in ultrapure water to obtain ZnCl with the concentration of 25mM respectively 2 And (3) solution. 25mM Na-CDH solution and 10mM HEPES buffer pH 8.0 were mixed well and 50mM ZnCl was added dropwise 2 And stirring the solution by using a magnetic stirrer during the period to ensure that the solution and the solution are fully contacted, and then placing the system at the normal temperature for 48 hours to enable the system to self-assemble into the active nano material N alpha-CDH-Zn.
The mixture formed by standing self-assembly was centrifuged at 9000rpm for 20min to discard the supernatant and take out the precipitate, and the collected precipitate was washed 5 times with deionized water to remove the unreacted reactants. And (3) putting the precipitate obtained after the last centrifugation in a constant-temperature vacuum drying oven, and drying at 40 ℃ for 72 hours to obtain solid powder.
Example 3ZnCl 2 And N alpha-CDH, wherein the molar ratio of the N alpha-CDH to the carboxylesterase-like enzyme and the carbonic anhydrase double-enzyme activity is 1.
2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was dissolved in 800mL of deionized water to give a 10mM HEPES buffer, which was then adjusted with NaOHThe pH value is 8.0, the volume is fixed to 1L, and the mixture is filtered and sterilized by a filter membrane with the diameter of 0.45 mu m for standby. 1446.46mg of Nalpha-CDH was dissolved in ultrapure water to obtain a solution of Nalpha-CDH having a concentration of 50mM, and 13.64g of ZnCl was weighed 2 Dissolving in ultrapure water to obtain ZnCl with the concentration of 100mM 2 And (3) solution. The 50mM Na-CDH solution and the 10mM HEPES buffer solution with the pH value of 8.0 are uniformly mixed, and then 100mM ZnCl is dropwise added 2 And stirring the solution by using a magnetic stirrer during the period to ensure that the solution and the solution are fully contacted, and then placing the system at the normal temperature for 72 hours to enable the system to self-assemble into the active nano material N alpha-CDH-Zn.
The mixture formed by self-assembly after standing for different times is centrifuged at 10000rpm for 10min, the supernatant is discarded, and the collected precipitate is washed with deionized water for 5 times to remove the unreacted reactants. And (3) putting the precipitate obtained after the last centrifugation in a constant-temperature vacuum drying oven, and drying for 24 hours at 80 ℃ to obtain solid powder.
Example 4CuCl 2 And N alpha-CDH, the molar ratio of the carboxylesterase-like enzyme to carbonic anhydrase double-enzyme activity is 1.
2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was weighed and dissolved in 800mL of deionized water to obtain 10mM HEPES buffer, and then adjusted to pH 8.0 with NaOH to a volume of 1L, which was then sterilized by filtration through a 0.45 μm filter. 578.584mg of Nalpha-CDH was dissolved in ultrapure water to obtain a 20mM Nalpha-CDH solution, and 341mg of CuCl was weighed 2 ·2H 2 Dissolving O in ultrapure water to obtain CuCl with the concentration of 20mM 2 The solution is ready for use. The N.alpha. -CDH solution and 10mM HEPES buffer solution having a pH of 8.0 were mixed uniformly, and 40mM CuCl was added dropwise 2 And stirring the solution by using a magnetic stirrer during the period to ensure that the solution is fully contacted with the solution, and then placing the system at the normal temperature for 36 hours to ensure that the system is self-assembled into the active nano material N alpha-CDH-Cu.
The mixture formed by standing for different time is centrifuged at 8000rpm for 30min to remove supernatant and take out precipitate, and the collected precipitate is washed with deionized water for 5 times to remove unreacted reactants. And (3) putting the precipitate obtained after the last centrifugation in a constant-temperature vacuum drying oven, and drying for 48 hours at 60 ℃ to obtain solid powder.
Example 5CuCl 2 And (3) synthesizing artificial enzyme with carboxylesterase-like enzyme and carbonic anhydrase double-enzyme activities, wherein the molar ratio of the artificial enzyme to the N alpha-CDH is 1.
2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was weighed and dissolved in 800mL of deionized water to obtain 10mM HEPES buffer, and then adjusted to pH 8.0 with NaOH to a volume of 1L, which was then sterilized by filtration through a 0.45 μm filter. 723.23mg of Nalpha-CDH was dissolved in ultrapure water to obtain a 25mM Nalpha-CDH solution, and 852.5mg of CuCl was added 2 ·2H 2 Dissolving O in ultrapure water to obtain CuCl with the concentration of 50mM 2 The solution is ready for use. The N.alpha. -CDH solution and 10mM HEPES buffer solution having a pH of 8.0 were mixed uniformly, and 50mM CuCl was added dropwise 2 And stirring the solution by using a magnetic stirrer to make the solution and the solution fully contact, and then placing the system at the normal temperature for 48 hours to enable the system to self-assemble into the active nano material N alpha-CDH-Cu.
The mixture formed by standing for various periods of time was centrifuged at 9000rpm for 20min to discard the supernatant and the precipitate collected was washed 5 times with deionized water to remove unreacted reactants. And (3) putting the precipitate obtained after the last centrifugation in a constant-temperature vacuum drying oven, and drying at 40 ℃ for 72h to obtain solid powder.
Example 6CuCl 2 And N alpha-CDH, wherein the molar ratio of the N alpha-CDH to the carboxylesterase-like enzyme and the carbonic anhydrase double-enzyme activity is 1.
2.383g of 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES) was weighed and dissolved in 800mL of deionized water to obtain 10mM HEPES buffer, then pH was adjusted to 8.0 using NaOH, and the volume was adjusted to 1L, and the solution was sterilized by filtration through a 0.45 μm filter. 1446.46mg of Na-CDH was dissolved in ultrapure water to obtain a 50mM Na-CDH solution, and 1705mg of CuCl was dissolved in ultrapure water 2 ·2H 2 Dissolving O in ultrapure water to obtain CuCl with the concentration of 100mM 2 The solution is ready for use. The N.alpha. -CDH solution and 10mM HEPES buffer solution having a pH of 8.0 were mixed uniformly, and 00mM CuCl was added dropwise 2 And stirring the solution by using a magnetic stirrer during the period to ensure that the solution and the solution are in full contact, and then placing the system at the normal temperature for 72 hours to enable the system to self-assemble into the active nano material N alpha-CDH-Cu.
The mixture formed by standing for different time is centrifuged at 10000rpm for 10min, the supernatant is discarded, the precipitate is taken out, and the collected precipitate is washed with deionized water for 5 times to remove the reactant which does not participate in the reaction. And (3) putting the precipitate obtained after the last centrifugation in a constant-temperature vacuum drying oven, and drying for 24 hours at 80 ℃ to obtain solid powder.
Example 7 carboxylesterase-like enzyme activity catalytic performance study of artificial enzyme.
Nitrobenzoate esters are one of the commonly used substrates for the verification of esterase activity. Therefore, in the invention, 4-nitrobenzene butyrate (pNPB) is used as a substrate to determine the catalytic activity of the N alpha-CDH-Zn mimic esterase, and the pNPB generates paranitrophenol (pNP) after hydrolysis and has a strong absorption peak at 405 nm. The prepared solid powder N α -CDH-Zn was ultrasonically dispersed in 10mM phosphate buffer solution (pH 7.0) to give a concentration of 1 mg. ML -1 The suspension of Na-CDH-Zn (K). mu.L of N.alpha. -CDH-Zn suspension (1 mg. ML) was taken -1 ) To 800. Mu.L of phosphate buffer (10mM, pH 7.0) was added, and 100. Mu.L of a pNPB solution (1 mM) was further added and mixed so as to be uniform, so that the total reaction volume was 1mL. Reacting for 1h at 25 ℃, then centrifuging for 5min at 12000rpm, separating N alpha-CDH-Zn, collecting the supernatant after the reaction, and measuring the absorbance of the solution at 405nm after the reaction by using a microplate reader. The catalytic capacity of na-CDH-Zn was calculated as the product concentration μ M from the pNP standard curve a =0.0068 c.
From fig. 2, it follows that: blank represents a Blank group, wherein no N alpha-CDH-Zn is added, the color of the solution is not obviously changed, weak absorption is generated at 405nm, the result shows that only a small amount of substrate pNPB in the reaction system is hydrolyzed, the colorless solution is remarkably changed after 1h of reaction after the 48h of synthesized catalyst N alpha-CDH-Zn is added, and strong absorption is generated at 405nm, which indicates that the rate of catalyzing ester hydrolase hydrolysis reaction is high. When pNPB with different concentrations is added, the catalytic reaction rates are different, and the dynamic parameters of N alpha-CDH-Zn catalysis are respectively calculated according to the difference of the reaction rates and are that Km is 0.49mM, vmax is 1.08 mu M.S -1 In comparison with other studies, the Km value of N.alpha. -CDH-Zn was lower, indicating that N.alpha. -CDH-Zn has a higher affinity for the substrate pNPB.
Example 8 self-assembly of carboxylesterase-like enzyme catalytic performance study of artificial enzymes at different times.
The invented N alpha-CDH-Zn was weighed to dissolve in PB (10 mM, pH 7.0) buffer solution at different times for self-assembly, and subjected to intense ultrasound for 20min to obtain a concentration of 1 mg. ML -1 The suspension is reserved; mu.L of N.alpha. -CDH-Zn suspension (1 mg. ML) was taken -1 ) To 800. Mu.L of phosphate buffer (10mM, pH 7.0) was added, and 100. Mu.L of a pNPB solution (1 mM) was further added and mixed so as to be uniform, so that the total reaction volume was 1mL. Reacting for 1h at 25 ℃, then centrifuging for 5min at 12000rpm, separating N alpha-CDH-Zn, collecting the supernatant after reaction, and measuring the absorbance of the solution after reaction at 405nm by using a microplate reader. The catalytic capacity of na-CDH-Zn was calculated as the product concentration μ M from the pNP standard curve a =0.0068 c.
From fig. 3, it can be derived: blank represents a Blank group in which no N.alpha. -CDH-Zn was added, the catalytic activity of the group was substantially 0, and the mimic enzyme (100. Mu.g.mL) formed when different assembly times were added -1 ) The catalyst is used for hydrolyzing pNPB and then detecting the light absorption value of a product pNP, the corresponding concentration is calculated according to a standard curve of the pNP to further calculate the catalytic efficiency, and experiments show that the activity of the catalyst shows a trend of increasing firstly and then decreasing along with the prolonging of the self-assembly time. The mimic enzyme formed by 48h self-assembly has the strongest catalytic capability, and the differences of the catalytic capabilities of the mimic enzymes formed by other conditions are not obvious.
Example 9 catalytic performance of artificial enzyme carboxylesterase activity under extreme pH conditions.
In order to test the catalytic performance of the prepared N alpha-CDH-Zn under different pH conditions, 100 mu L of 1 mg/mL -1 After incubating the N alpha-CDH-Zn suspension with 800 mu L of buffer solution with pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0 for 30min, adding 100 mu L of pNPB solution (1 mM) and mixing uniformly (total reaction volume is 1 mL), reacting for 1h at room temperature, centrifuging at 10000rpm for 5min to retain supernatant, and monitoring the light absorption value of the solution at 405nm by using a microplate reader. The catalytic capacity of na-CDH-Zn was calculated as the product concentration μ M from the pNP standard curve a =0.0068 c. Wherein the pH values of 3.0, 4.0 and 5.0 are prepared from 50mM glycine-hydrochloric acid buffer solution, the pH values of 6.0, 7.0 and 8.0 are prepared from 10mM PBS buffer solution, and the pH value of 9.0 is prepared from 50mM glycine-sodium hydroxide buffer solutionAnd (4) preparing.
As can be seen from FIG. 4, the N alpha-CDH-Zn of the present invention shows strong catalytic ability in the pH range of 5.0-9.0, and the activity thereof can maintain 80.1% of the maximum activity under the condition of pH 9.0. The artificial enzyme N alpha-CDH-Zn of the invention still has better catalytic capability under alkaline condition, and can solve the problem of poor stability of natural enzyme and the existing mimic enzyme.
Example 10 catalytic performance of carboxylesterase-like activity of artificial enzyme under high temperature conditions.
In order to test the catalytic performance of the prepared N alpha-CDH-Zn in the invention after incubation at high temperature, 100 mu L of 1 mg/mL -1 The Nalpha-CDH-Zn suspension is mixed with 800 mu L of 10mM PB (pH 7.0) buffer solution, then the mixture is respectively placed under the temperature conditions of 25 ℃, 30, 40, 50, 60, 70, 80 and 90 ℃ for incubation for 30min until the temperature is recovered to the room temperature, 100 mu L of pNPB solution (1 mM) is added and mixed evenly (the total reaction volume is 1 mL), the mixture is reacted for 1h at the room temperature, then the mixture is centrifuged at 10000rpm for 5min, the supernatant is retained, and the absorbance value of the solution at 405nm is monitored by a microplate reader. The catalytic capacity of na-CDH-Zn was calculated as the product concentration μ M from the pNP standard curve a =0.0068 c.
From FIG. 5, it can be seen that the change of temperature has little influence on the catalytic activity of N α -CDH-Zn, the optimum catalytic temperature of N α -CDH-Zn is 30 ℃, the activity of N α -CDH-Zn is gradually reduced with the increase of catalytic temperature, but the catalytic activity of N α -CDH-Zn can reach 90.1% of the highest activity at 80 ℃, which indicates that the mimic enzyme N α -CDH-Zn has better temperature stability. The N alpha-CDH-Zn still has good catalytic performance in a higher temperature range (60-90 ℃). Therefore, the higher reaction temperature of the N alpha-CDH-Zn shows higher catalytic capability similar to carboxylesterase.
Example 11 investigation of salt tolerance of artificial enzyme carboxylesterase.
In order to test the catalytic performance of the N alpha-CDH-Zn of the invention after incubation in the buffer solution with different ionic strengths, 100 mu L of 1 mg/mL -1 The suspension of Nalpha-CDH-Zn was mixed with 800. Mu.L of 10mM PB (pH 7.0) buffer, incubated in 50, 100, 200, 300, 400, 500, 600mM NaCl-containing buffer for 30min, and 100. Mu.L of pNPB solution (1 m NaCl) was addedM) are mixed evenly (the total reaction volume is 1 mL), after 1h of reaction at room temperature, the mixture is centrifuged at 10000rpm for 5min to retain supernatant, and the absorbance value of the solution at 405nm is monitored by using a microplate reader. The catalytic capacity of na-CDH-Zn was calculated as the product concentration μ M from the pNP standard curve a =0.0068 c.
From FIG. 6, it can be seen that the catalytic activity of N α -CDH-Zn is gradually decreased with the increase of ionic strength, and when the ionic strength reaches 600mM, the activity of N α -CDH-Zn can reach 50.5% of the highest activity, which indicates that the mimic enzyme N α -CDH-Zn has good stability of ionic strength, and can be applied to environments with high salt ions.
Example 12 recyclability of carboxylesterase-like activity of an artificial enzyme.
Weighing 1mg of the Nalpha-CDH-Zn of the invention, placing the Nalpha-CDH-Zn into a 1.5mL centrifuge tube, adding 900 mu L of 10mM PB (pH 7.0) buffer solution and 100 mu L of pNPB, reacting at room temperature for 1h after uniform mixing, centrifuging at 10000rpm for 5min at 4 ℃ after the reaction is finished, taking the supernatant, measuring the light absorption value at 405nm, cleaning the precipitate with ultrapure water for 3 times, adding the reactant, repeating the cycle for 10 times, and calculating the relative activity of the Nalpha-CDH-Zn.
The drawback of natural carboxylesterase enzymes, which are inherently water-soluble in proteins and therefore not recyclable after a single use, has limited their industrial application. The N alpha-CDH-Zn can be recovered and reused by centrifugation, and as can be seen from FIG. 7, the N alpha-CDH-Zn still maintains 65.1% of activity after 10 cycles. This indicates that N α -CDH-Zn has good recycling property. The loss of activity is due to the loss of mass of the catalyst which inevitably results from the washing process, thereby causing a reduction in activity.
EXAMPLE 13 CO of an Artificial enzyme of Carbonhydridase Activity 2 The transformation ability of (a).
Effective removal of CO 2 Has important significance for the catalytic performance and the application of carbonic anhydrase. Therefore, we will CO 2 Conversion to CaCO 3 The carbonic anhydrase performance of N.alpha. -CDH-Zn was evaluated. Weighing 11.915g HEPES, dissolving in 800mL deionized water, adjusting pH to 8.0 with NaOH buffer solution, diluting to 1L, filtering with 0.45 μm filter membrane for sterilization, weighing 4.44g anhydrous calcium chloride, and dissolving in 100mL of HEPES buffer solution at pH 8.0 to give 400mM CaCl 2 The solution was filter sterilized with a 0.45 μm filter for further use. Dissolving catalyst Nalpha-CDH-Zn in HEPES to obtain suspension with concentration of 1mg/L, adding 1mL of catalyst suspension, 5mL of CaCl 2 The solution, 5mL HEPES buffer, was mixed well in a 25mL beaker. Introducing CO into the system 2 Gas for 1h. Centrifugal collection of CaCO 3 And dried and finally the resulting CaCO is weighed 3 The yield was calculated.
Blank in FIG. 8 represents Blank group, in which no Na-CDH-Zn was added, blank control, zn 2+ CaCO of Nalpha-CDH-Zn 3 The mass of (A) was 5.1, 8.3, 42.7mg, respectively. CaCO for Nalpha-CDH-Zn formation 3 The weight of (a) was 8.4 times that of the blank. Thus showing that the N alpha-CDH-Zn can reduce the content of carbon dioxide in the environment.
Example 14 class carboxylesterase and carbonic anhydrase dual enzyme activity cytotoxicity of artificial enzymes.
Adopting thiazole blue colorimetry to carry out colorimetry on artificial enzymes Nalpha-CDH-Zn, nalpha-CDH and Zn 2+ The cytotoxicity of (a) was characterized. Adding 80 μ L mouse microglia BV-2 (8 × 10) into sterilized 96-well plate 3 One), and culturing for 24h to make the cells adhere to the wall. Then 20. Mu.L of samples to be tested at different concentrations were added and incubation continued for 24h. 5.5 mg. Multidot.mL of a sterilized PBS solution (containing 10mM PB and 10mM NaCl, pH 7.0) was prepared -1 MTT solution (2). mu.L of MTT solution was added to each well and incubation was continued for 3-4h. The cell culture plate was then centrifuged at 1500rpm for 10min and the supernatant discarded. 100 μ L of dimethyl sulfoxide (DMSO) was added to each well, and the 96-well plate was shaken in an air shaker at 150rpm at 37 ℃ until the blue-violet formazan particles were completely dissolved in the plate. Finally, the absorbance of each sample at 570nm was measured using a microplate reader. The cell group to which PBS buffer was added was used as a control group, and the sample containing only the culture medium without the cells was used as a blank group. The cell activity was calculated using equation 1.
From FIG. 9, it can be seen that the MTT method is usedThe in vitro cytotoxicity of the artificial enzyme N alpha-CDH-Zn is evaluated, the in vitro cytotoxicity of different catalytic materials is evaluated by an MTT method, and the research finds that various catalytic materials show the condition that the cytotoxicity is enhanced along with the increase of the material concentration after the catalytic materials are co-cultured with BV-2 cells for 24 hours. Blank represents a Blank group in which no Nalpha-CDH-Zn, zn and Nalpha-CDH were added, and it was found from the study that the toxicity of Nalpha-CDH-Zn to cells was gradually increased with the increase of the concentration, but when the concentration in the mixed solution reached 800. Mu.g.mL -1 At this time, the cell activity was maintained at 76.2%. After the addition of N alpha-CDH, the toxicity gradually increases with the increase of the concentration, and when the concentration reaches 100 mu g/mL -1 In this case, the cell activity was 68.4% of that of the control group, and the cytotoxicity of N.alpha. -CDH was stronger than that of N.alpha. -CDH-Zn. When 200. Mu.g/mL of the solution is added -1 Zn of (2) 2+ The post-cell activity decreased to 45.4% of the original value, indicating Zn 2+ Has strong toxicity to cells. Therefore, we will have a slightly toxic na-CDH and a more toxic Zn 2+ Through coordination interaction, a nano material with hydrolytic ester substances and good biocompatibility to the environment is formed.
While the methods and techniques of the present invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications of the methods and techniques described herein may be practiced without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.
Claims (7)
1. An artificial enzyme having both carboxylesterase-like and carbonic anhydrase-like activities; the supramolecular material with carboxylesterase-like and carbonic anhydrase activity is formed by coordination and combination of N alpha-carbobenzoxy-D-histidine and chloride salt consisting of transition metal, and is marked as N alpha-CDH-M, wherein N alpha-CDH represents N alpha-carbobenzoxy-D-histidine, and M represents transition metal copper and zinc.
2. A process for the preparation of an artificial enzyme of carboxylesterase-and carbonic anhydrase-like activity according to claim 1, characterized in that it comprises the steps of:
1) Adding the aqueous solution of the Nalpha-benzyloxycarbonyl-D-histidine Nalpha-CDH and the aqueous solution of the chloride salt of the transition metal copper or zinc into a weak alkaline buffer solution, and magnetically stirring to complete the coordination chelation reaction; standing at normal temperature for 24-72h to complete self-assembly.
2) Centrifuging at 8000-10000rpm for 10-30min, discarding supernatant, collecting precipitate, cleaning the precipitate with ultrapure water for 3-5 times, and vacuum drying the precipitate containing partial water after the last centrifugation to obtain N alpha-CDH-Zn or N alpha-CDH-Cu solid sample.
3. The method as set forth in claim 2, wherein the water specifications of the na-CDH and the transition metal salt solution prepared in step 1) are at least ultra pure water; the concentration of the N alpha-CDH solution is 20-50mM; the concentration of the transition metal salt solution is 20-100mM; the process of adding the transition metal salt solution to the na-CDH solution needs to be performed at normal temperature.
4. The method as set forth in claim 2, wherein the molar ratio of the transition metal salt solution to the na-CDH solution in step 1) is 1.
5. The method as set forth in claim 2, wherein the reaction conditions of step 1) after the addition of the transition metal salt solution are: self-assembling for 24-72h at normal temperature.
6. The method as set forth in claim 2, wherein the temperature of the vacuum drying oven of step 2) is set to 40-80 ℃ and the drying time is 24-72 hours until the precipitate containing a small amount of moisture is dried to powder na-CDH-Zn or na-CDH-Cu.
7. The use of a self-assembled nanomaterial of claim 1 to mimic carboxylesterase and carbonic anhydrase activity.
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Citations (5)
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| WO2015056858A1 (en) * | 2013-10-17 | 2015-04-23 | 포항공과대학교 산학협력단 | Carbonic anhydrase having high-temperature stability and carbon dioxide collector comprising same |
| KR20160096793A (en) * | 2015-02-05 | 2016-08-17 | 한국에너지기술연구원 | Carbonic anhydrase mimetic enzyme comprising transition metal and tridentate ligands comprising nitrogens and use thereof |
| CN114874449A (en) * | 2022-04-28 | 2022-08-09 | 南阳理工学院 | Preparation method and application of metal-organic framework compound containing single-atom catalytic site |
| CN114904581A (en) * | 2022-05-31 | 2022-08-16 | 杭州诺莘科技有限责任公司 | Oxidized mimic enzyme and preparation method and application thereof |
| CN117106189A (en) * | 2023-07-18 | 2023-11-24 | 华南理工大学 | Metal organic framework artificial hydrolase and preparation method and application thereof |
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| WO2015056858A1 (en) * | 2013-10-17 | 2015-04-23 | 포항공과대학교 산학협력단 | Carbonic anhydrase having high-temperature stability and carbon dioxide collector comprising same |
| KR20160096793A (en) * | 2015-02-05 | 2016-08-17 | 한국에너지기술연구원 | Carbonic anhydrase mimetic enzyme comprising transition metal and tridentate ligands comprising nitrogens and use thereof |
| CN114874449A (en) * | 2022-04-28 | 2022-08-09 | 南阳理工学院 | Preparation method and application of metal-organic framework compound containing single-atom catalytic site |
| CN114904581A (en) * | 2022-05-31 | 2022-08-16 | 杭州诺莘科技有限责任公司 | Oxidized mimic enzyme and preparation method and application thereof |
| CN117106189A (en) * | 2023-07-18 | 2023-11-24 | 华南理工大学 | Metal organic framework artificial hydrolase and preparation method and application thereof |
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