CN114807112B - Method for immobilizing laccase by magnetic graphite phase carbon nitride and application thereof - Google Patents
Method for immobilizing laccase by magnetic graphite phase carbon nitride and application thereof Download PDFInfo
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- CN114807112B CN114807112B CN202210480482.XA CN202210480482A CN114807112B CN 114807112 B CN114807112 B CN 114807112B CN 202210480482 A CN202210480482 A CN 202210480482A CN 114807112 B CN114807112 B CN 114807112B
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- carbon nitride
- phase carbon
- graphite phase
- laccase
- magnetic graphite
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- 108010029541 Laccase Proteins 0.000 title claims abstract description 137
- JMANVNJQNLATNU-UHFFFAOYSA-N oxalonitrile Chemical compound N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 title claims abstract description 126
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 title claims abstract description 125
- 229910002804 graphite Inorganic materials 0.000 title claims abstract description 124
- 239000010439 graphite Substances 0.000 title claims abstract description 124
- 238000000034 method Methods 0.000 title claims abstract description 18
- 230000003100 immobilizing effect Effects 0.000 title description 4
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims abstract description 44
- 229910001431 copper ion Inorganic materials 0.000 claims abstract description 44
- 238000006243 chemical reaction Methods 0.000 claims abstract description 18
- 230000009471 action Effects 0.000 claims abstract description 11
- FOINSAWEWXUXPQ-UHFFFAOYSA-N 4-acetamido-2-aminobenzenesulfonic acid Chemical compound CC(=O)NC1=CC=C(S(O)(=O)=O)C(N)=C1 FOINSAWEWXUXPQ-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000009920 chelation Effects 0.000 claims abstract description 7
- 238000012986 modification Methods 0.000 claims abstract description 7
- 230000004048 modification Effects 0.000 claims abstract description 7
- 238000006557 surface reaction Methods 0.000 claims abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 46
- 238000003756 stirring Methods 0.000 claims description 31
- 239000000203 mixture Substances 0.000 claims description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 25
- 239000002135 nanosheet Substances 0.000 claims description 24
- 239000003054 catalyst Substances 0.000 claims description 22
- 239000012445 acidic reagent Substances 0.000 claims description 21
- 239000002131 composite material Substances 0.000 claims description 21
- 238000002156 mixing Methods 0.000 claims description 19
- 239000004098 Tetracycline Substances 0.000 claims description 17
- 229960002180 tetracycline Drugs 0.000 claims description 17
- 229930101283 tetracycline Natural products 0.000 claims description 17
- 235000019364 tetracycline Nutrition 0.000 claims description 17
- 150000003522 tetracyclines Chemical class 0.000 claims description 17
- 238000010438 heat treatment Methods 0.000 claims description 15
- 238000004132 cross linking Methods 0.000 claims description 12
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- ODWXUNBKCRECNW-UHFFFAOYSA-M bromocopper(1+) Chemical compound Br[Cu+] ODWXUNBKCRECNW-UHFFFAOYSA-M 0.000 claims description 11
- 238000000944 Soxhlet extraction Methods 0.000 claims description 9
- 230000015556 catabolic process Effects 0.000 claims description 9
- 239000013522 chelant Substances 0.000 claims description 9
- 238000006731 degradation reaction Methods 0.000 claims description 9
- 239000006185 dispersion Substances 0.000 claims description 9
- 238000007885 magnetic separation Methods 0.000 claims description 9
- 238000010907 mechanical stirring Methods 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 230000035484 reaction time Effects 0.000 claims description 8
- 238000009210 therapy by ultrasound Methods 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 4
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 4
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 4
- OPQARKPSCNTWTJ-UHFFFAOYSA-L copper(ii) acetate Chemical compound [Cu+2].CC([O-])=O.CC([O-])=O OPQARKPSCNTWTJ-UHFFFAOYSA-L 0.000 claims description 3
- 230000002538 fungal effect Effects 0.000 claims description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 2
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims 1
- 230000003197 catalytic effect Effects 0.000 abstract description 24
- 239000000758 substrate Substances 0.000 abstract description 20
- 238000006555 catalytic reaction Methods 0.000 abstract description 7
- 230000003647 oxidation Effects 0.000 abstract description 7
- 238000007254 oxidation reaction Methods 0.000 abstract description 7
- 230000033116 oxidation-reduction process Effects 0.000 abstract description 6
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 230000001699 photocatalysis Effects 0.000 abstract description 4
- 230000002195 synergetic effect Effects 0.000 abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 229940088710 antibiotic agent Drugs 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000007146 photocatalysis Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 2
- 238000012546 transfer Methods 0.000 abstract description 2
- 230000000593 degrading effect Effects 0.000 abstract 1
- 125000000524 functional group Chemical group 0.000 abstract 1
- 239000002351 wastewater Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 75
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 21
- GWMKHUGTECCYGK-UHFFFAOYSA-N acetamidosulfamic acid Chemical group C(C)(=O)NNS(=O)(=O)O GWMKHUGTECCYGK-UHFFFAOYSA-N 0.000 description 19
- 235000019441 ethanol Nutrition 0.000 description 14
- 239000007853 buffer solution Substances 0.000 description 10
- 241000222355 Trametes versicolor Species 0.000 description 9
- 238000005259 measurement Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 230000027756 respiratory electron transport chain Effects 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 229910001882 dioxygen Inorganic materials 0.000 description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical group [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- DBXNUXBLKRLWFA-UHFFFAOYSA-N N-(2-acetamido)-2-aminoethanesulfonic acid Chemical compound NC(=O)CNCCS(O)(=O)=O DBXNUXBLKRLWFA-UHFFFAOYSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000001166 ammonium sulphate Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 150000001879 copper Chemical class 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
- 238000004042 decolorization Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- UQSXHKLRYXJYBZ-UHFFFAOYSA-N iron oxide Inorganic materials [Fe]=O UQSXHKLRYXJYBZ-UHFFFAOYSA-N 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 230000005415 magnetization Effects 0.000 description 1
- 239000005543 nano-size silicon particle Substances 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000643 oven drying Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- NDLPOXTZKUMGOV-UHFFFAOYSA-N oxo(oxoferriooxy)iron hydrate Chemical group O.O=[Fe]O[Fe]=O NDLPOXTZKUMGOV-UHFFFAOYSA-N 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000011941 photocatalyst Substances 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 238000004065 wastewater treatment Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- 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
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/22—Organic complexes
- B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
- B01J31/226—Sulfur, e.g. thiocarbamates
-
- B01J35/23—
-
- B01J35/33—
-
- B01J35/39—
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/02—Compositional aspects of complexes used, e.g. polynuclearity
- B01J2531/0213—Complexes without C-metal linkages
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/10—Complexes comprising metals of Group I (IA or IB) as the central metal
- B01J2531/16—Copper
-
- 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
-
- 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
- C02F2101/38—Organic compounds containing nitrogen
Abstract
The invention discloses a magnetic graphite phase carbon nitride immobilized laccase, which is characterized in that magnetic graphite phase carbon nitride is synthesized firstly, is grafted with 4-acetamido-2-aminobenzenesulfonic acid, is subjected to surface functionalization treatment of copper ion chelation modification, is subjected to immobilization reaction with laccase, and the active center of the laccase is directly connected with copper ions of a functional group on the surface of the magnetic graphite phase carbon nitride, so that electrons are generated between the laccase and Fe in the oxidation process 3 O 4 ‑g‑C 3 N 4 The transfer between the immobilized laccase and the immobilized laccase greatly improves the catalytic efficiency of the immobilized laccase, improves the catalytic oxidation rate by utilizing the synergistic effect of enzyme catalysis and photocatalysis, realizes the oxidation of a substrate with higher oxidation-reduction potential and a macromolecular substrate, enhances the affinity of the immobilized enzyme to the substrate, has superparamagnetism, can be conveniently and effectively controlled and separated under the action of an externally applied magnetic field, and has high catalytic performance of the prepared immobilized laccase and obvious effect in degrading intractable antibiotics in wastewater.
Description
Technical Field
The invention relates to the field of laccase immobilization, in particular to a method for immobilizing laccase by using magnetic graphite phase carbon nitride and application thereof.
Background
Laccase (EC 1.10.3.2), belongs to a group of oxidation-reduction enzymes with copper atoms in the catalytic center, and in recent years, the green and environment-friendly characteristics of laccase make the laccase play important roles in aspects of fruit juice clarification, wine production, wastewater treatment, bioremediation, dye decolorization, degradation of environmental pollutants and the like, but the industrial application of natural laccase has the limitations of high production cost, unstable operation conditions, difficult separation of products from reaction media, lack of reusability and the like, and brings a certain limiting effect in the industrial application, so that the immobilized laccase has some special industrial application advantages in the sense.
At present, the selection of immobilized laccase carrier materials mainly focuses on high polymer materials, and although the materials are widely available, the immobilized laccase has low efficiency and poor catalytic effect, so that the economic cost is increased to a certain extent, and certain obstruction is added for the commercial application of laccase, so that it is very important to select a proper carrier and allow the application of the laccase on an industrial scale. The oxidation-reduction potential of laccase is 0.5-0.8V, wherein the oxidation-reduction potential of the trametes versicolor laccase is 0.8V; taking laccase oxidized phenols as an example, the redox potential is mainly concentrated at 0.5-0.8V, which explains the reason that substrate electrons become rate limiting steps towards laccase natural intermediates (Kinetic and biochemical properties of high and low redox potential laccases from fungal and plant origin, marco Frasconi, etc., biochimica et Biophysica Acta,2010, 1804:899-908). At present, the carriers commonly used for laccase immobilization comprise magnetic nano silicon dioxide, magnetic ferroferric oxide nano particles and the like, but the obtained magnetic immobilized laccase has low affinity to a substrate, so that the catalytic efficiency is low.
Disclosure of Invention
The invention aims to provide a magnetic graphite phase carbon nitride immobilized laccase composite catalyst, which adopts magnetic graphite phase carbon nitride as an immobilization carrier, and forms a laccase and magnetic graphite phase carbon nitride complex through laccase immobilization, wherein laccase plays a role in electron transfer in the phenol catalysis process, and directly takes g-C as follows 3 N 4 Surface-accepted electrons (e) - ) Reducing molecular oxygen into water, accelerating H + And e - Is characterized by low electron accepting rate of laccase from substrate and photo-generated electron and O 2 Slow bonding.
The magnetic graphite phase carbon nitride immobilized laccase composite catalyst is laccase immobilized on magnetic graphite phase carbon nitride, and the magnetic graphite phase carbon nitride is magnetic graphite phase carbon nitride subjected to surface functionalization.
The preparation method of the graphite phase carbon nitride nano-sheet adopts the prior literature reported method (Ultratin g-C 3 N 4 with enriched surface carbon vacancies enables highly efficient photocatalytic nitrogen fixing. YI Zhang, J Colloid Interface Sci,2019.553: p.530-539.) a graphite phase carbon nitride nanosheet is prepared, comprising the main steps of: 6g of dicyandiamide (Sinopharm, chemically pure, 98%) are calcined in a tube furnace with a semi-closed crucible with a lid. The samples were processed by a temperature programming step. Heating to 350 deg.C for 1.5 hr, heating to 600 deg.C for 2 hr, cooling and grinding to obtain yellow powder with g-C 3 N 4 。
In order to enable laccase to be better immobilized on the magnetic graphite phase carbon nitride, the magnetic graphite phase carbon nitride is converted into the magnetic graphite phase carbon nitride nano-sheet decorated by copper ion chelation. There are various methods for converting the magnetic graphite phase carbon nitride into the copper ion chelating modified magnetic graphite phase carbon nitride nanosheets, and preferably, the magnetic graphite phase carbon nitride is firstly converted into the magnetic graphite phase carbon nitride with an acetamido-sulfamate function through a 4-acetamido-2-aminobenzenesulfonic acid grafting reagent, and then the magnetic graphite phase carbon nitride nanosheets chelating copper ions are obtained through mixing reaction with a copper ion solution.
The magnetic g-C3N4 has stronger absorption capacity under visible light than the g-C3N4, which can realize the catalysis of the substrate by the catalyst under the visible light. Photo-enzyme electron transfer can be achieved by using a linker and chelating copper ions on the surface. The magnetic g-C3N4 nanosheets are adopted as immobilized carriers, the combination of the laccase and laccase active centers is realized in an alcohol solution system, a photo-enzyme composite catalyst is formed, the laccase plays a role in electron transfer in the catalytic process, electrons (e-) are directly accepted from the surface of the g-C3N4 to reduce molecular oxygen into water, separation of holes (h+) and photo-generated electrons (e-) is accelerated, the problems that the electron accepting rate of the laccase from a substrate is slow and the e-and h+ are easy to be compounded are solved, the catalytic oxidation efficiency is improved by utilizing the synergistic effect of enzyme catalysis and photocatalysis, and oxidation of a substrate with higher oxidation-reduction potential and a macromolecular substrate is realized.
A method for preparing a magnetic graphite phase carbon nitride immobilized laccase composite catalyst comprises the steps of synthesizing magnetic graphite phase carbon nitride, carrying out grafting reaction and copper ion chelation modification on the magnetic graphite phase carbon nitride to obtain surface functionalized magnetic graphite phase carbon nitride, and immobilizing laccase on the surface functionalized magnetic graphite phase carbon nitride to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst. Specifically, the method comprises the following steps:
(1) Preparing magnetic graphite phase carbon nitride;
(2) Carrying out grafting reaction on the magnetic graphite phase carbon nitride;
(3) The grafted magnetic graphite phase carbon nitride is chelated with copper ions;
(4) Laccase immobilization reaction.
The preparation method of the magnetic graphite phase carbon nitride in the step (1) comprises the following steps: feCl is added 3 ·6H 2 O is dissolved in absolute ethyl alcohol and stirred uniformly, then graphite phase carbon nitride is added into a monohydric alcohol solution, the mixture is stirred uniformly in a beaker, and then the mixture is dried in an oven to obtain the magnetic graphite phase carbon nitride nano-sheet. The Fe isCl 3 ·6H 2 The content of O is 0.005-0.04% (w/v), preferably 0.01-0.03% (w/v), more preferably 0.015-0.025% (w/v).
The monohydric alcohol is selected from any 1 or at least 2 of methanol, ethanol, propanol, butanol, preferably ethanol. The polarity of the ethanol can meet the reaction requirement, ferric trichloride can be dissolved, a hydrophilic film can be formed on the surface of graphite-phase carbon nitride, and the deposition of the ferric oxide on the surface of the graphite-phase carbon nitride is ensured.
The content of carbon nitride in the graphite phase is 0.05 to 0.4% (w/v), preferably 0.1 to 0.3% (w/v), more preferably 0.15 to 0.25% (w/v).
The drying temperature of the oven is 40-80Preferably 50-70->More preferably 55-65->The oven drying time is 7 to 17 hours, preferably 9 to 15 hours, more preferably 11 to 13 hours. The temperature and time of drying will affect the crystalline form of the ferroferric oxide.
The grafting reaction flow in the step (2) mainly comprises the following steps: adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water, carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution under mechanical stirring, heating, carrying out Soxhlet extraction to remove the carrier-free acetamido-sulfamic acid reagent, and separating to obtain a product. The content of the magnetic graphite phase carbon nitride is 0.5 to 5% (w/v), preferably 1 to 4% (w/v), more preferably 2 to 3 (w/v). The water is any 1 of laboratory water, deionized water, distilled water, and secondary distilled water, preferably distilled water.
The acetamido-sulfamic acid reagent is selected from any 1 or 2 combinations of 4-acetamido-2-sulfamic acid and n- (2-acetamido) -2-aminoethanesulfonic acid, and is preferably 4-acetamido-2-sulfamic acid. The 4-acetamido-2-aminobenzenesulfonic acid has benzene ring, and the existence of large pi bond can more effectively realize the electron transfer of enzyme and photocatalyst.
The acetamido-sulfamic acid reagent is present in an amount of 1.2-2.5% (w/v), preferably 1.5-2.2% (w/v), more preferably 1.8-1.9 (w/v).
Heating at a temperature of 70-150 deg.fPreferably 80-130->More preferably 90-120->The heating time is 16-32h, preferably 20-28h, more preferably 23-25h.
The chelation of copper ions in the step (3) is as follows: dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution, stirring the mixture, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
And performing chelation modification on the magnetic graphite phase carbon nitride by copper ions to obtain the magnetic graphite phase carbon nitride with the chelation modification of the copper ions. The copper ion solution is a soluble solution containing divalent copper ions, such as any 1 or a combination of at least 2 of a copper chloride solution, a copper sulfate solution, a copper bromide solution, or a copper acetate solution, such as a copper chloride solution/copper sulfate solution, a copper nitrate solution/copper chloride solution, a copper chloride solution/copper sulfate solution/copper acetate solution, or the like. Preferably, the copper ion solution of step (3) is preferably a copper bromide solution.
Preferably, the copper salt content in step (3) is 0.5-1.2% (w/v), preferably 0.6-1.1% (w/v), more preferably 0.8-0.9 (w/v).
Preferably, the stirring speed of the mixture of step (3) is 130-260r/min, preferably 160-240r/min, more preferably 180-220r/min.
Preferably, the stirring time of the mixture of step (3) is from 18 to 30 hours, preferably from 20 to 28 hours, more preferably from 23 to 25h.
The laccase immobilization reaction in the step (4) is as follows: mixing the copper ion chelate modified magnetic graphite phase carbon nitride prepared in the step (3) with a trametes versicolor laccase solution, oscillating at room temperature, adding an ammonium sulfate solution, uniformly mixing, adding a glutaraldehyde solution, crosslinking and stirring, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst.
Preferably, the laccase solution of step (4) is free laccase dissolved in a buffer, typical but non-limiting examples of which are citric acid buffer, tartaric acid buffer, acetic acid buffer, phosphoric acid buffer, etc. The buffer solution is a buffer solution well known to the person skilled in the art, the preparation process is simple and easy to operate, in the invention, which buffer solution is used for dissolving laccase, and the person skilled in the art can select according to the expert knowledge and actual experimental conditions.
The laccase solution in step (4) has a concentration of 1.3-2.0g/L, preferably 1.5-1.8g/L, more preferably 1.6-1.7 g/L,
preferably, the mass ratio of laccase to copper ion chelating modified magnetic graphite phase carbon nitride in step (4) is 13-22, preferably 15-20, more preferably 16-17.
Preferably, the saturation of the ammonium sulphate in step (4) is 65-90% (v/v), preferably 70-86% (v/v), more preferably 76-82% (v/v).
Preferably, the glutaraldehyde concentration in step (4) is 0.8-1.8% (v/v), preferably 1.0-1.5% (v/v), more preferably 1.1-1.3% (v/v).
Preferably, the stirring speed in step (4) is 100-200r/min, preferably 120-180r/min, more preferably 140-160r/min.
Preferably, the time of the crosslinking stirring reaction in step (4) is 2.0 to 6.0 hours, preferably 3.0 to 5.0 hours, more preferably 3.5 to 4.5 hours.
Compared with the prior art, the invention has the following beneficial effects:
(1) The magnetic graphite phase carbon nitride immobilized laccase composite catalyst provided by the invention can realize the catalytic oxidation processElectron in laccase and Fe 3 O 4 -g-C 3 N 4 The transfer between the two steps eliminates the electron transfer speed limiting step in the catalytic reaction of the two steps, the affinity of the immobilized enzyme to the substrate is enhanced, and the catalytic oxidation efficiency is improved by utilizing the synergistic effect of enzyme catalysis and photocatalysis;
(2) The magnetic graphite phase carbon nitride immobilized laccase composite catalyst provided by the invention can be used for oxidizing a substrate with higher oxidation-reduction potential and a macromolecular substrate;
(3) The magnetic graphite phase carbon nitride immobilized laccase composite catalyst provided by the invention has superparamagnetism, and can be conveniently and effectively controlled and separated under the action of an externally applied magnetic field;
(4) The preparation process of the magnetic graphite phase carbon nitride immobilized laccase composite catalyst provided by the invention is simple and easy to amplify.
Drawings
The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:
FIG. 1 is a graph of g-C 3 N 4 、Fe 3 O 4 -g-C 3 N 4 And Fe (Fe) 3 O 4 -g-C 3 N 4 Hysteresis loop results of the immobilized laccase composite catalyst material at room temperature.
Detailed Description
To facilitate understanding of the present invention, examples are set forth below. It should be apparent to those skilled in the art that the examples are merely provided to aid in understanding the present invention and should not be construed as limiting the invention in any way.
Example 1
(1) 0.005% (w/v) FeCl 3 ·6H 2 O is dissolved in absolute ethyl alcohol and stirred uniformly, then 0.05% (w/v) of graphite phase carbon nitride is added into the ethanol solution, the mixture is stirred uniformly in a beaker, and then the mixture is dried in an oven at 40 ℃ for 7 hours, so that the magnetic graphite phase carbon nitride nano-sheet is obtained.
(2) The magnetic graphite phase carbon nitride obtained in the step (1) is processedAdding nano-sheet into water with content of 0.5% (w/v), ultrasonic treating to obtain dispersion solution, adding acetamido-sulfamic acid reagent into the solution under mechanical stirring with content of 1.2% (w/v), 70And heating for 16h, removing the carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain a product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper bromide solution until the content is 0.5% (w/v), stirring the mixture for 18h at the rotating speed of 130r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 1.3g/L, enabling the laccase to be 13 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable saturation to be 65% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable final concentration to be 0.8% (v/v), crosslinking and stirring for 2 hours at 100r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilization laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
Example 2
(1) 0.01% (w/v) FeCl 3 ·6H 2 Dissolving O in absolute ethanol, stirring, adding 0.1% (w/v) graphite phase carbon nitride in ethanol solution, stirring in beaker, and stirringDrying for 9 hours in a 50 ℃ oven to obtain the magnetic graphite phase carbon nitride nano-sheet.
(2) Adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water with the content of 1.0% (w/v), carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution with the content of 1.5% (w/v) and 80% under mechanical stirringAnd heating for 20h, removing the carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain a product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution until the content is 0.6% (w/v), stirring the mixture for 20h at the rotating speed of 160r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 1.5g/L, enabling the laccase to be 15 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable saturation to be 70% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable final concentration to be 1.0% (v/v), crosslinking and stirring for 3 hours at 120r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilization laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
Example 3
(1) 0.015% (w/v) FeCl 3 ·6H 2 O is dissolved in anhydrous BStirring uniformly in alcohol, adding 0.15% (w/v) of graphite phase carbon nitride into ethanol solution, stirring uniformly in a beaker, and drying in a 55 ℃ oven for 11 hours to obtain the magnetic graphite phase carbon nitride nano-sheet.
(2) Adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water with the content of 2.0% (w/v), carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution with the content of 1.8% (w/v) under mechanical stirring, and carrying out 90% treatmentAnd heating for 23h, removing the carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain a product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution until the content is 0.8% (w/v), stirring the mixture for 23h at the rotating speed of 180r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 1.6g/L, enabling the laccase to be 16 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable saturation to be 76% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable the final concentration to be 1.1% (v/v), crosslinking and stirring for 3.5h at 140r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
Example 4
(1) 0.02% (w/v) of FeCl to be added 3 ·6H 2 O is dissolved in absolute ethyl alcohol and stirred uniformly, then 0.2% (w/v) of graphite phase carbon nitride is added into ethanol solution, the mixture is stirred uniformly in a beaker, and then the mixture is dried in an oven at 60 ℃ for 12 hours, so that the magnetic graphite phase carbon nitride nano-sheet is obtained.
(2) Adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water with the content of 2.5% (w/v), carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution with the content of 1.875% (w/v) under mechanical stirring, and carrying out 100And heating for 24 hours, removing the carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain a product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution until the content is 0.86% (w/v), stirring the mixture for 24 hours at the rotating speed of 200r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 1.66g/L, enabling the laccase to be 16.6 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable the saturation to be 80% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable the final concentration to be 1.2% (v/v), carrying out crosslinking stirring for 4.0h at 150r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilization laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
Example 5
(1) 0.025% (w/v) FeCl to be added 3 ·6H 2 O is dissolved in absolute ethyl alcohol and stirred uniformly, then 0.25% (w/v) of graphite phase carbon nitride is added into the ethanol solution, the mixture is stirred uniformly in a beaker, and then the mixture is dried in a drying oven at 65 ℃ for 13 hours, so that the magnetic graphite phase carbon nitride nano-sheet is obtained.
(2) Adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water with the content of 3.0% (w/v), carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution with the content of 1.9% (w/v) under mechanical stirring, and carrying out 120And (5) heating for 25 hours, removing the carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain a product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution until the content is 0.9% (w/v), stirring the mixture for 25 hours at the rotating speed of 220r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 1.7g/L, enabling the laccase to be 17 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable saturation to be 82% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable the final concentration to be 1.3% (v/v), crosslinking and stirring for 4.5 hours at 160r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
Example 6
(1) 0.03% (w/v) FeCl 3 ·6H 2 O is dissolved in absolute ethyl alcohol and stirred uniformly, then 0.3% (w/v) of graphite phase carbon nitride is added into ethanol solution, the mixture is stirred uniformly in a beaker, and then the mixture is dried in an oven at 70 ℃ for 15 hours, so that the magnetic graphite phase carbon nitride nano-sheet is obtained.
(2) Adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water with the content of 4.0% (w/v), carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution with the content of 2.2% (w/v) under mechanical stirring, and carrying out 130Heating for 28h, removing carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain the product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution until the content is 1.1% (w/v), stirring the mixture for 28h at the rotating speed of 240r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 1.8g/L, enabling the laccase to be 20 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable saturation to be 86% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable the final concentration to be 1.5% (v/v), crosslinking and stirring for 5.0h under 180r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
Example 7
(1) 0.04% (w/v) FeCl 3 ·6H 2 O is dissolved in absolute ethyl alcohol and stirred uniformly, then 0.4% (w/v) of graphite phase carbon nitride is added into ethanol solution, the mixture is stirred uniformly in a beaker, and then the mixture is dried in an oven at 80 ℃ for 17 hours, so that the magnetic graphite phase carbon nitride nano-sheet is obtained.
(2) Adding the magnetic graphite phase carbon nitride nano-sheet obtained in the step (1) into water with the content of 5.0% (w/v), carrying out ultrasonic treatment to obtain a dispersion solution, adding an acetamido-sulfamic acid reagent into the solution with the content of 2.5% (w/v) under mechanical stirring, and carrying out 150And (3) heating for 32h, removing the carrier-free acetamido-sulfamic acid reagent by Soxhlet extraction, and separating to obtain a product.
(3) Dispersing the product obtained in the step (2) in water under the action of ultrasonic waves, dropwise adding a copper ion solution until the content is 1.2% (w/v), stirring the mixture for 30h at the rotating speed of 260r/min, and magnetically separating to obtain the copper ion chelating magnetic graphite phase carbon nitride.
(4) Mixing the copper ion chelate modified magnetic graphite phase carbon nitride of copper bromide prepared in the step (3) with a trametes versicolor laccase solution to enable the laccase final concentration to be 2.0g/L, enabling the laccase to be 22 in mass ratio to the magnetic graphite phase carbon nitride, oscillating at room temperature, adding an ammonium sulfate solution to enable saturation to be 90% (v/v), uniformly mixing, adding a glutaraldehyde solution to enable final concentration to be 1.8% (v/v), crosslinking and stirring for 6.0h under 200r/min, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst.
Catalytic activity test:
under the treatment condition of the method, the apparent Mie constant of the magnetic graphite phase carbon nitride immobilized laccase is obtained by taking tetracycline as a substrate to carry out catalytic activity measurement (see table 1).
Experiments were performed in a citric acid buffer system at pH 4.0, with tetracycline concentration of 150mg/L, immobilized laccase and immobilized carrier concentration of 1g/L, and an equal amount of active free laccase was added as a control experiment, the reaction time was 30min, and the full-wavelength light experiment was performed under a 40W power lamp tube to obtain the results (see Table 2).
TABLE 1 determination of Km values
TABLE 2 comparison of tetracycline degradation rates
As can be seen from the data in Table 2, in 7 examples, the sum of the degradation rate of the immobilized laccase under dark conditions and the degradation rate of the immobilized carrier under light conditions is smaller than the degradation rate of the immobilized laccase under light conditions, which means that the immobilized laccase under light conditions shows synergistic effect of photo-enzyme catalysis, wherein the degradation rate of the immobilized laccase under light conditions in example 4 is highest on tetracycline, which means that the electron transfer rate is fastest under the conditions.
TABLE 3 degradation effect of immobilized laccase on other antibiotics under light conditions
As can be seen from Table 3, the immobilized laccase and the carrier also have a certain degradation effect on other antibiotics.
FIG. 1 is g-C 3 N 4 、Fe 3 O 4 -g-C 3 N 4 And Fe (Fe) 3 O 4 -g-C 3 N 4 Hysteresis loop result, g-C, of immobilized laccase composite catalyst material at room temperature 3 N 4 The nanomaterial is not magnetic and Fe 3 O 4 -g-C 3 N 4 And Fe (Fe) 3 O 4 -g-C 3 N 4 Both materials of the immobilized laccase composite catalyst show superparamagnetism, and the maximum saturation magnetization is 76.22emu/g and 45.82emu/g respectively.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (6)
1. The magnetic graphite phase carbon nitride immobilized laccase is characterized by being prepared by the following steps:
s1, preparing magnetic graphite phase carbon nitride;
s2, surface functionalization of magnetic graphite phase carbon nitride, wherein the specific method comprises the following steps:
s21, grafting 4-acetamido-2-aminobenzenesulfonic acid on the magnetic graphite phase carbon nitride:
adding the prepared magnetic graphite phase carbon nitride nano-sheet into water, carrying out ultrasonic treatment to obtain a dispersion solution, adding a 4-acetamido-2-aminobenzenesulfonic acid reagent into the solution under mechanical stirring, heating, carrying out Soxhlet extraction to remove the carrier-free 4-acetamido-2-aminobenzenesulfonic acid reagent, and separating to obtain a product;
s22, performing chelation modification on the magnetic graphite phase carbon nitride grafted by the 4-acetamido-2-aminobenzenesulfonic acid obtained in the S21:
dispersing the product obtained in the step S21 in water under the action of ultrasonic waves, dropwise adding a copper ion solution, stirring the mixture, and magnetically separating to obtain copper ion chelated magnetic graphite phase carbon nitride;
the content of the magnetic graphite phase carbon nitride is 0.5-5% (w/v), the heating temperature is 70-150 ℃, and the heating time is 16-32h;
the content of the 4-acetamido-2-aminobenzenesulfonic acid reagent is 1.2-2.5% (w/v);
the copper ion solution is any one or more of copper chloride solution, copper sulfate solution, copper bromide solution or copper acetate solution, and the content of copper ions is 0.5-1.2% (w/v); the stirring speed of the mixture is 130-260r/min, and the stirring time is 18-30 h;
s3, mixing the surface functionalized magnetic graphite phase carbon nitride with laccase solution, oscillating at room temperature, adding ammonium sulfate solution, uniformly mixing, adding glutaraldehyde solution, crosslinking and stirring, carrying out laccase immobilization reaction, and then carrying out magnetic separation and washing to obtain the magnetic graphite phase carbon nitride immobilized laccase composite catalyst.
2. The magnetic graphite phase carbon nitride immobilized laccase according to claim 1, wherein the preparation method of the magnetic graphite phase carbon nitride in S1 comprises the following steps: and dissolving FeCl3.6H2O in monohydric alcohol, stirring uniformly, adding graphite phase carbon nitride into the monohydric alcohol solution, stirring uniformly the mixture in a beaker, and drying in an oven to obtain the magnetic graphite phase carbon nitride nano-sheet.
3. The magnetic graphite phase carbon nitride immobilized laccase of claim 2, wherein the amount of fecl3.6h2o in S1 is 0.005-0.04% (w/v), and the monohydric alcohol is selected from any one or more of methanol, ethanol, propanol, butanol; the content of the graphite phase carbon nitride is 0.05-0.4% (w/v).
4. The magnetic graphite phase carbon nitride immobilized laccase of claim 2, wherein the drying temperature of the oven in S1 is 40-80 ℃ and the drying time is 7-17 h.
5. The magnetic graphite phase carbon nitride immobilized laccase according to claim 1, wherein the laccase in S3 is selected from fungal laccase, the concentration of the laccase solution is 1.3-2.0g/L, the mass ratio of laccase to copper ion chelate modified magnetic graphite phase carbon nitride is 13-22, the saturation of ammonium sulfate is 65-90% (v/v), the concentration of glutaraldehyde is 0.8-1.8% (v/v), the crosslinking stirring speed is 100-200r/min, and the crosslinking stirring reaction time is 2.0-6.0 h.
6. The use of a magnetic graphite phase carbon nitride immobilized laccase according to claim 1 in tetracycline degradation.
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