CN114054101B - Phenylalanine-copper nano laccase and preparation method and application thereof - Google Patents
Phenylalanine-copper nano laccase and preparation method and application thereof Download PDFInfo
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 66
- VEWATHHQLVAIBB-QRPNPIFTSA-N (2S)-2-amino-3-phenylpropanoic acid copper Chemical compound [Cu].N[C@@H](CC1=CC=CC=C1)C(=O)O VEWATHHQLVAIBB-QRPNPIFTSA-N 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000010949 copper Substances 0.000 claims abstract description 99
- 229910052802 copper Inorganic materials 0.000 claims abstract description 30
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 29
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims abstract description 29
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims description 32
- 238000006243 chemical reaction Methods 0.000 claims description 18
- 238000001338 self-assembly Methods 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 239000000835 fiber Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 9
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 8
- 229910001431 copper ion Inorganic materials 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 6
- 239000003513 alkali Substances 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000004065 wastewater treatment Methods 0.000 claims description 4
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 3
- YXLXNENXOJSQEI-UHFFFAOYSA-L Oxine-copper Chemical compound [Cu+2].C1=CN=C2C([O-])=CC=CC2=C1.C1=CN=C2C([O-])=CC=CC2=C1 YXLXNENXOJSQEI-UHFFFAOYSA-L 0.000 claims description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 3
- 239000012670 alkaline solution Substances 0.000 claims description 3
- 150000001879 copper Chemical class 0.000 claims description 3
- 229910017604 nitric acid Inorganic materials 0.000 claims description 3
- 239000003002 pH adjusting agent Substances 0.000 claims description 2
- 239000003929 acidic solution Substances 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 abstract description 36
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 238000012360 testing method Methods 0.000 description 15
- 238000002835 absorbance Methods 0.000 description 13
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 12
- 239000007864 aqueous solution Substances 0.000 description 11
- 230000000694 effects Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000006185 dispersion Substances 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 description 5
- 238000013112 stability test Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000004061 bleaching Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000002023 wood Substances 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000000024 high-resolution transmission electron micrograph Methods 0.000 description 3
- 230000003278 mimic effect Effects 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 229910000365 copper sulfate Inorganic materials 0.000 description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 238000000879 optical micrograph Methods 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000012265 solid product Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- SHYPCGPEDNUPRA-QRPNPIFTSA-N (2s)-2-amino-3-phenylpropanoic acid;hydrate Chemical compound O.OC(=O)[C@@H](N)CC1=CC=CC=C1 SHYPCGPEDNUPRA-QRPNPIFTSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003575 carbonaceous material Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 150000004696 coordination complex Chemical class 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- XTVVROIMIGLXTD-UHFFFAOYSA-N copper(II) nitrate Chemical compound [Cu+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O XTVVROIMIGLXTD-UHFFFAOYSA-N 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005580 one pot reaction Methods 0.000 description 1
- 125000002524 organometallic group Chemical group 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004076 pulp bleaching Methods 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
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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
- B01J35/00—Catalysts, in general, characterised by their form or physical properties
- B01J35/50—Catalysts, in general, characterised by their form or physical properties characterised by their shape or configuration
- B01J35/58—Fabrics or filaments
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- 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
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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
- B01J31/1691—Coordination polymers, e.g. metal-organic frameworks [MOF]
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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
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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
- B01J35/00—Catalysts, in general, characterised by their form or physical properties
- B01J35/40—Catalysts, in general, characterised by their form or physical properties characterised by dimensions, e.g. grain size
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
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Abstract
The invention provides phenylalanine-copper nano laccase and a preparation method and application thereof, and relates to the technical field of biocatalysis. The phenylalanine-copper nano laccase provided by the invention is obtained by self-assembling phenylalanine and a water-soluble copper source in a water phase with the pH value of 5-10. The phenylalanine and water-soluble copper source self-assemble into a beta-sheet-like secondary structure with stable form in a water phase with the pH value of 5-10, the structure and the active site of the natural laccase are well simulated, and the obtained phenylalanine-copper nano laccase has strong stability, high catalytic activity and high crystallinity, and still maintains the high laccase catalytic activity under the environments of high temperature, acidity, alkalinity, high ionic strength and the like.
Description
Technical Field
The invention relates to the technical field of biocatalysis, in particular to phenylalanine-copper nano laccase and a preparation method and application thereof.
Background
Laccases are a class of polyphenol oxidoreductases comprising trinuclear copper cluster sites, which are widely found in bacterial, fungal and plant species. Laccase can oxidize various aromatic and non-aromatic compounds through a free radical catalytic reaction mechanism in the presence of oxygen, and has wide application in the fields of biosensing, bioremediation, wood delignification, green synthesis, textile dye bleaching, pulp bleaching, wastewater detoxification and the like. However, the existing laccase has the defects of long fermentation period, low yield and poor enzyme stability, and limits the application of the laccase. The laccase-like nano enzyme with simple synthesis, high activity and good stability is an important development direction of artificial simulation laccase.
Currently, laccase-like nanoenzymes mainly include organometallic complexes, colloidal metal particles, polymers, and carbon materials. However, the laccase-like nano-enzyme is usually obtained by covalent coordination construction in an organic solvent system, and the stability of the laccase-like nano-enzyme is poor.
Disclosure of Invention
In view of the above, the invention aims to provide phenylalanine-copper nano laccase, and a preparation method and application thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a phenylalanine-copper nano laccase which is prepared by self-assembling raw materials comprising phenylalanine and a water-soluble copper source in a water phase with a pH value of 5-10.
Preferably, the phenylalanine-copper nano laccase is in a needle-shaped fiber, the length of the needle-shaped fiber is 20-40 mu m, and the diameter of the needle-shaped fiber is 1.5-3.5 mu m;
each structural unit of the phenylalanine-copper nano laccase comprises 4 copper ions and 8 phenylalanine molecules.
The invention provides a preparation method of phenylalanine-copper nanometer laccase, which comprises the following steps:
and mixing phenylalanine, a water-soluble copper source and water, regulating the pH value to 5-10, and performing self-assembly reaction to obtain the phenylalanine-copper nano laccase.
Preferably, the molar ratio of copper to phenylalanine in the water-soluble copper source is 1:9-9:1.
Preferably, the water-soluble copper source is a water-soluble copper salt.
Preferably, the pH adjuster used for adjusting the pH value comprises an alkali solution and/or an acid solution.
Preferably, the alkaline solution comprises sodium hydroxide solution and/or potassium hydroxide solution; the concentration of the alkali solution is 0.8-1.2 mol/L.
Preferably, the acid solution comprises one or more of hydrochloric acid solution, sulfuric acid solution and nitric acid solution; the concentration of the acid solution is 0.8-1.2 mol/L.
Preferably, the temperature of the self-assembly reaction is 20-80 ℃ and the time is 0.5-3 h.
The invention provides application of the phenylalanine-copper nano laccase or the phenylalanine-copper nano laccase obtained by the preparation method in the technical scheme in biosensing, bioremediation, wood delignification, organic synthesis, bleaching or wastewater treatment.
The invention provides phenylalanine-copper nano laccase (F-Cu (II)), which is prepared by self-assembling raw materials comprising phenylalanine and a water-soluble copper source in a water phase with a pH value of 5-10. In the invention, phenylalanine and a water-soluble copper source are self-assembled into a beta-sheet-like secondary structure with stable form in a water phase with the pH value of 5-10, F-Cu (II) is used as a self-assembled amyloid macromolecule to simulate the beta-sheet secondary structure of natural laccase and an active site taking copper ions as an active center well, F-Cu (II) is used as a nano enzyme compared with the natural enzyme, the F-Cu (II) is different from the protein structure of the natural enzyme, F-Cu (II) is used as a metal complex, the environmental tolerance is better, the stability is higher, and the phenylalanine-copper nano laccase provided by the invention has strong stability, high catalytic activity and high crystallinity and still maintains high catalytic activity under the environments of high temperature, acidity, alkalinity, high ionic strength and the like.
The invention provides a preparation method of phenylalanine-copper nanometer laccase, which comprises the following steps: and mixing phenylalanine, a water-soluble copper source and water, regulating the pH value to 5-10, and performing self-assembly reaction to obtain the phenylalanine-copper nano laccase. In the invention, in the self-assembly reaction process, under the condition that the pH value is 5-10, the active center Cu 2+ The beta-sheet secondary structure with stable morphology is self-assembled with phenylalanine, the structure and the active site of the natural laccase are well simulated, and the prepared phenylalanine-copper nano laccase has strong stability, high catalytic activity and high crystallinity, and the phenylalanine-copper nano laccase still maintains high laccase catalytic activity under the environments of high temperature, acidity, alkalinity, high ionic strength and the like. The invention adopts a one-pot method, has simple process and high yield; in addition, the self-assembly is carried out in a water phase system, so that the method is safe and environment-friendly.
Drawings
FIG. 1 is a graph showing the results of crystallinity of F-Cu (II) prepared in examples 1 and 5 to 14;
FIG. 2 is an optical micrograph and an HRTEM image (interpolated diagram) of F-Cu (II) prepared in example 1;
FIG. 3 is an XRD spectrum of F-Cu (II) prepared in example 1;
FIG. 4 is an ultraviolet-visible absorption spectrum of F-Cu (II), copper ion and phenylalanine prepared in example 1;
FIG. 5 is Fourier infrared (FT-IR) of F-Cu (II) prepared in example 1;
FIG. 6 is a graph of the UV absorbance spectrum of F-Cu (II) prepared in example 1 as a laccase mimic for catalyzing 2, 4-DP;
FIG. 7 is a graph showing the results of the catalytic activity test of F-Cu (II) prepared in examples 1 to 2;
FIG. 8 is a graph showing the results of the catalytic activity test of F-Cu (II) prepared in example 1 and examples 3 to 4;
FIG. 9 is a graph showing steady state kinetics measurements of F-Cu (II) prepared in example 1 as laccase mimics;
FIG. 10 is a graph showing the results of the relative catalytic activity test of F-Cu (II) prepared in example 1 at different pH, temperature, ethanol volume fraction and storage days, wherein a is the relative catalytic activity at different pH, b is the relative catalytic activity at different temperature, c is the relative catalytic activity at different storage days, and d is the relative catalytic activity at different ethanol volume fraction.
Detailed Description
The invention provides a phenylalanine-copper nano laccase which is prepared by self-assembling raw materials comprising phenylalanine and a water-soluble copper source in a water phase with a pH value of 5-10. In the present invention, the pH is preferably 6 to 10, more preferably 7 to 10, and even more preferably 8 to 9.
In the present invention, the morphology of the phenylalanine-copper nano laccase is preferably needle-shaped fiber, and the length of the needle-shaped fiber is preferably 20-40 μm, more preferably 25-35 μm; the diameter of the needle-like fibers is preferably 1.5 to 3.5. Mu.m, more preferably 2 to 3. Mu.m. In the present invention, each structural unit of the phenylalanine-copper nano laccase preferably comprises 4 copper ions and 8 phenylalanine molecules.
The invention provides a preparation method of phenylalanine-copper nanometer laccase, which comprises the following steps:
and mixing phenylalanine, a water-soluble copper source and water, regulating the pH value to 5-10, and performing self-assembly reaction to obtain the phenylalanine-copper nano laccase.
In the present invention, all raw material components are commercially available products well known to those skilled in the art unless specified otherwise.
In the present invention, the water-soluble copper source is preferably a water-soluble copper salt, and more preferably includes one or more of copper sulfate, copper chloride, and copper nitrate. In the present invention, the water-soluble copper source is preferably used in the form of an aqueous solution of the water-soluble copper source, and the concentration of the water-soluble copper source is preferably 0.05 to 0.2mmol/L, more preferably 0.1mmol/L. In the present invention, the molar ratio of the water-soluble copper source to phenylalanine is preferably 1:9 to 9:1, more preferably 2:8 to 8:2, and particularly preferably 1:4, 3:7, 2:3, 7:3, 1:1, 3:2, 2:1 or 4:1.
In the embodiment of the invention, the phenylalanine, the water-soluble copper source and the water are mixed preferably by adding the phenylalanine water solution and the water-soluble copper source water solution into the water for mixing to obtain a primary mixture; the concentration of the phenylalanine aqueous solution is preferably 0.05 to 0.2mmol/L, more preferably 0.1mmol/L; the concentration of the aqueous solution of the water-soluble copper source is preferably 0.05 to 0.2mmol/L, more preferably 0.1mmol/L; the concentration of phenylalanine in the primary mixture is preferably 0.05 to 0.2mmol/L, more preferably 0.1mmol/L.
In the present invention, the pH adjustor used for adjusting the pH value preferably includes an alkali solution and/or an acid solution; the alkali solution preferably comprises sodium hydroxide solution and/or potassium hydroxide solution; the concentration of the alkali solution is preferably 0.8 to 1.2mmol/L, more preferably 0.9 to 1.1mmol/L, and still more preferably 1mmol/L; the acid solution preferably comprises one or more of hydrochloric acid solution, sulfuric acid solution and nitric acid solution; the concentration of the acid solution is preferably 0.8 to 1.2mmol/L, more preferably 0.9 to 1.1mmol/L, and even more preferably 1mmol/L. In the present invention, the pH is preferably 6 to 10, more preferably 7 to 10, and even more preferably 8 to 9.
The present invention is not particularly limited to the above-mentioned mixing, and the raw materials may be uniformly mixed, specifically, stirring and mixing.
In the present invention, the temperature of the self-assembly reaction is preferably 20 to 80 ℃, more preferably 30 to 70 ℃, further preferably 40 to 60 ℃, and most preferably 50 ℃; the time of the self-assembly reaction is preferably 0.5 to 3 hours,more preferably 1 to 2.5 hours, still more preferably 1.5 to 2 hours. In the invention, in the self-assembly reaction process, under the condition that the pH value is 5-10, the active center Cu 2+ Self-assembling with phenylalanine to form phenylalanine-copper nanometer laccase with beta-sheet secondary structure and needle-like fiber.
After the self-assembly reaction is completed, the invention preferably further comprises the steps of carrying out solid-liquid separation on a reaction system obtained by the self-assembly reaction, washing the obtained solid product with water, and drying to obtain the alanine-copper nano laccase. In the present invention, the solid-liquid separation method is not particularly limited, and any solid-liquid separation method known to those skilled in the art may be used, and specifically, filtration or centrifugation may be used. In the present invention, the water for washing preferably includes distilled water, and the number of times of washing is preferably 2 to 4, more preferably 3. In the present invention, the drying temperature is preferably 20 to 60 ℃, more preferably 40 ℃; the invention has no special limit to the drying time, and the drying is carried out until the weight is constant; the drying is preferably carried out in an oven.
The invention provides application of the phenylalanine-copper nano laccase or the phenylalanine-copper nano laccase obtained by the preparation method in the technical scheme in biosensing, bioremediation, wood delignification, organic synthesis, bleaching or wastewater treatment. The phenylalanine-copper nano laccase provided by the invention has stable form, high crystallinity and high catalytic activity, still maintains high catalytic activity under the environments of high temperature, extreme pH, high ionic strength and the like, and has good application prospects in biosensing, bioremediation, wood delignification, organic synthesis, bleaching and wastewater treatment.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
2mL of phenylalanine aqueous solution with concentration of 100mmol/L and 1mL of CuSO with concentration of 100mmol/L are mixed 4 Mixing the aqueous solution with 7mL of water, adding 100 mu L of 1mol/L NaOH aqueous solution to adjust the pH to 8, performing self-assembly reaction for 2h at 40 ℃, performing centrifugal separation, washing the obtained solid product with distilled water for 3 times, and drying in a baking oven at 40 ℃ until the weight is constant to obtain phenylalanine-copper nano laccase (blue needle-shaped powder, abbreviated as F-Cu (II)); wherein phenylalanine and CuSO 4 Molar ratio = 2:1.
Examples 2 to 14
Phenylalanine-copper nano laccase was prepared according to the method of example 1, and the preparation conditions are shown in table 1:
TABLE 1 preparation conditions for examples 1 to 14
Comparative example 1
Literature "Guan, m; wang, m.; qi, w; su, r.; he, Z.biomineralization-inspired marker-cystine nanoleaves capable of laccase-like catalysis for the colorimetric detection of epineephrine.front.chem.Sci.Eng.2021, 15,310-318 ".
Comparative example 2
Literature "Sun, h; zhou, y; ren, j.; qu, X.carbon nanozymes: enzymatic properties, catalytic mechanism, and applications. Angew. Chem. Int. Edit.2018,57,9224-9237, "native laccase disclosed herein
Comparative example 3
Literature "Chen, z; wang, z.; ren, j.; qu, X.enzyme mimicry for combating bacteria and biofms.Account chem.Res.2018,51,789-799 et al, discloses a native laccase
Comparative example 4
Literature "Jiang, d.; ni, d.; rosenkrans, z.t.; huang, p.; yan, x; cai, W.nanozyme: new horizons for responsive biomedical applications.chem.Soc.Rev.2019,48,3683-3704, "native laccase as disclosed herein
Test case
(1) Structure of the
FIG. 1 is a graph showing the results of crystallinity of laccase enzymes prepared in examples 1 and 5 to 14, and it is clear from FIG. 1 that the crystallinity of F-Cu (II) enzyme is best when the molar ratio of phenylalanine to copper sulfate is 2:1.
FIG. 2 is an optical micrograph and an HRTEM image (interpolated diagram) of F-Cu (II) prepared in example 1. As can be seen from fig. 2, phenylalanine and Cu (II) ions self-assemble into needle-like fibers several tens of micrometers long. The lattice spacing observed in the HRTEM image was 0.38nm, corresponding to the (080) plane of F-Cu (II).
FIG. 3 shows XRD patterns of F-Cu (II) prepared in example 1. As can be seen from FIG. 3, the F-Cu (II) structure is molecular packing of monoclinic crystal, and is P 21 Space group, each structural unit contains 8 phenylalanine molecules and 4 copper ions.
FIG. 4 is a UV-visible absorption spectrum of F-Cu (II), copper ions and phenylalanine prepared in example 1. As can be seen from FIG. 4, F-Cu (II) has a new absorption band at 614nm and a shoulder band at 300nm, which correspond to the characteristic absorption peaks of T1 copper and T3 copper of laccase, respectively, indicating that F-Cu (II) has an absorption spectrum similar to that of natural laccase.
FIG. 5 shows Fourier infrared (FT-IR) of F-Cu (II) prepared in example 1, and it can be seen from FIG. 5 that the stretching vibration peaks of C=O and N-H bonds of F-Cu (II) are shifted to 1614cm, respectively -1 And 3245cm -1 The method comprises the steps of carrying out a first treatment on the surface of the At 1103cm -1 A typical N-Cu-N bond corresponding stretching vibration peak was also observed, indicating that copper ions are involved in complex formation and that F-Cu (II) is very similar to the beta-sheet secondary structure of the native laccase, which makes F-Cu (II) possible to have laccase-like structure and surface chemistry.
(2) Catalytic Activity
The catalytic activities of F-Cu (II) prepared in examples 1 to 4 were determined by the chromogenic reaction of the phenolic compound 2, 4-dichlorophenol (2, 4-DP) with 4-aminoantipyrine (4-AAP), respectively. The test method is as follows: F-Cu (II) prepared in examples 1 to 4 were mixed with water to prepare aqueous F-Cu (II) dispersion having a concentration of 0.5 mg/mL. 2, 4-dichlorophenol aqueous solution (200. Mu.L, 10 mmol/L), 4-aminoantipyrine (100. Mu.L, 100 mmol/L) and PBS buffer (1500. Mu.L, 10mmol/L, pH=7.4) were mixed, and F-Cu (II) aqueous dispersion (200. Mu.L, 0.5 mg/mL) was added to the resulting mixture, and after incubation at room temperature (25 ℃) for 20 minutes, absorbance at 510nm was measured. The test results are shown in FIGS. 6 to 8.
FIG. 6 is a graph showing the UV total spectrum absorption of F-Cu (II) prepared in example 1 as laccase mimic for catalyzing 2,4-DP, and as can be seen from FIG. 6, the F-Cu (II) prepared in the invention has catalytic activity.
FIG. 7 is a graph showing the results of the catalytic activity test of F-Cu (II) prepared in examples 1 and 2, and it is understood from FIG. 7 that the catalytic activity of F-Cu (II) is not greatly affected by different copper sources.
FIG. 8 is a graph showing the results of the catalytic activity test of F-Cu (II) prepared in example 1 and examples 3 to 4. As can be seen from FIG. 8, the catalytic activity of F-Cu (II) laccase nanoenzyme is best when the self-assembly temperature is 40 ℃.
(3) Steady state kinetic test of catalytic activity
Laccase to be tested: F-Cu (II) prepared in example 1 and the natural laccase disclosed in comparative examples 1 to 4. The laccase to be tested is prepared into laccase water dispersion liquid with the concentration of 0.5 mg/mL.
The initial reaction rates were determined by reacting aqueous solutions of 2, 4-dichlorophenol at various concentrations (0.1, 0.5, 1, 2, 3 and 4 mmol/L), aqueous laccase dispersion to be tested (0.5 mg/mL, 200. Mu.L) and aqueous 4-aminoantipyrine (100 mmol/L, 100. Mu.L). Calculation of kinetic parameters K using Michaelis-Menten equation m And k cat /K m The test results are shown in fig. 9 and table 2.
FIG. 9 shows the steady state kinetics of F-Cu (II) prepared in example 1 as a laccase mimic, as can be seen from FIG. 9, the reaction rate versus 2,4-DP concentration is in accordance with a typical Michaelis-Menten model 1/v 0 ＝K m /v max -1/[S 0 ]+1/v max . Wherein v is 0 、K m 、v max Respectively apparent initial catalytic rate, michaelis constant and mostLarge reaction rate, [ S ] 0 ]Is the substrate concentration.
TABLE 2 results of steady state kinetic testing of the F-Cu (II) prepared in example 1 and the native laccase disclosed in comparative examples 1-4
As can be seen from Table 2, F-Cu (II) has better substrate affinity and higher catalytic efficiency than most of the natural laccase, which indicates that F-Cu (II) prepared by the invention has good catalytic activity.
(4) Stability test
The relative catalytic activity of F-Cu (II) prepared in example 1 was tested at different pH, temperature, ethanol volume fraction and days of storage, and the test results are shown in FIG. 10, wherein a is the relative catalytic activity at different pH, b is the relative catalytic activity at different temperature, c is the relative catalytic activity at different days of storage, d is the relative catalytic activity at different ethanol volume fraction, and the specific test procedure is as follows:
(4.1) the F-Cu (II) prepared in example 1 was mixed with water to prepare an aqueous F-Cu (II) dispersion having a concentration of 0.5 mg/mL. Mixing 2, 4-dichlorophenol aqueous solution (200 mu L,10 mmol/L) and 4-aminoantipyrine (100 mu L,100 mmol/L) aqueous solution, and adding F-Cu (II) aqueous dispersion (200 mu L,0.5 mg/mL) into the obtained mixed solution to obtain a solution to be tested for stability;
(4.1.1) incubating the stable test solution for 2 hours at room temperature and different pH values (4, 5, 6, 7, 8, 9 and 10), and measuring absorbance at 510 nm; relative activity of F-Cu (II) =absorbance at different pH values/absorbance at pH value 7. The test results are shown in table 3 and fig. 10 a.
TABLE 3 stability test results of F-Cu (II) at different pH values
| pH | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| Relative activity/% | 89 | 90.9 | 92.3 | 100 | 105 | 92.1 | 54.5 |
As can be seen from Table 3 and FIG. 10, the F-Cu (II) prepared by the method has high catalytic activity under the condition that the pH value is 4-10, which indicates that the pH value of the F-Cu (II) prepared by the method has wide application range.
(4.1.2) incubating the stable test solution for 2 hours under the conditions of pH=7 and different temperatures (25 ℃, 50 ℃,100 ℃, 150 ℃ and 200 ℃) respectively, and then measuring absorbance at 510 nm; relative activity of F-Cu (II) =absorbance at different temperatures/absorbance at 25 ℃. The test results are shown in fig. 10 b and table 4.
TABLE 4 stability test results of F-Cu (II) at different temperatures
| Temperature/. Degree.C | 25 | 50 | 100 | 150 | 200 |
| Relative activity/% | 100 | 100.5 | 101.6 | 92.4 | 187.4 |
As can be seen from FIG. 10 b and Table 4, the F-Cu (II) prepared by the present invention has high catalytic activity at 25-200 ℃, which indicates that the F-Cu (II) prepared by the present invention has excellent high temperature resistance.
(4.1.3) after incubating the stable test solution for 2 hours at room temperature at ph=7 for different times of storage (0, 1, 3, 5, 7,9, 11, 13 and 15 days), absorbance was measured at 510 nm; relative activity of F-Cu (II) =absorbance at different storage times/absorbance at 0 days of storage. The test results are shown in table 5 and fig. 10 c.
TABLE 5 stability test results of F-Cu (II) at different storage times
| Storage time/day | 0 | 1 | 3 | 5 | 7 | 9 | 11 | 13 | 15 |
| Relative activity/% | 100 | 100 | 95 | 92 | 90 | 93 | 88 | 86 | 86 |
As is clear from Table 5 and FIG. 10 c, the F-Cu (II) prepared in the present invention maintained high catalytic activity even after 15 days of storage, demonstrating that the F-Cu (II) prepared in the present invention is excellent in long-term storage stability.
(4.2) F-Cu (II) prepared in example 1 was placed in a water-ethanol solvent to prepare F-Cu (II) dispersion having a concentration of 0.5mg/mL, and the volume fractions of ethanol in the water-ethanol solvent were 0%, 25%, 50%, 75% and 100%, respectively.
2, 4-dichlorophenol aqueous solution (200. Mu.L, 10 mmol/L), 4-aminoantipyrine (100. Mu.L, 100 mmol/L) and PBS buffer (1500. Mu.L, 10mmol/L, pH=7.4) were mixed, F-Cu (II) dispersion (200. Mu.L, 0.5 mg/mL) was added to the obtained mixture, and the resulting stable solution was incubated at room temperature and pH=7 for 20 minutes, and then absorbance at 510nm was measured; relative activity of F-Cu (II) =absorbance at different ethanol volume concentrations/absorbance at 0% ethanol volume concentration. The test results are shown in table 6 and d in fig. 10.
TABLE 6 stability test results of F-Cu (II) at different ethanol concentrations
| Ethanol volume fraction/% | 0 | 25 | 50 | 75 | 100 |
| Relative activity/% | 100 | 105.3 | 104.3 | 86.1 | 80 |
As can be seen from the graph d in Table 6 and FIG. 10, the F-Cu (II) prepared by the method still has high catalytic activity in the presence of the organic solvent, which indicates that the F-Cu (II) prepared by the method has excellent organic solvent resistance.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (6)
1. Application of phenylalanine-copper nano laccase in wastewater treatment; the phenylalanine-copper nano laccase is obtained by self-assembling raw materials comprising phenylalanine and a water-soluble copper source in a water phase with the pH value of 5-10; the phenylalanine-copper nano laccase is in a needle-shaped fiber, the length of the needle-shaped fiber is 20-40 mu m, and the diameter of the needle-shaped fiber is 1.5-3.5 mu m; each structural unit of the phenylalanine-copper nano laccase comprises 4 copper ions and 8 phenylalanine molecules;
the preparation method of the phenylalanine-copper nano laccase comprises the following steps: mixing phenylalanine, a water-soluble copper source and water, regulating the pH value to 5-10, and performing self-assembly reaction to obtain phenylalanine-copper nano laccase; the temperature of the self-assembly reaction is 20-80 ℃ and the time is 0.5-3 h.
2. The use according to claim 1, wherein the molar ratio of copper to phenylalanine in the water-soluble copper source is 1:9 to 9:1.
3. The use according to claim 1 or 2, characterized in that the water-soluble copper source is a water-soluble copper salt.
4. The use according to claim 1, wherein the pH adjusting agent used for adjusting the pH value comprises an alkaline solution and/or an acidic solution.
5. Use according to claim 4, characterized in that the alkaline solution comprises sodium hydroxide solution and/or potassium hydroxide solution; the concentration of the alkali solution is 0.8-1.2 mol/L.
6. The use according to claim 4, wherein the acid solution comprises one or more of a hydrochloric acid solution, a sulfuric acid solution and a nitric acid solution; the concentration of the acid solution is 0.8-1.2 mol/L.
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| EP3329778A1 (en) * | 2016-11-30 | 2018-06-06 | Univerza v Mariboru Fakulteta za strojnistvo | Process for synthesis of antimicrobial copper nanoparticles |
| CN113000069A (en) * | 2021-02-25 | 2021-06-22 | 广西大学 | Preparation method and application of bionic laccase functionalized imine covalent organic framework nanoenzyme |
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| EP3329778A1 (en) * | 2016-11-30 | 2018-06-06 | Univerza v Mariboru Fakulteta za strojnistvo | Process for synthesis of antimicrobial copper nanoparticles |
| CN113000069A (en) * | 2021-02-25 | 2021-06-22 | 广西大学 | Preparation method and application of bionic laccase functionalized imine covalent organic framework nanoenzyme |
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