CN115283000A - Preparation method of manganese/carbon nano enzyme with high laccase activity - Google Patents
Preparation method of manganese/carbon nano enzyme with high laccase activity Download PDFInfo
- Publication number
- CN115283000A CN115283000A CN202210954140.7A CN202210954140A CN115283000A CN 115283000 A CN115283000 A CN 115283000A CN 202210954140 A CN202210954140 A CN 202210954140A CN 115283000 A CN115283000 A CN 115283000A
- Authority
- CN
- China
- Prior art keywords
- manganese
- carbon
- nanoenzyme
- laccase
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 229910052748 manganese Inorganic materials 0.000 title claims abstract description 83
- 239000011572 manganese Substances 0.000 title claims abstract description 83
- 229910052799 carbon Inorganic materials 0.000 title claims abstract description 77
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 72
- 108010029541 Laccase Proteins 0.000 title claims abstract description 62
- 230000000694 effects Effects 0.000 title claims abstract description 60
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 title abstract description 20
- 102000004190 Enzymes Human genes 0.000 title abstract description 20
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 43
- 229940082328 manganese acetate tetrahydrate Drugs 0.000 claims abstract description 22
- CESXSDZNZGSWSP-UHFFFAOYSA-L manganese(2+);diacetate;tetrahydrate Chemical compound O.O.O.O.[Mn+2].CC([O-])=O.CC([O-])=O CESXSDZNZGSWSP-UHFFFAOYSA-L 0.000 claims abstract description 22
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 8
- 239000008098 formaldehyde solution Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 34
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 25
- 238000010438 heat treatment Methods 0.000 claims description 21
- 239000007864 aqueous solution Substances 0.000 claims description 12
- 238000003756 stirring Methods 0.000 claims description 11
- 238000010000 carbonizing Methods 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 8
- 239000012299 nitrogen atmosphere Substances 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 238000010992 reflux Methods 0.000 claims description 8
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 8
- 239000012498 ultrapure water Substances 0.000 claims description 8
- 239000003344 environmental pollutant Substances 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 231100000719 pollutant Toxicity 0.000 claims description 5
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 claims description 4
- 238000011068 loading method Methods 0.000 claims description 4
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
- 230000003647 oxidation Effects 0.000 claims description 3
- 238000007254 oxidation reaction Methods 0.000 claims description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000000593 degrading effect Effects 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 abstract description 16
- 241000222355 Trametes versicolor Species 0.000 abstract description 5
- 150000002989 phenols Chemical class 0.000 abstract description 3
- 239000002957 persistent organic pollutant Substances 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000003837 high-temperature calcination Methods 0.000 abstract 1
- 238000005580 one pot reaction Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 16
- 230000015556 catabolic process Effects 0.000 description 13
- 238000006731 degradation reaction Methods 0.000 description 13
- 238000006555 catalytic reaction Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000002086 nanomaterial Substances 0.000 description 8
- 239000007853 buffer solution Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000009835 boiling Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- DOTMOQHOJINYBL-UHFFFAOYSA-N molecular nitrogen;molecular oxygen Chemical compound N#N.O=O DOTMOQHOJINYBL-UHFFFAOYSA-N 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 238000009826 distribution Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- AMWRITDGCCNYAT-UHFFFAOYSA-L hydroxy(oxo)manganese;manganese Chemical compound [Mn].O[Mn]=O.O[Mn]=O AMWRITDGCCNYAT-UHFFFAOYSA-L 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 241000252892 Pycnopsyche gentilis Species 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 238000002441 X-ray diffraction Methods 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 239000003575 carbonaceous material Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000013335 mesoporous material Substances 0.000 description 1
- 239000002082 metal nanoparticle Substances 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000013105 nano metal-organic framework Substances 0.000 description 1
- 239000013289 nano-metal-organic framework Substances 0.000 description 1
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Images
Classifications
-
- 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
- B01J27/00—Catalysts comprising the elements or compounds of halogens, sulfur, selenium, tellurium, phosphorus or nitrogen; Catalysts comprising carbon compounds
- B01J27/24—Nitrogen compounds
-
- 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
- B01J35/20—Catalysts, in general, characterised by their form or physical properties characterised by their non-solid state
- B01J35/23—Catalysts, in general, characterised by their form or physical properties characterised by their non-solid state in a colloidal state
-
- 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
- B01J35/60—Catalysts, in general, characterised by their form or physical properties characterised by their surface properties or porosity
- B01J35/61—Surface area
- B01J35/613—10-100 m2/g
-
- 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
- B01J35/60—Catalysts, in general, characterised by their form or physical properties characterised by their surface properties or porosity
- B01J35/64—Pore diameter
- B01J35/647—2-50 nm
-
- 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
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
-
- 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
- C02F2101/36—Organic compounds containing halogen
Landscapes
- 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)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a preparation method of manganese/carbon nano enzyme with high laccase activity. The preparation of the nano enzyme is synthesized by taking manganese acetate tetrahydrate, dicyandiamide and formaldehyde as raw materials through a one-pot high-temperature calcination method. The manganese acetate tetrahydrate comprises 0.5196-2.5980g, the dicyandiamide comprises 5.0000g, and the aqueous formaldehyde solution comprises 4.4-6.6 mL. The manganese/carbon nanoenzyme prepared by the method can catalyze and oxidize phenols and other various refractory organic pollutants, and shows excellent laccase catalytic activity. The specific activity (0.48U/mg) of the nano laccase is close to that of the natural laccase sold in the market (from coriolus versicolor, more than or equal to 0.5U/mg), and is greatly superior to other nano laccases reported at present. The invention has the advantages of high activity, low cost, easy storage and the like.
Description
Technical Field
The invention belongs to the technical field of novel environment-friendly materials, relates to preparation of nano enzyme, and particularly relates to a preparation method of nano enzyme with high laccase activity. The nano-laccase exhibits high laccase activity, has specific activity equivalent to that of a natural laccase (from coriolus versicolor) sold in the market, and is far superior to other nano-laccases reported at present. The nano laccase has high enzymology activity and low cost, so that the nano laccase has great application value in the environmental protection fields of environmental pollutant removal and the like.
Background
Laccase (p-diphenol: dioxygen oxidase, EC 1.10.3.2) is a polyphenol oxidase, which catalyzes the oxidation of organic substrates, and mainly comprises phenolic compounds and polyamines. Natural laccase is considered as an environmentally friendly catalyst due to its excellent ability to degrade organic pollutants, no toxic products generated during the catalytic process and water as the final product. Has wide application value in the aspects of dye decoloration, biological bleaching, paper making, wastewater treatment, food processing, environmental pollutant detoxification, biological detection and the like. However, the practical applications of natural laccases are severely restricted by problems of high cost, difficult preparation, instability, etc. (c. Gali, c. Madzak, r. Vadal a, c. Jolivalt and p. Gentili,ChemBioChem, 2013, 14, 2500-2505.；M. Fernandez-Fernandez, M. Á. Sanromán and D. Moldes, Biotechnology advances, 2013, 31, 1808-1825.）。
the nano enzyme is a nano material with enzyme activity, and is a novel artificial mimic enzyme. Compared with natural enzymes, the nano-enzyme has the advantages of low cost, high stability, easy recovery and the like, and can be well applied to a plurality of fields. Therefore, nanoenzymes have attracted considerable attention as potential substitutes for natural enzymes (J. Wu, X. Wang, Q. Wang, Z. Lou, S. Li, Y. Zhu, L. Qin and H. Wei,Chem. Soc. Rev., 2019, 48, 1004；Y. Chen, Y. Xianyu, M. Dong, J. Zhang, W. Zheng, Z. Qian and X. Jiang, Anal. Chem., 2018, 90, 6906）。
the nano-enzyme which is discovered at present comprises nano-materials such as metal nano-particles, alloy nano-materials, carbon-based nano-materials, graphene quantum dots, carbon nitride nano-materials, metal organic frameworks and the like. The enzyme mainly simulated by these nanoenzymes is peroxidase, and the research on nanolaccases is still less. Although the nanoenzyme has made remarkable progress, the development of nanoenzyme is still hindered by the problems of low activity and poor specificity, so the design and development of novel high-efficiency nanoenzyme are necessary. (S, ji, B, jiang, H, hao, Y, chen, J, dong, Y, mao, Z, zhang, R, gao, W, chen and R. Zhang, Nature Catalysis, 2021, 4, 407-417.）
The metal-loaded carbon-based nano material can be applied to various catalytic reactions, wherein the metal part can be used as an active site of the catalytic reaction, and the metal-loaded carbon-based nano material is a research hotspot in the field of catalysis in recent years. The manganese/carbon nanoenzyme prepared by the method has high-content manganese loaded on a carbon material, so that the catalytic activity (0.48U/mg) of the manganese/carbon nanoenzyme is greatly improved, and the catalytic specific activity of the manganese/carbon nanoenzyme is equivalent to that of a commercially available natural laccase (from Coriolus versicolor, more than or equal to 0.5U/mg, sigma-Aldrich brand). Finally, the manganese/carbon nano enzyme which can be compared with natural laccase is obtained.
Disclosure of Invention
The invention aims to solve the problems of few researches on nano laccase and unsatisfactory activity and the like in the prior art, and provides a preparation method of manganese/carbon nano enzyme with high laccase activity.
In order to achieve the purpose, the technical scheme adopted by the invention for achieving the purpose is as follows:
a preparation method of manganese/carbon nanoenzyme with high laccase activity is characterized by comprising the following steps:
(1) Dissolving manganese acetate tetrahydrate, formaldehyde aqueous solution (37 wt%) and dicyandiamide in ultrapure water at normal temperature, violently stirring for 5 min, heating the solution to slightly boil and refluxing for 6-12 h, and then putting the solution into a vacuum drying oven to dry for 12 h at 100 ℃; the manganese acetate tetrahydrate comprises 0.5196-2.5980g, the dicyandiamide comprises 5 g, and the formaldehyde aqueous solution comprises 4.4-6.6 mL.
(2) And putting the obtained sample into a tube furnace, carbonizing in nitrogen atmosphere, keeping at 800 ℃ for 2 h at the heating rate of 2-5 ℃/min, cooling to room temperature, taking out the obtained product, and grinding into powder.
The manganese acetate tetrahydrate comprises 2.5980g, the dicyandiamide comprises 5.0000g and the formaldehyde aqueous solution comprises 6.6 mL. The reflux time was 12 h. The heating rate was 3.5 ℃/min.
The invention further discloses application of the manganese/carbon nanoenzyme with high laccase activity prepared by the method in coating a carbon layer on the manganese surface and improving the manganese loading capacity; the application of coating a carbon layer on the surface of manganese and improving the manganese loading refers to that: catalyze the oxidation of pollutants such as phenols, such as hydroquinone and 2, 4-dichlorophenol, and further degrade the phenol pollutants. The experimental results show that: the manganese/carbon nano material with high laccase activity is successfully prepared, the laccase activity is greatly increased, the specific activity is close to that of the laccase in the market (from coriolus versicolor), and the activity far exceeds that of other nano laccase reported at present.
The invention is described in more detail below:
a preparation method of manganese/carbon nanoenzyme with high laccase activity comprises the following steps:
(1) Dissolving manganese acetate tetrahydrate, formaldehyde aqueous solution (37 wt%) and dicyandiamide in ultrapure water at normal temperature, vigorously stirring for 5 min, heating the solution to slightly boil and refluxing for 6-12 h, and then putting the solution into a vacuum drier to dry for 12 h at 100 ℃;
(2) Putting the obtained sample into a tube furnace, carbonizing the sample in nitrogen atmosphere, keeping the temperature at 800 ℃ for 2 hours at the heating rate of 2 to 5 ℃/min, cooling the sample to room temperature, taking out the obtained product, and grinding the product into powder;
further, the manganese acetate tetrahydrate in the step (1) is 0.5196-2.5980g, the dicyandiamide is 5 g, and the formaldehyde aqueous solution is 4.4-6.6 mL;
further, the manganese acetate tetrahydrate in the step (1) is preferably 2.5980g, and the aqueous formaldehyde solution is preferably 6.6 mL;
further, the reflux time in the step (1) is 12 hours;
further, the heating rate in the step (2) is 2-5 ℃/min, preferably 3.5 ℃/min.
The invention mainly solves the problem of low activity of the existing nano laccase, mainly inspects the influence of the mass ratio of manganese acetate tetrahydrate to formaldehyde aqueous solution and dicyandiamide on the performance of the material, and has the main difficulty that the manganese surface coated carbon layer material is prepared, high load capacity and good dispersity are realized, and the laccase activity of the material is greatly improved.
Compared with the prior art, the preparation method of the manganese/carbon nano enzyme with high laccase activity disclosed by the invention has the positive effects that:
1. the preparation method of the manganese/carbon nanoenzyme provided by the invention has the advantages of simple synthetic route and good repeatability.
2. Compared with natural laccase enzyme, the manganese/carbon nanoenzyme can be stored at normal temperature and can be repeatedly used.
3. The catalytic specific activity (0.48U/mg) of the prepared manganese/carbon nano-enzyme is close to that of the natural laccase (from coriolus versicolor, which is greater than or equal to 0.5U/mg) sold in the market, and is greatly superior to other nano-laccase (such as CH-Cu nanozymes, 0.02U/mg, J. Wang, R. Huang, W. Qi, R. Su, B.P. Binks and Z. He,Applied Catalysis B: Environmental, 2019, 254, 452-462）。
4. as a novel simulated laccase, the manganese/carbon nanoenzyme can replace natural laccase and can be applied to the fields of textile industry, paper industry, sewage treatment and the like. Compared with the natural laccase sold in the market (such as Sigma-Aldrich brand, 1377.47 yuan/1 g), the production cost is only one third of the selling price, and the application cost is greatly reduced.
Drawings
FIG. 1 is a TEM image of the prepared manganese/carbon nanoenzyme with high laccase activity;
FIG. 2 is an element distribution diagram of the prepared manganese/carbon nanoenzyme with high laccase activity; a-d are the distribution of manganese, oxygen, carbon and nitrogen elements, respectively;
FIG. 3 is an XRD pattern of the manganese/carbon nanoenzyme prepared with high laccase activity;
FIG. 4 is a BET plot of the manganese/carbon nanoenzymes prepared with high laccase activity;
FIG. 5 is a UV-Vis diagram of manganese/carbon nanoenzyme simulated laccase activity test;
FIG. 6 is a graph showing the effect of different manganese contents on the activity of manganese/carbon nanoenzymes;
FIG. 7 is the effect of pH on the catalytic activity of manganese/carbon nanoenzymes with high laccase activity;
FIG. 8 is a graph of manganese/carbon nanoenzyme specific activity test for optimal laccase activity;
FIG. 9 is a graph of the degradation of manganese/carbon nanoenzymes for optimal laccase activity applied to 2,4-DP, where a: high performance liquid chromatogram under different time; b: degradation rate at different times;
fig. 10 is a graph of manganese/carbon nanoenzyme degradation for hydroquinone with optimal laccase activity, wherein a: high performance liquid chromatograms at different times; b: degradation rate at different times.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. In this specification, the term "2,4-DP" is the abbreviated name for the compound "2, 4-dichlorophenol", which are used interchangeably; the term "4-AP" is the abbreviated name for the compound "4-aminoantipyrine", which are used interchangeably. The reactants and reagents used in the invention are all commercially available.
Example 1
A preparation method of manganese/carbon nano enzyme with high laccase activity comprises the following steps:
(1) 2.5980g of manganese acetate tetrahydrate and 5.0000g of dicyandiamide are added into 25 mL of ultrapure water at normal temperature, 6.6 mL of formaldehyde aqueous solution (37 wt%) is added under stirring, then the mixture is stirred vigorously for 5 min, the temperature is slowly increased when the heating is started until the solution is dark brown, then the solution is heated to slight boiling and reflows for 12 h, and then the solution is placed into a vacuum drying oven to be dried for 12 h at 100 ℃;
(2) And putting the obtained sample into a tube furnace, carbonizing the sample in nitrogen atmosphere, keeping the temperature at 800 ℃ for 2 h at the heating rate of 3.5 ℃/min, cooling the sample to room temperature, taking out the obtained product, and grinding the product into powder, thereby obtaining the manganese/carbon nanoenzyme.
The obtained product was TEM characterized as oxides of manganese wrapped in a carbon and nitrogen material (fig. 1); the element distribution test chart shows that the manganese, oxygen, carbon and nitrogen elements are uniformly distributed (figure 2); XRD test pattern shows that the crystal phase in the product contains graphite carbon, manganese oxide and manganese carbide (figure 3)(ii) a Warp N 2 The specific surface area is 74.3 m in an adsorption and desorption test 2 The curve is IV class adsorption isotherm, which indicates that the product is a mesoporous material (figure 4).
Example 2
A preparation method of manganese/carbon nanoenzyme with high laccase activity comprises the following steps:
(1) 2.0784g of manganese acetate tetrahydrate and 5.0000g of dicyandiamide are added into 25 mL of ultrapure water at normal temperature, 6.6 mL of formaldehyde aqueous solution (37 wt%) is added under stirring, then the mixture is stirred vigorously for 5 min, the temperature is slowly increased when the heating is started until the solution is dark brown, then the solution is heated to slight boiling and reflows for 12 h, and then the solution is placed into a vacuum drying oven to be dried for 12 h at 100 ℃;
(2) And (3) putting the obtained sample into a tube furnace, carbonizing in nitrogen atmosphere, keeping at 800 ℃ for 2 h at the heating rate of 3.5 ℃/min, cooling to room temperature, taking out the obtained product, and grinding into powder to obtain the manganese/carbon nanoenzyme.
Example 3
A preparation method of manganese/carbon nanoenzyme with high laccase activity comprises the following steps:
(1) 1.5588 g of manganese acetate tetrahydrate and 5.0000g of dicyandiamide are added to 25 mL of ultrapure water at normal temperature, 6.6 mL of an aqueous formaldehyde solution (37 wt%) is added with stirring, followed by vigorous stirring for 5 min, the temperature is slowly raised when heating is started until the solution is dark brown, then the solution is heated to slight boiling and refluxed for 12 h, and then the solution is placed into a vacuum drying oven to be dried for 12 h at 100 ℃;
(2) And putting the obtained sample into a tube furnace, carbonizing the sample in nitrogen atmosphere, keeping the temperature at 800 ℃ for 2 h at the heating rate of 3.5 ℃/min, cooling the sample to room temperature, taking out the obtained product, and grinding the product into powder, thereby obtaining the manganese/carbon nanoenzyme.
Example 4
A preparation method of manganese/carbon nano enzyme with high laccase activity comprises the following steps:
(1) Adding 1.0392 g of manganese acetate tetrahydrate and 5.0000g of dicyandiamide into 25 mL of ultrapure water at normal temperature, adding 6.6 mL of formaldehyde aqueous solution (37 wt%) under stirring, then stirring vigorously for 5 min, slowly raising the temperature when heating is started until the solution is dark brown, then heating the solution to slight boiling and refluxing for 12 h, and then placing the solution into a vacuum drying oven to dry for 12 h at 100 ℃;
(2) And putting the obtained sample into a tube furnace, carbonizing the sample in nitrogen atmosphere, keeping the temperature at 800 ℃ for 2 h at the heating rate of 3.5 ℃/min, cooling the sample to room temperature, taking out the obtained product, and grinding the product into powder, thereby obtaining the manganese/carbon nanoenzyme.
Example 5
A preparation method of manganese/carbon nanoenzyme with high laccase activity comprises the following steps:
(1) Adding 0.5196 g of manganese acetate tetrahydrate and 5.0000g of dicyandiamide into 25 mL of ultrapure water at normal temperature, adding 6.6 mL of formaldehyde aqueous solution (37 wt%) under stirring, then stirring vigorously for 5 min, slowly heating when heating is started until the solution is dark brown, then heating the solution to slight boiling and refluxing for 12 h, and then placing the solution into a vacuum drying oven to dry for 12 h at 100 ℃;
(2) And putting the obtained sample into a tube furnace, carbonizing the sample in nitrogen atmosphere, keeping the temperature at 800 ℃ for 2 h at the heating rate of 3.5 ℃/min, cooling the sample to room temperature, taking out the obtained product, and grinding the product into powder, thereby obtaining the manganese/carbon nanoenzyme.
Example 6
A manganese/carbon nanoenzyme simulated laccase performance test experiment comprises the following steps:
(1) 2550 muL of pH 6.8 buffer solution, 150 muL of 2mg/mL 2,4-DP solution, 150 muL of 2mg/mL 4-AP solution and 150 muL of 2mg/mL manganese/carbon nanoenzyme (2.5980 g of manganese acetate tetrahydrate synthetic sample) solution are uniformly mixed in a centrifuge tube.
(2) The catalytic kinetics curves at 510 nm were examined using the time-driven mode of the UV-Vis spectrometer. Or after reacting for 60 min, measuring the ultraviolet-visible spectrum of the reaction solution by using the spectrum mode of an ultraviolet-visible spectrometer. And determining the catalytic capacity according to the absorbance difference value in a certain time period.
FIG. 5 records UV-Vis graphs of the prepared manganese/nitrogen-oxygen-carbon nanoenzyme with high activity in different systems for catalyzing and oxidizing 2,4-DP by oxygen and performing coupled color reaction with 4-AP. As shown in the figure, no color reaction occurred when there was only 2,4-DP and 4-AP; when 2,4-DP, 4-AP and manganese/nitrogen oxygen carbon nano-enzyme all exist, color reaction occurs. The color change is caused by the manganese/nitrogen oxygen carbon nano enzyme to oxidize 2,4-DP through oxygen catalysis and generate color reaction with 4-AP, and the catalytic reaction is the same as natural laccase, so that the prepared manganese/nitrogen oxygen carbon nano enzyme has laccase activity.
Example 7
The influence of different manganese contents on the catalytic activity of the manganese/carbon nanoenzyme with high laccase activity is as follows:
(1) And uniformly mixing 2900 muL of pH 3 buffer solution, 55 muL of 2mM ABTS solution and 45 muL of 1 mg/mL manganese/carbon nanoenzyme solution with different manganese contents in a centrifuge tube, wherein the time is controlled within 30 s.
(2) The catalytic kinetics curve at 420 nm was examined using the time-driven mode of the UV-Vis spectrometer.
As shown in FIG. 6, the catalytic activity of the manganese/nitrogen-oxygen-carbon nanoenzyme increases with the increase of the manganese content, and the highest laccase activity is exhibited when the manganese raw material content reaches 2.5980 g.
Example 8
The influence of different pH values on the catalytic activity of the manganese/carbon nano-enzyme with high laccase activity comprises the following steps:
(1) Preparing buffer solutions with pH values of 3, 4, 5, 6, 6.4, 6.6, 6.8, 7 and 8 respectively.
(2) 2550 muL of buffer solutions with different pH values, 150 muL of 2mg/mL 2,4-DP solution, 150 muL of 2mg/mL 4-AP solution and 150 muL of 2mg/mL manganese/carbon nanoenzyme solution (2.5980 g of manganese acetate tetrahydrate synthetic sample) are uniformly mixed in a centrifuge tube.
(3) The catalytic kinetics curve was measured over 1 h at 550 nm using the time-driven mode of the UV-Vis spectrometer.
As shown in FIG. 7, the absorbance value tended to increase and decrease with increasing pH, and peaked at pH 6.8. Therefore, it is found that the catalytic activity of the manganese/carbon nanoenzyme is best when the pH is controlled to 6.8.
Example 9
The determination of the optimal manganese/carbon nanoenzyme specific activity comprises the following steps:
(1) 2.875 mL of solutions containing different amounts (60-360. Mu.g) of nanoenzyme (2.5980 g manganese acetate tetrahydrate synthetic sample) were placed in 0.2 mol/L Na 2 HPO 4 -NaH 2 PO 4 The pH of the buffer solution was 6.8.
(2) And adding 125 mu L of catechol solution into the centrifuge tube and mixing, wherein the final concentration is 0.02 mol/L and the total volume is 3 mL.
(3) The reaction mixture was placed in a thermostatic water bath and incubated at 25 ℃ for 1 minute. The water bath is connected with an ultraviolet/visible spectrometer.
(4) The catalytic kinetics curves at 410 nm were examined using the time-driven mode of the UV-Vis spectrometer.
The result is shown in FIG. 8, the specific enzyme activity of the manganese/carbon nanoenzyme is 0.48U/mg, which is close to the activity (not less than 0.5U/mg) of the natural laccase sold in the market, and is greatly higher than the activity of other nanoenzymes reported at present, and the manganese/carbon nanoenzyme shows excellent catalytic performance.
Example 10
Application of optimal manganese/carbon nanoenzyme in degradation of 2, 4-dichlorophenol
(1) To a buffer solution of pH =6.8, 2.4-DP at a final concentration of 100 μ g/mL and nanoenzyme (2.5980 g of manganese acetate tetrahydrate synthesis sample) at a final concentration of 100 μ g/mL were added and reacted in a water bath at 25 ℃.
(2) 1 mL of the reaction solution was taken at 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h and 8 h of reaction, and the supernatant was centrifuged.
(3) The residual 2.4-DP concentration was determined by high performance liquid chromatography. Using the formula: x = (C) 0 －C t )/C 0 The degradation rate of the catalytic reaction was calculated. Wherein C is 0 At an initial concentration of 2.4-DP, C t The concentration of 2.4-DP at the t-hour is indicated.
The result is shown in FIG. 9, FIG. 9a corresponds to the chromatogram under different times, the main chromatographic peak belongs to 2.4-DP, and the area of the chromatographic peak of 2.4-DP is gradually reduced along with the continuous increase of time, which indicates that the manganese/carbon nanoenzyme gradually catalyzes and degrades the 2.4-DP. As shown in FIG. 9B, after 8 hours, the degradation rate of the manganese/carbon nanoenzyme on 2,4-DP reached 91.80%, which is superior to the degradation effect reported in the above documents (J.H. Wang, R.L. Huang, W.Qi, R.X. Su, B.P. Binks and Z.M. He, applied Catalysis B-Environmental, 2019, 254, 452-462).
Example 11
Application of optimal manganese/carbon nanoenzyme to degradation of hydroquinone
(1) Hydroquinone at a final concentration of 100 μ g/mL and nanoenzyme (2.5980 g manganese acetate tetrahydrate synthetic sample) at a final concentration of 100 μ g/mL were added to a buffer solution at pH =6.8 and reacted at 25 ℃ in a water bath.
(2) 1 mL of the reaction solution was taken at 1 hour, 2 hours, 3 hours, and 4 hours of reaction, and the supernatant was centrifuged.
(3) The residual hydroquinone concentration was determined by high performance liquid chromatography. Using the formula: x = (C) 0 －C t )/C 0 The degradation rate of the catalytic reaction was calculated. Wherein C is 0 Is the initial concentration of hydroquinone, C t It represents the hydroquinone concentration at the t-hour.
The result is shown in fig. 10, fig. 10a corresponds to the chromatogram under different times, the chromatographic peak with the retention time of 3.942 minutes belongs to hydroquinone, and the area of the chromatographic peak of hydroquinone is gradually reduced along with the continuous increase of time, which indicates that the manganese/carbon nanoenzyme gradually catalyzes and degrades the hydroquinone. As shown in FIG. 10 b, after a short period of 2 hours, the degradation rate of the manganese/nitrogen oxygen carbon nanoenzyme on hydroquinone reached 88%, and after 4 hours, the degradation rate reached 94%, which was superior to the degradation effect reported in the literature (J. Wang, R. Huang, W. Qi, R. Su, B.P. Binks and Z. He,Applied Catalysis B: Environmental, 2019, 254, 452-462). In conclusion, the manganese/carbon nanoenzyme which is stable, cheap, easy to prepare and efficient has great application value in the environmental protection fields of pollutant treatment and the like.
The foregoing is only a preferred embodiment of the present invention. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and it is therefore intended that all such equivalent changes and modifications as would be obvious to one skilled in the art be included within the scope of the appended claims.
Claims (5)
1. A preparation method of manganese/carbon nanoenzyme with high laccase activity is characterized by comprising the following steps:
(1) Co-dissolving manganese acetate tetrahydrate, 37wt% of formaldehyde aqueous solution and dicyandiamide in ultrapure water at normal temperature, violently stirring for 5 min, heating the solution to slightly boil and refluxing for 6-12 h, and then putting the solution into a vacuum drying oven to dry for 12 h at 100 ℃; the manganese acetate tetrahydrate accounts for 0.5196-2.5980g, the dicyandiamide accounts for 5.0000g, and the formaldehyde aqueous solution accounts for 4.4-6.6 mL;
(2) And (3) putting the obtained sample into a tube furnace, carbonizing the sample in a nitrogen atmosphere, keeping the temperature at 800 ℃ for 2 hours at the heating rate of 2 to 5 ℃/min, then cooling the sample to room temperature, taking out the obtained product, and grinding the product into powder.
2. The method for preparing manganese/carbon nanoenzyme with high laccase activity according to claim 1, wherein the manganese acetate tetrahydrate is 2.5980g, the dicyandiamide is 5.0000g, and the aqueous formaldehyde solution is 6.6 mL.
3. The method for preparing manganese/carbon nanoenzyme with high laccase activity according to claim 1, wherein the reflux time is 12 h.
4. The method for preparing manganese/carbon nanoenzyme with high laccase activity according to claim 1, wherein the temperature increase rate is 3.5 ℃/min.
5. The application of the preparation method of the manganese/carbon nanoenzyme with high laccase activity in the aspects of manganese surface coating of a carbon layer and manganese loading capacity improvement of the manganese nano-enzyme with high laccase activity in the aspects of claim 1; the application of the manganese surface coated with the carbon layer and the aspect of improving the manganese loading amount refers to that: catalyzing the oxidation of hydroquinone and 2, 4-dichlorophenol which are phenolic pollutants, and further degrading the phenolic pollutants.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210954140.7A CN115283000B (en) | 2022-08-10 | 2022-08-10 | Preparation method of manganese/carbon nano enzyme with high laccase activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210954140.7A CN115283000B (en) | 2022-08-10 | 2022-08-10 | Preparation method of manganese/carbon nano enzyme with high laccase activity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115283000A true CN115283000A (en) | 2022-11-04 |
| CN115283000B CN115283000B (en) | 2024-09-20 |
Family
ID=83828922
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210954140.7A Active CN115283000B (en) | 2022-08-10 | 2022-08-10 | Preparation method of manganese/carbon nano enzyme with high laccase activity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115283000B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5240893A (en) * | 1992-06-05 | 1993-08-31 | General Motors Corporation | Method of preparing metal-heterocarbon-nitrogen catalyst for electrochemical cells |
| CN106430146A (en) * | 2016-11-22 | 2017-02-22 | 重庆文理学院 | Nitrogen-manganese co-doped hierarchical porous carbon material preparation method |
-
2022
- 2022-08-10 CN CN202210954140.7A patent/CN115283000B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5240893A (en) * | 1992-06-05 | 1993-08-31 | General Motors Corporation | Method of preparing metal-heterocarbon-nitrogen catalyst for electrochemical cells |
| CN106430146A (en) * | 2016-11-22 | 2017-02-22 | 重庆文理学院 | Nitrogen-manganese co-doped hierarchical porous carbon material preparation method |
Non-Patent Citations (2)
| Title |
|---|
| HAORAN GE ET AL: "Fungus-Based MnO/Porous Carbon Nanohybrid as Efficient Laccase Mimic for Oxygen Reduction Catalysis and Hydroquinone Detection", NANOMATERIALS, 8 May 2022 (2022-05-08), pages 2 * |
| YU XIONG ET AL.: "Gram-Scale Synthesis of High-Loading Single-Atomic-Site Fe Catalysts for Effective Epoxidation of Styrene", ADV. MATER, 19 July 2020 (2020-07-19), pages 1 - 2 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115283000B (en) | 2024-09-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fan et al. | Mn-doped g-C3N4 composite to activate peroxymonosulfate for acetaminophen degradation: the role of superoxide anion and singlet oxygen | |
| Silva et al. | Immobilization of soybean peroxidase on silica-coated magnetic particles: a magnetically recoverable biocatalyst for pollutant removal | |
| Feng et al. | Soybean peroxidase trapped in product precipitate during phenol polymerization retains activity and may be recycled | |
| CN114904535B (en) | Preparation method of nano enzyme with activity superior to that of natural laccase | |
| Sani et al. | Design, synthesis and activity study of tyrosinase encapsulated silica aerogel (TESA) biosensor for phenol removal in aqueous solution | |
| CN113244958B (en) | Mn-MOF cold-fit nano enzyme and preparation method and application thereof | |
| Gao et al. | Enhancing the catalytic performance of chloroperoxidase by co-immobilization with glucose oxidase on magnetic graphene oxide | |
| Shi et al. | Pd nanoparticles supported on nitrogen, sulfur-doped three-dimensional hierarchical nanostructures as peroxidase-like catalysts for colorimetric detection of xanthine | |
| CN115920890A (en) | Preparation method of iron monoatomic-doped fluorescent carbon dot nanoenzyme | |
| Huang et al. | Oxygen oxidation of ethylbenzene over manganese porphyrin is promoted by the axial nitrogen coordination in powdered chitosan | |
| CN112007679A (en) | Co/V bimetal doped g-C3N4Photocatalyst and preparation method and application thereof | |
| CN111534505A (en) | Immobilized laccase and preparation method and application thereof | |
| Wang et al. | A Cu (II) MOF with laccase-like activity for colorimetric detection of 2, 4-dichlorophenol and p-nitrophenol | |
| CN113332990B (en) | Perovskite catalytic material and green synthesis method and application thereof | |
| CN114100586A (en) | Composite nano enzyme and preparation method and application thereof | |
| CN115283000A (en) | Preparation method of manganese/carbon nano enzyme with high laccase activity | |
| CN106582770B (en) | A kind of Zn/g-C3N4The preparation method and application of ozone catalyst | |
| Liu et al. | Mn-CDs with oxidase activity produces ROS to inhibit cell activity | |
| Liu et al. | Aspartic acid based metal–organic frameworks with dual function of NADH peroxidase and glycerol dehydrogenase-mimicking activities | |
| Wu et al. | Characterization of immobilized tyrosinase–an enzyme that is stable in organic solvent at 100° C | |
| CN112844431A (en) | Graphite phase single layer C chelated with copper ions3N4Nano enzyme and preparation method thereof | |
| CN110605133A (en) | Nitrogen-doped titanium-carbon composite catalyst and preparation method and application thereof | |
| CN114377717B (en) | Lignin-based carbon composite graphite phase carbon nitride/Mxene heterojunction photocatalyst and preparation method and application thereof | |
| Soria-Sánchez et al. | Efficient catalytic wet oxidation of phenol using iron acetylacetonate complexes anchored on carbon nanofibres | |
| Ma et al. | Ni-Zn supported defective carbon with multi-functional catalytic sites for Baeyer–Villiger reaction using air as oxidant |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |