CN111185229B - Preparation method and application of dipeptide-based bionic laccase - Google Patents
Preparation method and application of dipeptide-based bionic laccase Download PDFInfo
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Abstract
The invention relates to a preparation method and application of a bionic laccase based on dipeptide. The bionic laccase based on the dipeptide takes metal ions as catalytic active centers and takes the dipeptide as an organic framework, and has laccase-like activity; the bionic laccase based on the dipeptide is a dipeptide-cross linker-metal compound. The prepared bionic laccase has the following advantages: the preparation process is simple and the preparation conditions are mild; the prepared bionic laccase has the catalytic activity of simulating natural laccase; the catalytic efficiency is far higher than that of natural laccase; high stability and good biocompatibility. The preparation of the series of biomimetic laccase based on the dipeptide has potential application in the aspects of developing industrial application such as wastewater treatment, textile bleaching, food safety and the like.
Description
Technical Field
The invention belongs to the field of materials, and particularly relates to a preparation method and application of a bionic laccase based on dipeptide.
Background
The bionic enzyme is defined as an artificially constructed material with an enzyme function, and the construction of the bionic enzyme by taking peptide molecules as primitives draws wide attention in basic research and practical application. Compared with the natural enzyme, the biomimetic enzyme has the advantages of higher catalytic activity, higher stability and the like, and compared with the biomimetic enzyme constructed by metal oxide or carbon-based materials, the peptidyl biomimetic enzyme has the advantages of good biocompatibility, mild preparation conditions and the like, and has wide application in the aspects of biosensing, immunoassay, cancer diagnosis, treatment and the like.
On the basis of realizing the function of simulating the catalysis of natural enzymes, the peptidyl covalent assembly nano enzyme realizes high affinity and strong stability to substrates, and at present, the structures are relatively reported to be used in the construction of biomimetic enzymes (Wei H and Wang E K, chem.Soc.Rev.,2013,42, 6060). The Youli research group takes cysteine-histidine dipeptide as a ligand, and forms a metal organic nano-composite with copper ions by a hydrothermal method to construct the nano-enzyme with the activity of natural laccase. Compared with natural laccase, the nano-enzyme has wide substrate applicability and certain catalytic activity. In addition, the nanoenzymes retain stability under both high temperature and long term storage conditions (Wang J H, Huang R L, Qi W, Su R X, Bernard P, He Z M, appl.Catal.B-Environ,2019,254,452); similarly, the Allen research group introduced a preparation and characterization of redoxSimple method for preparing original biomaterial electrocatalyst by using poly-L-histidine as matrix and Cu2+Performing coordination to simulate active sites of laccase, and preparing a biomaterial electrocatalyst, compared with a bare electrode, Cu2+Polymeric histidine complex modified Glassy Carbon (GC) electrodes reduce redox overpotentials. Cu of different proportion composition2+Polymeric histidine complex array spots were characterized by deposition on GC electrodes and catalytic activity of mimic enzymes by scanning electrochemical microscopy (Weng Y C, Fan F R, Allen J B, j.am.chem.soc.,2005,127, 50).
Although a certain progress is made, the current preparation method based on the short peptide molecular bionic laccase still has the limitation of a hydrothermal method, and compared with natural laccase, the method has low catalytic efficiency and poor stability.
Disclosure of Invention
Aiming at the problems of high preparation difficulty, low catalytic efficiency and poor stability of the existing bionic enzyme (bionic laccase), the invention provides a bionic laccase based on dipeptide and a preparation method and application thereof.
In a first aspect, the invention provides a dipeptide based biomimetic laccase,
the bionic laccase based on the dipeptide takes metal ions as catalytic active centers and takes the dipeptide as an organic framework, and has laccase-like activity;
the bionic laccase based on the dipeptide is a dipeptide-cross linker-metal compound;
the dipeptide is obtained by condensing two amino acid molecules;
wherein the two amino acid molecules are the same or different amino acid molecules;
such amino acid molecules include, but are not limited to: phenylalanine molecule, histidine molecule, tyrosine molecule, glutamic acid molecule, lysine molecule, cysteine molecule, tryptophan molecule, leucine molecule, aspartic acid molecule, glutamine molecule, etc. with or without protecting group modification;
the protecting group may be: any one or mixture of at least two of acetyl (Ac-), phenyl, N-fluorenylmethyloxycarbonyl and ferrocenyl carbonyl;
in particular, the dipeptide may be: one of phenylalanine-phenylalanine with or without a protecting group modification, histidine-tyrosine with or without a protecting group modification, phenylalanine-tyrosine with or without a protecting group modification, and the like;
the dipeptide may specifically be a cationic phenylalanine dipeptide (CDP);
the crosslinking agent may be: one or a combination of at least two of glutaraldehyde, acetic anhydride and genipin, and the glutaraldehyde can be used specifically;
the metal may be Fe3+、Fe2+、Co2+、Ni2+、Cu2+、Zn2+、Mn2+、Pt2+、Ag+Any one or a combination of at least two of; specifically Cu2+。
In the dipeptide-crosslinking agent-metal complex, the molar ratio of the dipeptide to the crosslinking agent to the metal ions can be as follows in sequence: 1:5:10-1:5:100.
In a second aspect, the invention provides a method for preparing the dipeptide based biomimetic laccase.
The method for preparing the dipeptide-based bionic laccase provided by the invention comprises the following steps: 1) assembling the dipeptide, the cross-linking agent and the metal ions together to obtain the dipeptide-based bionic laccase; or
1') forming a dipeptide-crosslinking agent assembly by covalent interaction of the dipeptide and a crosslinking agent, and then coordinating the dipeptide-crosslinking agent assembly with metal ions to obtain the dipeptide-based bionic laccase.
Specifically, the operation of 1) is:
1.1) dissolving dipeptide in a strong polar solvent to obtain a dipeptide solution;
1.2) preparing a cross-linking agent aqueous solution;
1.3) dissolving metal salt in water to obtain a metal salt water solution;
1.4) mixing the dipeptide solution, the cross-linking agent aqueous solution and the metal salt aqueous solution, and reacting under stirring;
1.5) centrifugally separating the reaction system to obtain a precipitate to obtain a dipeptide-crosslinking agent-metal compound, namely the bionic laccase;
specifically, 1') is operated as:
1' 1) dissolving dipeptide in a strong polar solvent to obtain a dipeptide solution;
1' 2) preparing a cross-linking agent aqueous solution;
1 ' 3) mixing the solution prepared in the step 1 ' 1) and the solution prepared in the step 1 ' 2), standing in a dark place, incubating and curing to obtain a dipeptide-crosslinking agent suspension;
1' 4) centrifugally separating the obtained dipeptide-crosslinking agent suspension to obtain a precipitate, washing the precipitate, and adding water to disperse to obtain a dipeptide-crosslinking agent assembly dispersion liquid;
1' 5) adding a metal salt solution into the dipeptide-crosslinking agent assembly dispersion liquid, uniformly mixing, and standing in a dark place;
1' 6) centrifugally separating the system to obtain a precipitate to obtain a dipeptide-crosslinking agent-metal compound, namely the bionic laccase;
in the above method 1.1), 1' 1), the concentration of the dipeptide in the dipeptide solution may be 0.1 to 100mM, specifically 1 to 20 mM;
in the above method 1.2), 1' 2), the concentration of the crosslinking agent in the aqueous solution of the crosslinking agent may be 0.5 to 50mM, specifically 1 to 50 mM;
in the above method 1.3), 1' 5), the concentration of the metal salt in the aqueous solution of the metal salt may be 1 to 100mM, specifically 10 to 80 mM;
in the above method 1.4), the volume ratios of the dipeptide solution, the cross-linking agent aqueous solution and the metal salt aqueous solution may be: 1:5:10-1:20: 50;
the molar ratio of the dipeptide in the dipeptide solution, the cross-linking agent in the cross-linking agent aqueous solution and the metal in the metal salt aqueous solution can be 1:5:10-1:5: 100;
in the method 1.4), the reaction temperature may be 20-60 ℃, specifically 30-60 ℃, and the reaction time may be 10h-14h, specifically 12 h;
in the method 1.5), the centrifugal separation speed is 6000-;
the diameter of the dipeptide-crosslinking agent-metal complex can be 1-10 μm, specifically 3-6 μm;
in the above method 1' 3), the volume ratio of the dipeptide solution to the cross-linking agent aqueous solution may be 1:1 to 1: 20;
the temperature for the light-proof standing incubation curing can be 30-60 ℃, particularly 40-50 ℃, and the time can be 10-30 h;
in the method 1' 4), the rotation speed of the centrifugal separation can be 8000-10000rpm, and the washing can be performed for multiple times, specifically 3-6 times;
in the above method 1' 5), the concentration of the dipeptide-crosslinking agent assembly in the dipeptide-crosslinking agent assembly dispersion liquid may be 1 to 50 mM;
the volume ratio of the dipeptide-crosslinker assembly dispersion to the aqueous solution of the metal salt may be: 1:1-1: 40;
the molar ratio of the dipeptide, the cross-linking agent and the metal salt of the metal salt aqueous solution in the dipeptide-cross-linking agent assembly can be as follows in sequence: 1:5:10-1:5: 100;
the temperature for keeping standing away from light can be room temperature, and the time can be 1-18h, specifically 6-15 h;
in the above method 1' 6), the rotation speed of the centrifugal separation may be 5000-;
the diameter of the resulting dipeptide-crosslinker-metal complex may be 1-10 μm; specifically, it may be 3 to 6 μm.
The application of the dipeptide-based bionic laccase as an oxidation reaction catalyst also belongs to the protection scope of the invention.
The oxidation reaction may specifically be a reaction of oxidizing phenols to quinones.
The bionic laccase based on the dipeptide can be used as an oxidation reaction catalyst in the fields of phenol removal, wastewater treatment, textile bleaching, food safety and the like in industry.
Compared with the prior art, the invention has the following advantages:
(1) the dipeptide is used as a minimum recognition sequence for forming a protein polypeptide chain, has the advantages of good biocompatibility, no toxicity, definite metabolic mechanism and the like, and has no report of constructing the bionic laccase by using the dipeptide at present;
(2) compared with natural enzymes, the dipeptide molecular assembly bionic enzyme has the advantages of high catalytic efficiency, good stability and the like;
(3) the bionic laccase can catalyze and oxidize phenols, so that the bionic laccase has certain application prospects in the aspects of phenol removal, wastewater treatment, textile bleaching, food safety detection and the like in industry.
The bionic laccase prepared by the invention has the following advantages: 1) the preparation method is simple, and the preparation conditions are mild; 2) the biological molecules are used as construction elements, so that the biocompatibility is good; 3) the catalytic oxidation reaction efficiency is high; 4) the bionic enzyme has good stability. The preparation of the series of biomimetic laccase based on the dipeptide has potential application in the aspects of developing industrial application such as wastewater treatment, textile bleaching, food safety and the like.
The metal ions are used as the active center for transmitting electrons, dipeptide molecules are used as the building elements, and the dipeptide-cross-linking agent-metal ion composite assembly is prepared by a one-pot method and used as the bionic laccase, so that the bionic laccase has high catalytic efficiency and high stability, and has application potential.
Drawings
FIG. 1 is a transmission electron microscope image of a dipeptide-crosslinker-metal co-assembly structure.
FIG. 2 is a graph of the UV absorbance at 412nm of the reaction system as a function of time of adding the biomimetic laccase.
FIG. 3 is a comparison graph of catalytic activity of biomimetic laccase and natural laccase in the same reaction time.
FIG. 4 is a graph comparing the stability of biomimetic laccases and natural enzymes.
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
The method for testing the activity of the bionic laccase adopted in the following examples is as follows:
(1) weighing 1-20mg of 5, 5' -dithiobis (2-nitrobenzoic acid) powder, dissolving the powder in deionized water, adding equivalent sodium borohydride powder into the deionized water, and standing the mixture for 1 hour in a dark place to obtain 5-nitro-2-mercaptobenzoic acid (TNB for short) aqueous solution with the concentration of 1-50 mM;
(2) the 5-nitro-2-mercaptobenzoic acid solution obtained in the step (1) is used as an indicator, the indicator has strong ultraviolet absorption at a specific wavelength of 412nm, and the concentration of the indicator solution is 1-20 mM;
(3) using hydroquinone as a substrate of an oxidation reaction, weighing a certain amount of hydroquinone powder, and directly adding the hydroquinone powder into water for dissolving to obtain a hydroquinone solution, wherein the concentration of the hydroquinone solution is 1-20 mM;
(4) mixing the solution obtained in the step (1) and the solution obtained in the step (3) in a volume ratio of 1:1, adding the mixture into a certain amount of phosphoric acid buffer solution to ensure that the concentration of TNB and hydroquinone is 0-10mM, and adjusting the pH value of the solution to be 6.5-8.0;
(5) sucking 1-100 mul of the prepared bionic laccase assembly by using a pipette gun, and adding the bionic laccase assembly into the phosphoric acid buffer solution prepared in the step (4) to enable the concentration of the bionic laccase to be 0-50 mM;
(6) and (4) carrying out ultraviolet spectrum test on the mixed solution obtained in the step (5), recording the absorbance of the mixed solution after the bionic laccase is added for different time, and drawing a curve to obtain the catalytic activity of the bionic enzyme.
Example 1 preparation of cationic dipeptide-glutaraldehyde-copper Assembly biomimetic laccase
(1) Weighing 10mg of freeze-dried powder of cation phenylalanine dipeptide (CDP), dissolving CDP by using 1,1,1,3,3, 3-hexafluoroisopropanol (HFIP for short) as a solvent, and ultrasonically oscillating for 5min to completely dissolve the powder, wherein the concentration of CDP is 10 mM;
(2) sucking a certain amount of CDP solution by using a pipette gun, adding the CDP solution into a glutaraldehyde (GA for short) aqueous solution with the concentration of 5mM, wherein the volume ratio of the CDP solution to the GA solution is 1:10, shaking, incubating for 20h in a dark place to obtain a light yellow suspension, namely a CDP-GA nano suspension, centrifuging and washing for 3 times, and adding water for dispersion to obtain a water dispersion of nanoparticles (the concentration of a dipeptide-cross-linking agent assembly is 2 mM);
(3) and (3) adding a copper sulfate aqueous solution with the concentration of 20mM into the CDP-GA nanoparticle dispersion liquid in the step (2), so that the molar ratio of CDP to GA and Cu in the obtained mixed liquid is 1:5:10, stirring the mixed liquid at a dark state at room temperature to obtain the cation phenylalanine dipeptide-glutaraldehyde-copper ion co-assembled bionic laccase, wherein the molar ratio of CDP to GA and Cu in the assembly is 1:5: 10.
Testing the activity of the bionic laccase:
(4) 6mg of 5, 5' -dithiobis (2-nitrobenzoic acid) powder was weighed and dissolved in deionized water, and then 6mg of sodium borohydride powder was added thereto, and the mixture was left to stand in the dark for 1 hour to obtain an aqueous solution of 5-nitro-2-mercaptobenzoic acid (TNB for short) at a concentration of 10 mM.
(5) Weighing 10mg of hydroquinone powder, and directly adding the hydroquinone powder into water for dissolving to obtain a hydroquinone solution, wherein the concentration of the hydroquinone solution is 10 mM;
(6) mixing the solution obtained in the step (4) and the solution obtained in the step (5) in a volume ratio of 1:1, adding the mixture into a certain amount of phosphoric acid buffer solution to ensure that the concentration of TNB and hydroquinone is 1mM, and adjusting the pH value of the solution to be 6.5;
(7) adding 20 mu l of the bionic laccase assembly prepared in the step (3) into the mixed solution prepared in the step (6) by sucking with a pipette to enable the concentration of the bionic laccase to be 10 mM;
(8) and (4) testing the mixed solution obtained in the step (7) in an ultraviolet spectrometer, recording the absorbance of the mixed solution at 412nm after the bionic laccase is added for different time, and drawing a curve to obtain the catalytic activity of the bionic laccase.
FIG. 1 is a transmission electron microscope image of the bionic laccase prepared, and the diameter of the assembly is 3-5 μm.
FIG. 2 is a graph showing the change of the UV absorption value of the reaction system at 412nm with the time of adding the bionic laccase.
As can be seen from the figure, the ultraviolet absorption value of the substrate 5-nitro-2-mercaptobenzoic acid TNB at 412nm is reduced along with the time after the bionic laccase is added, the absorption value is reduced very rapidly in the initial stage, and the result proves that the bionic laccase rapidly catalyzes and oxidizes phenol into benzoquinone, and the product is combined with the indicator to cause the reduction of the absorption value of the indicator.
FIG. 3 is a comparison graph of catalytic activities of the bionic laccase and the natural laccase in the same reaction time, and it can be known from FIG. 3 that the apparent catalytic activities of the bionic laccase are all higher than those of the natural laccase in the same reaction time, and the catalytic efficiency of the prepared bionic laccase is more than twice that of the natural laccase.
FIG. 4 is a graph comparing the stability of the biomimetic laccase and the natural laccase, and it can be known from FIG. 4 that: after natural placement for a week, the bionic laccase still keeps good catalytic activity, but the catalytic action of the natural laccase almost completely disappears.
Example 2 preparation of cationic dipeptide-glutaraldehyde-copper Assembly biomimetic laccase
(1) Weighing 10mg of freeze-dried powder of cation phenylalanine dipeptide (CDP), dissolving CDP by using 1,1,1,3,3, 3-hexafluoroisopropanol (HFIP for short) as a solvent, and ultrasonically oscillating for 5min to completely dissolve the powder, wherein the concentration of CDP is 20 mM;
(2) sucking a certain amount of CDP solvent by using a liquid transfer gun, adding the CDP solvent into 5mM glutaraldehyde (GA for short) aqueous solution, wherein the volume ratio of the CDP solution to the GA solution is 1:20, oscillating, incubating for 30h in a dark place to obtain a light yellow suspension, namely CDP-GA nano suspension, centrifuging and washing for 3 times to obtain nano-particle aqueous dispersion, wherein the concentration of the dispersion is 4 mM;
(3) and (3) adding a copper sulfate aqueous solution with the concentration of 50mM into the CDP-GA nanoparticle dispersion liquid obtained in the step (2) to enable the molar ratio of CDP to GA and Cu in the obtained mixed liquid to be 1:10:25, stirring the mixed liquid at room temperature in a dark state to obtain the cationic phenylalanine dipeptide-glutaraldehyde-copper ion co-assembled bionic laccase, wherein the molar ratio of CDP to GA and Cu in the assembly is 1:10: 25.
(4) 6mg of 5, 5' -dithiobis (2-nitrobenzoic acid) powder was weighed and dissolved in deionized water, and then 6mg of sodium borohydride powder was added thereto, and the mixture was left to stand in the dark for 1 hour to obtain an aqueous solution of 5-nitro-2-mercaptobenzoic acid (TNB for short) at a concentration of 10 mM.
(5) Weighing 10mg of hydroquinone powder, and directly adding the hydroquinone powder into water for dissolving to obtain a hydroquinone solution, wherein the concentration of the hydroquinone solution is 10 mM;
(6) mixing the solution obtained in the step (4) and the solution obtained in the step (5) in a volume ratio of 1:1, adding the mixture into a certain amount of phosphoric acid buffer solution to ensure that the concentration of TNB and hydroquinone is 1mM, and adjusting the pH value of the solution to be 6.5;
(7) absorbing 40 mu l of the bionic laccase assembly prepared in the step (3) by using a pipette and adding the bionic laccase assembly into the mixed solution prepared in the step (6) to enable the concentration of the bionic laccase to be 20 mM;
(8) and (4) testing the mixed solution obtained in the step (7) in an ultraviolet spectrometer, recording the absorbance of the mixed solution at 412nm after the bionic laccase is added for different time, and drawing a curve to obtain the catalytic activity of the bionic laccase.
Example 3 preparation of cationic dipeptide-glutaraldehyde-copper Assembly biomimetic laccase (preparation of biomimetic laccase by directly mixing three components)
(1) Weighing 10mg of freeze-dried powder of cation phenylalanine dipeptide (CDP), dissolving CDP by using 1,1,1,3,3, 3-hexafluoroisopropanol (HFIP for short) as a solvent, and ultrasonically oscillating for 5min to completely dissolve the powder, wherein the concentration of CDP is 10 mM;
(2) diluting Glutaraldehyde (GA) water solution with initial concentration of 0.1M by a dilution factor of 20 times to obtain GA water solution with concentration of 5 mM;
(3) weighing 0.2g of copper sulfate pentahydrate powder, dissolving in deionized water, and preparing into 40mM copper sulfate aqueous solution;
(4) sucking the prepared solutions in the steps (1), (2) and (3) by using a liquid-transfering gun, and mixing, wherein the volume ratio of the taken CDP solution, GA solution and copper sulfate solution is 1:10: 20;
(5) incubating the mixed solution in the step (4) in a water bath at 37 ℃, keeping out of the sun, and reacting for 12 hours under stirring to obtain a light yellow suspension;
(6) and (4) carrying out centrifugal separation on the suspension obtained in the step (5), wherein the rotating speed is 7500rpm, washing the precipitate with deionized water after separating the precipitate, and washing for 4 times in total to obtain the cationic phenylalanine dipeptide-glutaraldehyde-copper co-assembled bionic laccase.
Testing the activity of the bionic laccase:
(7) 6mg of 5, 5' -dithiobis (2-nitrobenzoic acid) powder was weighed and dissolved in deionized water, and then 6mg of sodium borohydride powder was added thereto, and the mixture was left to stand in the dark for 1 hour to obtain an aqueous solution of 5-nitro-2-mercaptobenzoic acid (TNB for short) at a concentration of 10 mM.
(8) Weighing 10mg of hydroquinone powder, and directly adding the hydroquinone powder into water for dissolving to obtain a hydroquinone solution, wherein the concentration of the hydroquinone solution is 10 mM;
(9) mixing the solution obtained in the step (7) and the solution obtained in the step (8) in a volume ratio of 1:1, adding the mixture into a certain amount of phosphoric acid buffer solution to ensure that the concentration of TNB and hydroquinone is 1mM, and adjusting the pH value of the solution to be 7.0;
(10) absorbing 60 mu l of the bionic laccase assembly prepared in the step (6) by using a pipette and adding the bionic laccase assembly into the mixed solution prepared in the step (6) to enable the concentration of the bionic laccase to be 30 mM;
(11) and (3) testing the mixed solution obtained in the step (10) in an ultraviolet spectrometer, recording the absorbance of the mixed solution at 412nm after the bionic laccase is added for different time, and drawing a curve to obtain the catalytic activity of the bionic laccase.
The catalyst activity and stability of the bionic laccase prepared in the examples 2 and 3 are similar to those of the bionic laccase prepared in the example 1.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (9)
1. A bionic laccase based on dipeptide takes metal ions as catalytic active centers and dipeptide as an organic framework, and has laccase-like activity; the dipeptide-based biomimetic laccase is a dipeptide-crosslinker-metal complex.
2. The dipeptide-based biomimetic laccase according to claim 1, wherein: the dipeptide is composed of two amino acid molecules selected from the following classes: phenylalanine molecule, histidine molecule, tyrosine molecule, glutamic acid molecule, lysine molecule, cysteine molecule, tryptophan molecule, leucine molecule, aspartic acid molecule, and glutamine molecule with or without protecting group modification;
wherein the protecting group is: any one or mixture of at least two of acetyl, phenyl, N-fluorenylmethyloxycarbonyl and ferrocenyl carbonyl;
the cross-linking agent is: any one or combination of at least two of glutaraldehyde, acetic anhydride and genipin;
the metal is Fe3+、Fe2+、Co2+、Ni2+、Cu2+、Zn2+、Mn2+、Pt2+、Ag+Any one or a combination of at least two of them.
3. A process for preparing the dipeptide based biomimetic laccase described in claim 1 or 2, comprising:
1) assembling dipeptide, cross-linking agent and metal ions together to obtain the dipeptide-based bionic laccase; or
1') forming a dipeptide-crosslinking agent assembly by covalent interaction of the dipeptide and a crosslinking agent, and then coordinating the dipeptide-crosslinking agent assembly with metal ions to obtain the dipeptide-based bionic laccase.
4. The method of claim 3, wherein: 1) the operation of (1) is as follows:
1.1) dissolving dipeptide in a strong polar solvent to obtain a dipeptide solution;
1.2) preparing a cross-linking agent aqueous solution;
1.3) dissolving metal salt in water to obtain a metal salt water solution;
1.4) mixing the dipeptide solution, the cross-linking agent aqueous solution and the metal salt aqueous solution, and reacting under stirring;
1.5) centrifugally separating the reaction system to obtain precipitate, thus obtaining the dipeptide-cross-linking agent-metal compound, namely the bionic laccase.
5. The method of claim 3, wherein: 1') is operated as follows:
1' 1) dissolving dipeptide in a strong polar solvent to obtain a dipeptide solution;
1' 2) preparing a cross-linking agent aqueous solution;
1 ' 3) mixing the solution prepared in the step 1 ' 1) and the solution prepared in the step 1 ' 2), standing in a dark place, incubating and curing to obtain a dipeptide-crosslinking agent suspension;
1' 4) centrifugally separating the obtained dipeptide-crosslinking agent suspension to obtain a precipitate, washing the precipitate, and adding water to disperse to obtain a dipeptide-crosslinking agent assembly dispersion liquid;
1' 5) adding a metal salt solution into the dipeptide-crosslinking agent assembly dispersion liquid, uniformly mixing, and standing in a dark place;
1' 6) centrifugally separating the system to obtain a precipitate to obtain a dipeptide-crosslinking agent-metal compound, namely the bionic laccase.
6. The method of claim 4, wherein: 1.1), the concentration of dipeptide in the dipeptide solution is 0.1-100 mM;
1.2), the concentration of the cross-linking agent in the cross-linking agent aqueous solution is 0.5-50 mM;
1.3), the concentration of the metal salt in the metal salt aqueous solution is 1-100 mM;
1.4), the volume ratio of the dipeptide solution, the cross-linking agent aqueous solution and the metal salt aqueous solution is as follows in sequence: 1:5:10-1:20: 50;
the molar ratio of the dipeptide in the dipeptide solution, the cross-linking agent in the cross-linking agent aqueous solution and the metal in the metal salt aqueous solution can be 1:5:10-1:5: 100;
1.4), the reaction temperature is 20-60 ℃, and the reaction time is 10-14 h.
7. The method of claim 5, wherein: 1' 1), the concentration of the dipeptide in the dipeptide solution is 0.1-100 mM;
1' 2), the concentration of the cross-linking agent in the cross-linking agent aqueous solution is 0.5-50 mM;
1' 3), the volume ratio of the dipeptide solution to the cross-linking agent aqueous solution can be 1:1-1: 20;
1' 3), keeping away from light, standing, incubating and curing at the temperature of 30-60 ℃ for 10-30 h;
1' 4), the rotation speed of the centrifugal separation is 8000-10000rpm, and the washing can be carried out for a plurality of times, particularly 3-6 times;
1' 5), the concentration of the metal salt in the aqueous metal salt solution is 1 to 100 mM;
the concentration of the dipeptide-crosslinking agent assembly in the dipeptide-crosslinking agent assembly dispersion liquid is 1-50 mM;
the volume ratio of the dipeptide-crosslinking agent assembly dispersion liquid to the metal salt aqueous solution is as follows: 1:1-1: 40;
the molar ratio of the dipeptide to the cross-linking agent to the metal salt of the metal salt aqueous solution in the dipeptide-cross-linking agent assembly is as follows in sequence: 1:5:10-1:5: 100;
the temperature for keeping standing away from light is room temperature, and the time is 1-18 h;
1' 6), the rotation speed of the centrifugal separation is 5000-.
8. Use of the dipeptide based biomimetic laccase of claim 1 or 2 as an oxidation reaction catalyst; the oxidation reaction is a reaction in which phenols are oxidized to quinones.
9. Use according to claim 8, characterized in that: the application is that the bionic laccase based on dipeptide is used as an oxidation reaction catalyst for phenol removal, wastewater treatment, textile bleaching and food safety in industry.
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