CN115518691A - Artificial enzyme with laccase-like enzyme activity, preparation method and application thereof - Google Patents
Artificial enzyme with laccase-like enzyme activity, preparation method and application thereof Download PDFInfo
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- CN115518691A CN115518691A CN202211264345.9A CN202211264345A CN115518691A CN 115518691 A CN115518691 A CN 115518691A CN 202211264345 A CN202211264345 A CN 202211264345A CN 115518691 A CN115518691 A CN 115518691A
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- laccase
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- alanine
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 63
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 63
- 230000000694 effects Effects 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title abstract description 15
- 239000010949 copper Substances 0.000 claims abstract description 83
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 33
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 27
- 235000004279 alanine Nutrition 0.000 claims abstract description 27
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000012965 benzophenone Substances 0.000 claims abstract description 27
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 27
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- 239000002244 precipitate Substances 0.000 claims abstract description 25
- 239000011701 zinc Substances 0.000 claims abstract description 23
- 239000000203 mixture Substances 0.000 claims abstract description 18
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- 150000003624 transition metals Chemical class 0.000 claims abstract description 14
- 229910052802 copper Inorganic materials 0.000 claims abstract description 12
- 238000001291 vacuum drying Methods 0.000 claims abstract description 12
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 11
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 9
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- 238000004140 cleaning Methods 0.000 claims abstract description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims abstract description 3
- 230000009920 chelation Effects 0.000 claims abstract description 3
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
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- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 16
- 238000011534 incubation Methods 0.000 description 16
- MZHCENGPTKEIGP-UHFFFAOYSA-N 2-(2,4-dichlorophenoxy)propanoic acid Chemical compound OC(=O)C(C)OC1=CC=C(Cl)C=C1Cl MZHCENGPTKEIGP-UHFFFAOYSA-N 0.000 description 15
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 15
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 15
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- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 12
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- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 8
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- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
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- 230000007613 environmental effect Effects 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
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- 150000003839 salts Chemical class 0.000 description 5
- LINPIYWFGCPVIE-UHFFFAOYSA-N 2,4,6-trichlorophenol Chemical compound OC1=C(Cl)C=C(Cl)C=C1Cl LINPIYWFGCPVIE-UHFFFAOYSA-N 0.000 description 4
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 description 4
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- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 4
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- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 3
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- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
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- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
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- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
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- 239000013148 Cu-BTC MOF Substances 0.000 description 1
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- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-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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Classifications
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- 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
- B01J31/2226—Anionic ligands, i.e. the overall ligand carries at least one formal negative charge
- B01J31/2243—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
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
-
- 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
- B01J31/1691—Coordination polymers, e.g. metal-organic frameworks [MOF]
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/72—Treatment of water, waste water, or sewage by oxidation
- C02F1/725—Treatment of water, waste water, or sewage by oxidation by catalytic oxidation
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/72—Treatment of water, waste water, or sewage by oxidation
- C02F1/74—Treatment of water, waste water, or sewage by oxidation with air
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Life Sciences & Earth Sciences (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Inorganic Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to an artificial enzyme with laccase-like activity and a preparation method and application thereof, wherein a chloride aqueous solution of transition metal copper or zinc is added into an alkaline aqueous solution of benzophenone alanine, and is stirred to be uniformly mixed so as to complete coordination chelation reaction, and the mixture is placed in a water bath kettle at the temperature of 30-80 ℃ for 0-60h to complete self-assembly; centrifuging at 8000-10000rpm for 10-20min, discarding supernatant, collecting precipitate, cleaning precipitate with ultrapure water, and vacuum drying the precipitate to obtain BpA-Cu and BpA-Zn solid. The preparation condition of the invention is simple, the synthesis condition of the unnatural amino acid is mature and the price is low, and the source is wide; the activity of the catalyst can keep 50.2 percent of the original activity under the alkaline condition and keep 89.9 percent of the activity at the high temperature of 90 ℃, and the catalyst can be applied to the removal of phenol organic pollutants in water bodies in the fields of printing and dyeing and the like.
Description
Technical Field
The invention relates to a preparation method and application of a laccase-like activity simulant formed by self-assembly of benzophenone alanine and transition metal (such as copper, zinc and the like) ions through coordination, belongs to the technical field of artificial enzyme preparation and application, and particularly relates to an artificial enzyme with laccase-like activity, a preparation method and application.
Background
Enzymes are biomacromolecules with catalytic functions, and are mainly used in Nature to catalyze chemical reactions in organisms, which are generally performed under relatively mild conditions (Nature 2012,485, 185-194). However, in practical applications, natural enzymes have disadvantages of difficulty in preparation and purification, poor stability, sensitivity of catalytic activity to environmental conditions, difficulty in recovery and reuse, limited sources, limited types of catalytic reactions, etc., which have prompted the design and development of a variety of novel artificial catalysts to replace the functions of natural enzymes (Chemical Society Reviews 2019,48, 1004-1076).
Laccase (laccanase, EC 1.10.3.2) is an extracellular single-molecule glycoprotein, which was first found in lacca lacquer sap, widely distributed in fungi, higher plants and bacteria. Laccases are copper-containing polyphenol oxidase, can react with phenols, ascorbic acid, amines and other substances (microbiological Biotechnology 2017,10, 1457-1467), can catalyze and oxidize various organic pollutants which are difficult to degrade, and have wide application in the aspects of wastewater treatment, biosensors and biofuel cells. Laccase catalytic oxidation only generates water, and is a green biocatalyst. However, natural laccases, as natural proteins, still have the significant disadvantage of being easily denatured. 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, natural laccase is easily soluble in water, laccase free in water cannot be recycled and cannot be stored for a long time, and the two limits are limited to the application of laccase in industrial environment to a great extent. Therefore, there is a need to develop new mimic enzymes that have laccase activity and meet the stability requirements.
Currently, MOFs materials, noble metal nanoparticles, carbon-based nanomaterials, proteins, amino acids, etc. have been used to mimic laccases. Cysteine-histidine (Cys-His) dipeptide co-assembly has been used to construct nano-enzyme CH-Cu (Cellular and Molecular Life Sciences 2015,72, 869-883) with laccase catalytic activity. And are used to degrade phenolic contaminants and to detect epinephrine (Applied Catalysis B-Environmental,2019,254, 452-462). MOFs materials GMP-Cu, cu-Cys (cystine), and HKUST-1 also exhibit laccase activity and are applied to the degradation of phenolic contaminants and dyes (Frontiers of Chemical Science and Engineering,2021,15, 310-318). Pt nanoparticles with good dispersity, which are closed by nucleotides, are synthesized by using four nucleotides, and are used for catalyzing substrates of various laccases, and the Pt nanoparticles show good catalytic activity (Catalysis Letters,2017,147, 2144-2152). Copper-containing carbon-point CuCDs with laccase activity and fluorescence property are synthesized by a hydrothermal method, show good photoluminescence property under high salt and wide pH range, have good stability and can be used as a fluorescent probe for detecting hydroquinone (Nanoscale, 2015,7, 19641-19646). However, the reported related materials have poor performances in terms of complex preparation, reusability, catalytic activity and the like.
Disclosure of Invention
The invention aims to solve the problems of poor biocompatibility, complex preparation process and the like of materials used by the conventional simulated laccase, and provides application of a laccase-like activity mimic formed by coordination of benzophenone alanine and transition metal (such as copper, zinc and the like) ions in the field of treatment of phenolic pollutants. The invention also aims to provide a preparation method of the laccase-like activity mimic formed by coordination of benzophenone alanine and transition metal (such as copper, zinc and the like) ions. The preparation method is simple, the reaction condition is mild, the operation is easy, and the catalytic nano material with laccase activity is synthesized by using abundant, cheap and easily-obtained unnatural amino acids and transition metal ions as ligands, so that the cost for treating phenol pollution generated by industrial production, large-scale use of bactericides and herbicides in the agricultural production process and degradation of certain organic matters is saved.
The technical scheme of the invention is summarized as follows:
a supermolecular material with laccase-like activity is formed by the coordination combination of chloride salt consisting of amino and carboxyl of artificial enzyme benzophenone alanine with laccase-like activity and transition metal, and is recorded as BpA-M, wherein BpA is benzophenone alanine, and M represents transition metal copper and zinc.
The preparation method of the artificial enzyme with laccase-like activity comprises the following steps:
1) Adding a chloride aqueous solution of transition metal copper or zinc into an alkaline aqueous solution of benzophenone alanine, stirring to uniformly mix the two solutions to complete coordination chelation reaction, and standing in a water bath kettle at the temperature of 30-80 ℃ for 0-60h to complete self-assembly;
2) Centrifuging at 8000-10000rpm for 10-20min, discarding supernatant, collecting precipitate, cleaning precipitate with ultrapure water, and vacuum drying the precipitate to obtain BpA-Cu and BpA-Zn solid.
The alkaline solution of benzophenone alanine in the step 1) is prepared by dissolving 5-50mM of benzophenone alanine in 10mM of sodium hydroxide; preparing NaOH and transition metal salt solution with water specification at least being ultrapure water; the concentration of the transition metal chloride solution is 10-20mM.
The benzophenone alanine alkaline aqueous solution and the transition metal chloride solution in the step 1) need to be incubated for 30-60min in a water bath kettle at the temperature of 40-80 ℃ before being mixed; the process of adding the transition metal chloride solution into the benzophenone alanine alkaline water solution is carried out under the condition of water bath at 40-80 ℃.
The mole ratio of the transition metal chloride solution in the step 1) to the benzophenone alanine is 1-1.
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 moisture is dried to be powder BpA-Cu and BpA-Zn.
The invention relates to application of an artificial enzyme with laccase-like activity in the field of simulating laccase activity and solving phenol pollutants.
The invention uses benzophenone alanine and transition metal such as copper, zinc and other ions to form a laccase-like activity simulant through coordination, and utilizes the self-assembly nano material prepared by the method to simulate partial functions of laccase. The activity and stability of the simulated laccase and the natural laccase are measured by using 2, 4-dichlorophenol (2.4-DP) as a substrate and 4-aminoantipyrine (4-AP) as a color developing agent. This is mainly due to the fact that laccase oxidizes 2,4-DP in the presence of oxygen into free radicals, which can couple with 4-AP to a substance with a color change, which has a distinct absorption peak at 510 nm. Compared with natural laccase, the simulated laccase of the invention has higher substrate affinity compared with natural laccase. The Km values of the compounds are 0.07 and 0.27), the catalytic efficiency under extreme conditions (high temperature, strong acid and strong base) is higher, the catalytic efficiency is respectively 89.9%, 39.5% and 50.2%, and the biocompatibility is good, wherein the damage of 800 mu g/mL mimic enzyme BpA-Cu to mouse microglia BV-2 is lower, and the survival rate of cells can reach 83.84%. The mimic enzyme with laccase-like activity is applied to the field of catalytic nano materials, and can solve the problems of poor reutilization property and disappearance or reduction of activity under extreme conditions (strong acid, strong alkali, high temperature and high salt) of natural laccase.
Besides, in order to research the degradation capability of the simulated enzymes BpA-Cu and laccase on environmental phenolic pollutants, a plurality of phenolic pollutants such as hydroquinone, 2-aminophenol, 2,4, 6-trichlorophenol, catechol, phenol, 2-naphthol, 2-nitrophenol and 2,4-DP are selected as model substrates of the phenolic pollutants. The catalytic activity of the BpA-Cu and the laccase on various substrates is standardized and calculated by taking the activity of the BpA-Cu on 2,4-DP as 100%, and the relative catalytic capability of the mimic enzyme BpA-Cu on 7 phenolic pollutants as substrates is higher than that of the natural laccase except for 2, 4-DP. Especially, the oxidizing abilities of the pyrocatechol, the phenol and the 2-nitrophenol can respectively reach 97.1 percent, 70.1 percent and 81.2 percent of the catalytic ability of the 2,4-DP as the substrate. However, the catalytic capacity of natural laccases for these substrates is only 23.3%, 0.1% and 1.7%. Although the relative catalytic abilities of the mimic enzyme and the laccase are low when hydroquinone and 2,4, 6-trichlorophenol are used as substrates, the activity of the mimic enzyme is still higher than that of the laccase. The mimic enzyme BpA-Cu has oxidation capacity on various environmental phenolic pollutants, which indicates that the mimic enzyme BpA-Cu has substrate universality.
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 uv-vis spectrophotometers and microplate readers. Compared with natural laccase and laccase imitating materials reported in the literature, the invention has the advantages that:
1) The prepared mimic enzyme has higher capability of catalyzing substrate oxidation and lower Km, and the numerical value corresponding to the Km of the natural enzyme is 0.07mM VS 0.27mM;
2) The catalytic ability of the prepared mimic enzyme is enhanced along with the increase of the salt concentration, so that the mimic enzyme can play a role in the fields of treating lignin and organic phenol pollution in seawater and the like in the future;
3) The activity of the prepared mimic enzyme can keep 50.2 percent of the original activity under the alkaline condition and 89.9 percent of the activity at the high temperature of 90 ℃, so that the mimic enzyme can be used for removing phenol organic pollutants in water in the fields of printing and dyeing and the like.
Drawings
FIG. 1 is a scanning electron micrograph (b) of the self-assembly artificial enzyme BpA-Cu of example 2, which is an enlarged view of the micrograph (a).
FIG. 2 is a graph of the relative catalytic activity of the self-assembled artificial enzyme and the laccase from example 7.
FIG. 3 is a graph of the relative catalytic activity of the self-assembled artificial enzyme of example 8 in buffers containing different salt concentrations.
FIG. 4 is a graph of the relative catalytic activities of the self-assembled artificial enzyme and laccase of example 9 after incubation for 30min at different temperatures.
FIG. 5 is a graph of the relative catalytic activities of the self-assembled artificial enzyme and laccase in different pH buffers of example 10.
FIG. 6 shows the catalytic activity of the nanoenzyme BpA-Cu of example 11 after several cycles.
FIG. 7 is a graph showing the cytotoxicity of the self-assembling artificial enzyme BpA-Cu and the starting material in example 12.
FIG. 8 is a graph of the relative catalytic activity of the self-assembled artificial enzymes BpA-Cu and laccase for various phenolic contaminants of example 13.
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 nano enzyme with laccase activity 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 10-50mM BpA alkaline aqueous solution, and preparing 10-20mM CuCl with ultrapure water 2 、ZnCl 2 And (2) solution, namely placing the two obtained solutions in a water bath kettle at 40-80 ℃ for heat preservation for 30-60min, then slowly adding the heat-preserved transition metal salt solution into the BpA alkaline solution, wherein magnetic stirring is required in the process to ensure that the transition metal chloride solution is fully and uniformly mixed until the transition metal chloride solution is completely added, and the ratio of the BpA alkaline solution to the transition metal chloride solution is controlled to be 1-1. Fully mixing, and self-assembling in water bath at 40-80 deg.C for 0-60 hr to obtain final self-assembled supramolecular aggregate suspension.
2) Centrifuging the suspension obtained after the reaction at high speed, namely centrifuging at 4-25 deg.C and 8000-10000rpm for 10-20min, discarding the supernatant, retaining the precipitate, and washing the precipitate with ultrapure water for 3-5 times; and (3) putting the cleaned precipitate in a vacuum drying oven at 40-80 ℃ for 24-72h to obtain self-assembled nano enzyme powder with laccase-like activity. The activity, stability and biocompatibility of the nano enzyme powder are measured.
Example 1 BpA with CuCl at 40 ℃ 2 The molar ratio of (1).
40mg of solid NaOH was dissolved in 100mL of ultrapure water to obtain a 10mM NaOH solution, and 59.264mg of benzophenone alanine BpA was dissolved in the NaOH solution to obtain 10mM B20mL of pA alkaline solution; 170.5mg of cupric chloride dihydrate was dissolved in 100mL of ultrapure water to give CuCl at a concentration of 10mM 2 The solution is reserved, and 20mL, 4 mL and 2mL CuCl are respectively measured by a measuring cylinder 2 Respectively placing the solutions in a water bath kettle at 40 deg.C, and incubating for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated CuCl is added 2 The solution is slowly added into the BpA alkaline solution, magnetic stirring is adopted in the process until the solution is completely and uniformly mixed, and the mixture is kept stand for 60 hours in a water bath kettle at the temperature of 40 ℃ after being uniformly mixed so as to meet the self-assembly process of the mixture to obtain the BpA-Cu suspension.
And centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 8000rpm for 20min, discarding the supernatant which is a reactant not participating in the reaction, washing and precipitating for 5 times by using ultrapure water to further remove a reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 40 ℃ for 72h to obtain the nano enzyme solid powder BpA-Cu synthesized under different conditions.
Example 2 BpA with CuCl at 60 ℃ 2 The molar ratio of (1).
Dissolving 40mg of sodium hydroxide solid (NaOH) in 100mL of ultrapure water to obtain a 10mM NaOH solution for later use, and dissolving 118.528mg of benzophenone alanine (BpA) in the NaOH solution to obtain 20mL of a 20mM BpA alkaline solution; 341mg of copper chloride dihydrate (CuCl) 2 ·2H 2 O) was dissolved in 100mL of ultrapure water to give CuCl at a concentration of 20mM 2 The solution is reserved, and 20, 10 and 2mL of CuCl are measured by a measuring cylinder 2 Respectively placing the solutions in a water bath kettle at 60 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches the set value, the incubated CuCl is added 2 The solution is slowly added into the BpA alkaline solution, magnetic stirring is adopted in the process until the solution is completely and uniformly mixed, and the mixture is kept stand for 48 hours in a water bath kettle at 60 ℃ after being uniformly mixed so as to meet the self-assembly process of the mixture to obtain the BpA-Cu turbid liquid.
And centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 9000rpm for 18min, discarding the supernatant which is a reactant not participating in the reaction, washing the precipitate for 5 times by using ultrapure water to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 60 ℃ for 48h to obtain the nano enzyme solid powder BpA-Cu.
The morphology of the synthesized nanoenzyme is observed by using a scanning electron microscope, and the structure formed when the standing time is 0h is not obvious, and the structure of the nanoenzyme is gradually complete along with the prolonging of the self-assembly time as can be seen from figure 1.
Example 3 BpA with CuCl at 80 ℃ 2 The molar ratio of (1).
Dissolving 40mg of sodium hydroxide solid (NaOH) in 100mL of ultrapure water to obtain a 10mM NaOH solution for later use, and dissolving 296.32mg of benzophenone alanine (BpA) in the NaOH solution to obtain 20mL of a 50mM BpA alkaline solution; 852.5mg of cupric chloride dihydrate was dissolved in 100mL of ultra-pure water to give CuCl at a concentration of 50mM 2 The solution is reserved, and 20, 4 and 2mL of CuCl are measured by a measuring cylinder 2 Respectively placing the solutions in a water bath kettle at 80 ℃ for incubation for 30min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches the set value, the incubated CuCl is added 2 The solution is slowly added into the BpA alkaline solution, magnetic stirring is adopted in the process until the solution is completely and uniformly mixed, and the mixture is kept stand for 24 hours in a water bath kettle at 80 ℃ after being uniformly mixed so as to meet the self-assembly process of the mixture to obtain the BpA-Cu.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 10000rpm for 20min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate for 5 times by using ultrapure water to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 80 ℃ for 24h to obtain the nano enzyme solid powder BpA-Cu synthesized under different conditions.
Example 4 BpA and ZnCl at 40 ℃ 2 The molar ratio of (1).
40mg of solid NaOH was dissolved in 100mL of ultrapure water to obtain a 10mM NaOH solution, and 59.264mg of NaOH solution was addedDissolving benzophenone alanine BpA in the NaOH solution to obtain 20mL of 10mM BpA alkaline solution; 136.315mg of zinc chloride was dissolved in 100mL of ultrapure water to obtain ZnCl with a concentration of 10mM 2 The solution is reserved, and 20mL, 4 mL and 2mL ZnCl are respectively measured by a measuring cylinder 2 Respectively placing the solutions in a water bath kettle at 40 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated ZnCl is used 2 The solution is slowly added into the BpA alkaline solution, magnetic stirring is adopted in the process until the solution is completely and uniformly mixed, and the mixture is kept stand for 60 hours in a water bath kettle at the temperature of 40 ℃ after being uniformly mixed so as to meet the self-assembly process of the mixture to obtain the BpA-Zn suspension.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 8000rpm for 20min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate for 5 times by using ultrapure water to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 40 ℃ for 72h to obtain the nano enzyme solid powder BpA-Zn synthesized under different conditions.
Example 5 BpA and ZnCl at 60 ℃ 2 The molar ratio of (1)
Dissolving 40mg of sodium hydroxide solid (NaOH) in 100mL of ultra-pure water to obtain a 10mM NaOH solution for standby, and dissolving 118.528mg of benzophenone alanine (BpA) in the NaOH solution to obtain 20mL of a 20mM BpA alkaline solution; 272.63mg of ZnCl 2 Dissolved in 100mL of ultrapure water to obtain ZnCl with a concentration of 20mM 2 The solution is reserved, and 20, 10 and 2mL of ZnCl are measured by a measuring cylinder 2 Respectively placing the solutions in a water bath kettle at 60 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated ZnCl is used 2 The solution is slowly added into the BpA alkaline solution, magnetic stirring is adopted in the process until the solution is completely and uniformly mixed, and the mixture is kept stand for 48 hours in a water bath kettle at 60 ℃ after being uniformly mixed so as to meet the self-assembly process of the mixture to obtain the BpA-Zn suspension.
Centrifuging the suspension obtained by the reaction for 18min at 4 ℃ and 9000rpm in a low-temperature refrigerated centrifuge, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate for 5 times by using ultrapure water to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 60 ℃ for 48h to obtain the nano enzyme solid powder BpA-Zn.
Example 6 BpA with CuCl at 80 ℃ 2 The molar ratio of (1).
Dissolving 40mg of sodium hydroxide solid (NaOH) in 100mL of ultra-pure water to obtain a 10mM NaOH solution for later use, and dissolving 296.32mg of benzophenone alanine (BpA) in the NaOH solution to obtain 20mL of a 50mM BpA alkaline solution; 681.575mg of zinc chloride was dissolved in 100mL of ultrapure water to obtain ZnCl at a concentration of 50mM 2 The solution is reserved, and 20, 4 and 2mL of ZnCl is measured by a measuring cylinder 2 Respectively placing the solutions in a water bath kettle at 80 ℃ for incubation for 30min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated ZnCl is used 2 The solution is slowly added into the BpA alkaline solution, magnetic stirring is adopted in the process until the solution is completely and uniformly mixed, and the mixture is kept stand for 24 hours in a water bath kettle at 80 ℃ after being uniformly mixed so as to meet the self-assembly process of the mixture to obtain the BpA-Zn.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 10000rpm for 15min, discarding the supernatant which is a reactant not participating in the reaction, washing and precipitating with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 80 ℃ for 24h to obtain the nano enzyme solid powder BpA-Zn synthesized under different conditions.
Example 7 laccase-like activity catalytic performance study of nanoenzymes.
Weighing BpA-Cu and BpA-Zn of the invention, respectively dissolving in a PB (10mM, pH7.0) buffer solution, and performing strong ultrasound for 20min to obtain a suspension with the concentration of 1mg/mL for later use; 163.0014mg of 2, 4-dichlorophenol (2, 4-DP) is weighed and dissolved in 100mL of absolute ethyl alcohol to obtain a 2,4-DP solution with the concentration of 10mM for standby; weighing 203.24mg of 4-aminoantipyrine (4-AP) and dissolving in 100mL of absolute ethanol to obtain a 4-AP solution with the concentration of 10mM for later use; 100 mu L of 2,4-DP,100 mu L of 4-AP,100 mu L of LBpA-Cu or BpA-Zn and 700 mu L of PB buffer solution are uniformly mixed in a centrifuge tube of 1.5mL (the total reaction volume is 1 mL), the mixture is reacted for 1h at room temperature and centrifuged at 10000rpm for 5min to retain supernatant, and the solution is monitored for light absorption value (indophenol amino antipyrine dye) at 510nm by using a microplate reader to determine the oxidation capacity of the 2,4-DP by using the molar extinction coefficient of the indophenol amino antipyrine dye.
From FIG. 2, it can be seen that BpA and BpA-Zn have laccase-like activity, whereas BpA-Cu of the present invention can oxidize 2,4-DP and bind to 4-AP to darken the solution. As shown and when the rate of catalytic reaction is different when different concentrations of 2,4-DP are added, the kinetic parameters of catalysis of the BpA-Cu are respectively calculated to be Km of 0.07mM and Vmax of 2.1X 10 according to the difference of the reaction rates -5 mM·S -1 The Km value of BpA-Cu was lower compared to other studies, indicating that BpA-Cu has a higher affinity for the substrate 2,4-DP.
Example 8 investigation of salt tolerance of the activity of nanoenzyme laccase.
In order to test the catalytic performance of the BpA-Cu of the invention after incubation under the buffer solution condition containing different ionic strengths, 100 muL of 1mg/mL BpA-Cu suspension and 700 muL of 10mM PB (pH 7.0) buffer solution are mixed and then placed in the buffer solution condition containing 50, 100, 200, 300, 400, 500 and 600mM NaCl respectively for incubation for 30min, 100 muL of 10mM2,4-DP and 100 muL of 10mM 4-AP are added and mixed uniformly (total reaction volume is 1 mL), after 1h of reaction at room temperature, the supernatant is retained by centrifugation at 10000rpm for 5min, and the absorbance value of the solution at 510nm is monitored by using a microplate reader to determine the oxidation capacity of 2,4-DP by utilizing the molar coefficient of indophenol amino antipyrine dye.
From FIG. 3, it can be seen that the catalytic activity of BpA-Cu of the present invention increases with the salt ion concentration, and the activity can reach 3.62 times of that of the NaCl-free condition in the presence of 600mM NaCl, whereas the activity of the natural laccase decreases with the increase of the salt concentration. The nano enzyme can be applied to the treatment of the field of seawater phenol pollution.
Example 9 catalytic performance of laccase-like activity of nanoenzymes at high temperature.
In order to test the catalytic performance of BpA-Cu prepared by the invention after incubation at high temperature, 100 muL of 1mg/mL BpA-Cu suspension and 700 muL of 10mM PB (pH 7.0) buffer solution are mixed and then are respectively placed under the temperature conditions of 0, 30, 40, 50, 60, 70, 80 and 90 ℃ for incubation for 30min until the temperature is returned to room temperature, 100 muL of 10mM2,4-DP and 100 muL of 10mM 4-AP are added and uniformly mixed (the total reaction volume is 1 mL), after 1h of reaction at room temperature, the supernatant is retained by centrifugation at 10000rpm for 5min, an extinction instrument is used for monitoring the light absorption value of the solution at 510nm, the oxidation capacity of 2,4-DP is measured by utilizing the molar coefficient of indophenol amino antipyrine dye, and the relative activity under other conditions is calculated by taking the catalytic activity after incubation at 0 ℃ for 30min as 100%.
From FIG. 4, it can be seen that the change of temperature has little influence on the catalytic activity of BpA-Cu, the activity can be maintained to 89.98% after incubation for 30min at 90 ℃, the catalytic activity of laccase is reduced along with the increase of temperature, the catalytic activity is reduced to 36.85% when the incubation temperature is 50 ℃, and the laccase basically and completely loses the catalytic activity when the incubation temperature is 80 ℃. Compared with laccase, the BpA-Cu still has good catalytic performance in a higher temperature range (60-90 ℃). Therefore, the BpA-Cu shows higher laccase-like catalytic capability at higher reaction temperature, and can be used for removing phenol organic pollutants in water in the fields of printing and dyeing and the like.
Example 10 catalytic performance of laccase-like activity of nanoenzymes under extreme pH conditions.
In order to detect the catalytic performance of the BpA-Cu prepared by the invention under different pH conditions, 100 mu L of 1mg/mL BpA-Cu suspension and 700 mu L of buffer solution with pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 are incubated for 30min, 100 mu L of 2,4-DP and 100 mu L of 4-AP are mixed uniformly (the total reaction volume is 1 mL), the mixture reacts for 1h at room temperature, then the mixture is centrifuged at 10000rpm for 5min to retain supernatant, and an enzyme reader is used for monitoring the light absorption value of the solution at 510nm and the molar extinction coefficient of indophenol amino antipyrine dye is used for measuring the oxidation capacity of 2, 4-DP. Wherein the conditions of pH 3.0, 4.0 and 5.0 are prepared from 50mM glycine-hydrochloric acid buffer solution, the conditions of pH 6.0, 7.0 and 8.0 are prepared from 10mM PBS buffer solution, and the conditions of pH 9.0 and 10.0 are prepared from 50mM glycine-sodium hydroxide buffer solution.
As can be seen from FIG. 5, bpA-Cu shows a strong catalytic ability in the pH range of 5.0 to 8.0. In addition, laccase does not show catalytic activity at a pH value of 3.0-4.0, which shows that natural enzyme is easy to inactivate at an acidic extreme pH value, and the activity of BpA-Cu can keep the highest activity of 39.46% and 54.37% at the pH value, laccase does not show catalytic activity at a pH value of 9.0-10.0, and BpA-Cu still has certain activity at the pH value, so that natural laccase is easy to inactivate at the extreme pH value, and the nano enzyme BpA-Cu still has good catalytic capability at a harsh pH value, so that the nano enzyme BpA-Cu can be used for removing phenolic organic pollutants in water bodies in the fields of printing and dyeing and the like.
Example 11 recycling of laccase-like activity of nanoenzymes.
Weighing 1mg of the BpA-Cu of the invention, placing the BpA-Cu into a 1.5mL centrifuge tube, adding 800 muL of 10mM PB (pH 7.0) buffer solution, 100 muL of 10mM2,4-DP and 100 muL of 10mM 4-AP, uniformly mixing, reacting at room temperature for 1h, centrifuging at 10000rpm for 5min at 4 ℃ after the reaction is finished, taking supernate, measuring the light absorption value at 510nm, washing the precipitate with ultrapure water for 3 times, adding the reactant, repeating the cycle for 10 times, and calculating the relative activity of the BpA-Cu.
Natural laccases are limited in their industrial application due to the fact that proteins are very soluble in water and therefore cannot be recycled after a single use. The BpA-Cu can be recovered and reused by centrifugation, and the activity of the BpA-Cu is still 83.96 percent after 10 cycles as can be seen from figure 6. This indicates that BpA-Cu has good recyclability. The activity loss is caused by the mass loss of the catalyst inevitably caused by the cleaning process, so that the activity is reduced, and the recycling advantage of the BpA-Cu can be utilized to treat the environmental sewage.
Example 12 cytotoxicity of laccase-like activity nanoenzymes.
Uses thiazole blue colorimetric method to treat artificial enzymes BpA-Cu, bpA and Cu 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. By sterilisation5.5mg/mL MTT solution was prepared in PBS solution (containing 10mM PB and 10mM NaCl, pH 7.0). 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. 7, it can be seen that the MTT method was used to evaluate the cytotoxicity of the artificial enzyme BpA-Cu in vitro, and the research found that various catalytic materials showed increased cytotoxicity with increased material concentration after 24h of culture with BV-2 cells. From the study, the toxicity of the BpA-Cu to the cells is gradually enhanced along with the increase of the concentration, but when the concentration in the mixed solution reaches 800 mu g/mL, the cell activity can be maintained at 83.84%. After BpA is added, the toxicity is gradually increased along with the increase of the concentration, and when the concentration reaches 100 mu g/mL, the cell activity is 58.58 percent of that of the control group, and the toxicity of the cytotoxic BpA is stronger compared with that of BpA-Cu. While when Cu of 12.5. Mu.g/mL was added 2+ The post-cell activity decreased to 39.28% of the original value, indicating that Cu was present 2+ Has strong toxicity to cells. Therefore, we will have a slightly toxic BpA and a more toxic Cu 2+ The toxic phenolic pollutants in the environmental sewage are oxidized into non-toxic oxides through coordination interaction, and the oxides are harmless to the environment and have oxidation effect.
Example 13 laccase-like Activity the catalytic ability of nanoenzymes on various phenolic pollutants.
In order to study the catalytic ability of BpA-Cu and natural laccase on various phenolic substrates, the content of generated product free radicals is calculated by utilizing the beer-Lambert law and the molar extinction coefficient so as to obtain the catalytic ability. Ultrasonically dispersing the prepared solid powder BpA-Cu in 1In 0mM phosphate buffer (pH 7.0), a suspension of BpA-Cu was obtained at a concentration of 1 mg/mL. mu.L of the BpA-Cu suspension (1 mg/mL) was added to 700. Mu.L of a phosphate buffer (10 mM, pH 7.0), and 100. Mu.L of an ethanol solution (10 mM) of hydroquinone, 2-aminophenol, 2,4, 6-trichlorophenol, catechol, phenol, 2-naphthol, 2-nitrophenol and 2, 4-dichlorophenol and 100. Mu.L of an aqueous 4-AP solution (10 mM) were further added and mixed so that the total reaction volume became 1mL. Reacting for 1h at 25 ℃, then centrifuging for 5min at 12000rpm, separating BpA-Cu, collecting supernatant after reaction, and measuring the absorbance of the solution at 510nm after reaction by using a microplate reader. The molar extinction coefficient is 13.6mM -1 ·cm -1 (2, 4-dichlorophenol), 9.2mM -1 ·cm -1 (Hydroquinone) 7.8mM -1 ·cm -1 (phenol). The other relative catalytic capacities were calculated by taking the catalytic capacity of the mimic enzyme BpA-Cu for catalyzing 2, 4-dichlorophenol as 100%. Catalytic activities of BpA-Cu and laccase on various substrates were normalized with the activity of BpA-Cu on 2,4-DP as 100%.
As can be seen from FIG. 8, the relative catalytic ability of the mimic enzyme BpA-Cu on 7 phenolic pollutants as substrates is higher than that of the natural laccase except for 2,4-DP. Especially, the oxidizing capacities of the pyrocatechol, the phenol and the 2-nitrophenol can respectively reach 97.1 percent, 70.1 percent and 81.2 percent of the catalytic capacity of taking the 2, 4-dichlorophenol as a substrate. However, the catalytic capacity of natural laccases for these substrates is only 23.3%, 0.1% and 1.7%. Although the relative catalytic abilities of the mimic enzyme and the laccase are lower when hydroquinone and 2,4, 6-trichlorophenol are used as substrates, the activity of the mimic enzyme is still higher than that of the laccase. Therefore, the mimic enzyme BpA-Cu has the oxidizing capability on various environmental phenolic pollutants, which indicates that the mimic enzyme BpA-Cu has substrate universality. The laccase analogue enzyme BpA-Cu provides effective catalytic sites for various substrate molecules due to the simple supramolecular structure, so that the laccase analogue has universality of substrates.
While the methods and techniques of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and/or modifications of the methods and techniques described herein may be made 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 laccase-like activity; the method is characterized in that amino and carboxyl of benzophenone alanine and chloride salt composed of transition metal are combined through coordination to form a supermolecular material with laccase-like activity, and the supermolecular material is marked as BpA-M, wherein BpA is benzophenone alanine, and M represents transition metal copper and zinc.
2. The process for preparing the artificial enzyme having laccase-like activity according to claim 1, which comprises the steps of:
1) Adding a chloride aqueous solution of transition metal copper or zinc into an alkaline aqueous solution of benzophenone alanine, stirring to uniformly mix the two solutions to complete coordination chelation reaction, and standing in a water bath kettle at the temperature of 30-80 ℃ for 0-60h to complete self-assembly;
2) Centrifuging at 8000-10000rpm for 10-20min, discarding supernatant, collecting precipitate, cleaning precipitate with ultrapure water, and vacuum drying the precipitate to obtain BpA-Cu and BpA-Zn solid.
3. The method as set forth in claim 2, wherein the alkaline solution of benzophenone alanine of step 1) is prepared by dissolving benzophenone alanine in 5-50mM sodium hydroxide 10 mM; preparing NaOH and transition metal salt solution with water specification at least being ultrapure water; the concentration of the transition metal chloride solution is 10-20mM.
4. The method as set forth in claim 2, wherein the alkaline aqueous benzophenone alanine solution and the transition metal chloride solution of step 1) are incubated in a water bath at 40-80 ℃ for 30-60min before mixing; the process of adding the transition metal chloride solution into the benzophenone alanine alkaline water solution is carried out under the condition of water bath at 40-80 ℃.
5. The process as claimed in claim 2, wherein the molar ratio of transition metal chloride solution to benzophenone alanine in step 1) is from 1 to 1.
6. The method as set forth in claim 2, wherein the temperature of the vacuum drying oven in the step 2) is set to 40 to 80 ℃ and the drying time is 24 to 72 hours until the precipitate containing a small amount of moisture is dried to powders of BpA-Cu and BpA-Zn.
7. The use of the artificial enzyme with laccase-like activity of claim 1 in the field of laccase activity simulation and phenolic pollutant resolution.
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