CN115518691B - Artificial enzyme with laccase-like activity, and preparation method and application thereof - Google Patents
Artificial enzyme with laccase-like activity, and preparation method and application thereof Download PDFInfo
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- CN115518691B CN115518691B CN202211264345.9A CN202211264345A CN115518691B CN 115518691 B CN115518691 B CN 115518691B CN 202211264345 A CN202211264345 A CN 202211264345A CN 115518691 B CN115518691 B CN 115518691B
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- laccase
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- transition metal
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 61
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 61
- 230000000694 effects Effects 0.000 title claims abstract description 58
- 238000002360 preparation method Methods 0.000 title abstract description 19
- 239000010949 copper Substances 0.000 claims abstract description 78
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims abstract description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000006243 chemical reaction Methods 0.000 claims abstract description 31
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 27
- 235000004279 alanine Nutrition 0.000 claims abstract description 27
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000012965 benzophenone Substances 0.000 claims abstract description 27
- 229910021642 ultra pure water Inorganic materials 0.000 claims abstract description 27
- 239000012498 ultrapure water Substances 0.000 claims abstract description 27
- 239000011701 zinc Substances 0.000 claims abstract description 23
- 239000002244 precipitate Substances 0.000 claims abstract description 22
- 239000006228 supernatant Substances 0.000 claims abstract description 17
- 239000007787 solid Substances 0.000 claims abstract description 16
- 229910052723 transition metal Inorganic materials 0.000 claims abstract description 16
- 150000003624 transition metals Chemical class 0.000 claims abstract description 16
- 239000007864 aqueous solution Substances 0.000 claims abstract description 12
- 229910052802 copper Inorganic materials 0.000 claims abstract description 12
- 238000001291 vacuum drying Methods 0.000 claims abstract description 12
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000001338 self-assembly Methods 0.000 claims abstract description 11
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 9
- 229910052725 zinc Inorganic materials 0.000 claims abstract description 9
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims abstract description 3
- 230000009920 chelation Effects 0.000 claims abstract description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 93
- 239000000243 solution Substances 0.000 claims description 74
- 108010029541 Laccase Proteins 0.000 claims description 59
- 238000000034 method Methods 0.000 claims description 18
- 239000012670 alkaline solution Substances 0.000 claims description 15
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 13
- 239000000356 contaminant Substances 0.000 claims description 11
- 239000000843 powder Substances 0.000 claims description 11
- 239000012266 salt solution Substances 0.000 claims description 11
- 229910021381 transition metal chloride Inorganic materials 0.000 claims description 11
- 150000003841 chloride salts Chemical class 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- HFZWRUODUSTPEG-UHFFFAOYSA-N 2,4-dichlorophenol Chemical compound OC1=CC=C(Cl)C=C1Cl HFZWRUODUSTPEG-UHFFFAOYSA-N 0.000 claims description 7
- CDAWCLOXVUBKRW-UHFFFAOYSA-N 2-aminophenol Chemical compound NC1=CC=CC=C1O CDAWCLOXVUBKRW-UHFFFAOYSA-N 0.000 claims description 6
- JWAZRIHNYRIHIV-UHFFFAOYSA-N 2-naphthol Chemical compound C1=CC=CC2=CC(O)=CC=C21 JWAZRIHNYRIHIV-UHFFFAOYSA-N 0.000 claims description 6
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 claims description 6
- LINPIYWFGCPVIE-UHFFFAOYSA-N 2,4,6-trichlorophenol Chemical compound OC1=C(Cl)C=C(Cl)C=C1Cl LINPIYWFGCPVIE-UHFFFAOYSA-N 0.000 claims description 5
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 claims description 5
- 229950011260 betanaphthol Drugs 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims 1
- 235000001014 amino acid Nutrition 0.000 abstract description 5
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 239000002957 persistent organic pollutant Substances 0.000 abstract description 5
- 238000004043 dyeing Methods 0.000 abstract description 4
- 238000007639 printing Methods 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 230000003197 catalytic effect Effects 0.000 description 53
- 230000003278 mimic effect Effects 0.000 description 26
- 239000000376 reactant Substances 0.000 description 25
- 239000000758 substrate Substances 0.000 description 22
- 239000000725 suspension Substances 0.000 description 19
- 235000013824 polyphenols Nutrition 0.000 description 18
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 15
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 15
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 14
- 238000011534 incubation Methods 0.000 description 14
- MZHCENGPTKEIGP-UHFFFAOYSA-N 2-(2,4-dichlorophenoxy)propanoic acid Chemical compound OC(=O)C(C)OC1=CC=C(Cl)C=C1Cl MZHCENGPTKEIGP-UHFFFAOYSA-N 0.000 description 13
- 239000000872 buffer Substances 0.000 description 11
- 238000005119 centrifugation Methods 0.000 description 9
- 238000007254 oxidation reaction Methods 0.000 description 9
- 230000003647 oxidation Effects 0.000 description 8
- 238000005406 washing Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 6
- 230000008033 biological extinction Effects 0.000 description 6
- 231100000135 cytotoxicity Toxicity 0.000 description 6
- 230000003013 cytotoxicity Effects 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- 230000007613 environmental effect Effects 0.000 description 6
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000003344 environmental pollutant Substances 0.000 description 5
- 231100000719 pollutant Toxicity 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 239000003054 catalyst Substances 0.000 description 4
- 238000006555 catalytic reaction Methods 0.000 description 4
- 239000002086 nanomaterial Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 239000003513 alkali Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- MPTQRFCYZCXJFQ-UHFFFAOYSA-L copper(II) chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Cu+2] MPTQRFCYZCXJFQ-UHFFFAOYSA-L 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- RSAZYXZUJROYKR-UHFFFAOYSA-N indophenol Chemical compound C1=CC(O)=CC=C1N=C1C=CC(=O)C=C1 RSAZYXZUJROYKR-UHFFFAOYSA-N 0.000 description 3
- -1 indoxyl aminoantipyrine Chemical compound 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- 230000001988 toxicity Effects 0.000 description 3
- 235000005074 zinc chloride Nutrition 0.000 description 3
- 239000011592 zinc chloride Substances 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- PCKPVGOLPKLUHR-UHFFFAOYSA-N OH-Indolxyl Natural products C1=CC=C2C(O)=CNC2=C1 PCKPVGOLPKLUHR-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000012621 metal-organic framework Substances 0.000 description 2
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- 239000002105 nanoparticle Substances 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 229910001428 transition metal ion Inorganic materials 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- IVLXQGJVBGMLRR-UHFFFAOYSA-N 2-aminoacetic acid;hydron;chloride Chemical compound Cl.NCC(O)=O IVLXQGJVBGMLRR-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 239000013148 Cu-BTC MOF Substances 0.000 description 1
- 108010016626 Dipeptides Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 235000005811 Viola adunca Nutrition 0.000 description 1
- 240000009038 Viola odorata Species 0.000 description 1
- 235000013487 Viola odorata Nutrition 0.000 description 1
- 235000002254 Viola papilionacea Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001553 co-assembly Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 238000001027 hydrothermal synthesis Methods 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000004922 lacquer Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000005424 photoluminescence Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
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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
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/22—Organic complexes
- B01J31/2204—Organic complexes the ligands containing oxygen or sulfur as complexing atoms
- B01J31/2208—Oxygen, e.g. acetylacetonates
- B01J31/2226—Anionic ligands, i.e. the overall ligand carries at least one formal negative charge
- B01J31/2243—At least one oxygen and one nitrogen atom present as complexing atoms in an at least bidentate or bridging ligand
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/003—Catalysts comprising hydrides, coordination complexes or organic compounds containing enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/16—Catalysts comprising hydrides, coordination complexes or organic compounds containing coordination complexes
- B01J31/1691—Coordination polymers, e.g. metal-organic frameworks [MOF]
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/72—Treatment of water, waste water, or sewage by oxidation
- C02F1/725—Treatment of water, waste water, or sewage by oxidation by catalytic oxidation
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/72—Treatment of water, waste water, or sewage by oxidation
- C02F1/74—Treatment of water, waste water, or sewage by oxidation with air
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/10—Complexes comprising metals of Group I (IA or IB) as the central metal
- B01J2531/16—Copper
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2531/00—Additional information regarding catalytic systems classified in B01J31/00
- B01J2531/20—Complexes comprising metals of Group II (IIA or IIB) as the central metal
- B01J2531/26—Zinc
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/34—Organic compounds containing oxygen
- C02F2101/345—Phenols
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)
- Inorganic Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to an artificial enzyme with laccase-like activity and a preparation method and application thereof, wherein, a chloride aqueous solution of transition metal copper or zinc is added into an alkaline aqueous solution of benzophenone alanine, and the mixture is stirred to be uniformly mixed to complete coordination chelation reaction, and the mixture is placed in a water bath kettle at 30-80 ℃ for standing for 0-60h to complete self-assembly; centrifuging at 8000-10000rpm for 10-20min, discarding supernatant, collecting precipitate, cleaning with ultrapure water, and vacuum drying to obtain BpA-Cu and BpA-Zn solid. The preparation condition of the invention is simple, the synthesis condition of the unnatural amino acid is mature, the price is low, and the source is wide; the activity under alkaline condition can keep 50.2% of the original activity and 89.9% of the activity at high temperature of 90 ℃, and can be applied to the removal of phenolic organic pollutants in water bodies in the fields of printing and dyeing and the like.
Description
Technical Field
The invention relates to a preparation method and application of a laccase-like activity mimic formed by self-assembly of benzophenone alanine and transition metal (such as copper, zinc and the like) ions through coordination, belongs to the technical field of artificial enzyme preparation and application thereof, and in particular relates to an artificial enzyme with laccase-like activity, and a preparation method and application thereof.
Background
Enzymes are a kind of biomacromolecules with catalytic functions, and in Nature enzymes are mainly used to catalyze chemical reactions in living beings, which are usually performed under relatively mild conditions (Nature 2012,485,185-194). However, in practical applications, natural enzymes have disadvantages of difficulty in preparation and purification, poor stability, sensitivity of catalytic activity to environmental conditions, difficulty in recovery and reuse, limited sources, limited types of catalytic reactions, and the like, which have prompted the design and development of various novel artificial catalysts to replace the functions of natural enzymes (Chemical Society Reviews 2019,48,1004-1076).
Laccase (lacase, EC 1.10.3.2) is an extracellular single-molecule glycoprotein, which was first found in lac lacquer solutions and is widely distributed in fungi, higher plants and bacteria. Laccase is a kind of copper-containing polyphenol oxidase, can react with phenols, ascorbic acid, amines and other substances (Microbial Biotechnology 2017,10,1457-1467) and can catalyze and oxidize various refractory organic pollutants, and has wide application in the aspects of wastewater treatment, biosensors and biofuel cells. The laccase catalytic oxidation reaction only generates water, and is a green biocatalyst. However, natural laccase as a natural protein still has the significant disadvantage of being easily denatured. On the one hand, under the conditions of high temperature or polar acid, polar alkali and the like, the protein higher structure is easy to unfold, so that the active structure is damaged, and the activity is irreversibly lost. On the other hand, the natural laccase is easily dissolved in water, the laccase in the free water is not recoverable and can not be stored for a long time, and the application of the laccase in industrial environment is limited to a large extent by the two aspects. Thus, there is a need to study new mimic enzymes that have both laccase activity and meet stability requirements.
Currently, MOFs materials, noble metal nanoparticles, carbon-based nanomaterials, proteins, amino acids, etc. have all been used to mimic laccase. The protease CH-Cu (Cellular and Molecular Life Sciences 2015,72,869-883) with laccase catalytic activity has been constructed by utilizing cysteine-histidine (Cys-His) dipeptide co-assembly. And is used to degrade phenolic contaminants and detect epinephrine (Applied Catalysis B-Environmental,2019,254,452-462). MOFs materials GMP-Cu, cu-Cys (cystine) and HKUST-1 also showed laccase activity and were applied to degradation of phenolic contaminants and dyes (Frontiers of Chemical Science and Engineering,2021,15,310-318). Pt nanoparticles with good dispersibility, which are blocked by nucleotides, are synthesized by using four nucleotides, and are used for catalyzing substrates of various laccase, and the Pt nanoparticles show good catalytic activity (Catalysis Letters,2017,147,2144-2152). Copper-containing carbon point CuCDs with laccase activity and fluorescence performance are synthesized by a hydrothermal method, have good photoluminescence performance under a wide pH range and high salt content, have good stability, and can be used as a fluorescent probe for detecting hydroquinone (nanoscales, 2015,7,19641-19646). However, the reported related materials have poor performances in the aspects of complex preparation, reusability, catalytic activity and the like.
Disclosure of Invention
The invention aims to solve the problems of poor biocompatibility, complex preparation process and the like of materials used by the existing laccase simulation, and provides an application of a laccase-like activity simulator formed by coordination of benzophenone alanine and transition metal (such as copper, zinc and the like) ions in the field of phenolic pollutant treatment. The invention also aims to provide a preparation method of the laccase-like activity mimic formed by coordination of benzophenone alanine and transition metal (such as copper, zinc and the like) ions. The preparation method provided by the invention is simple, mild in reaction condition, easy to operate, and capable of synthesizing the catalytic nano material with laccase activity by taking the unnatural amino acid and the transition metal ion which are abundant in variety, low in cost and easy to obtain as ligands, thereby being beneficial to saving the cost of treating phenol pollution generated by industrial production, generated by using a large amount of bactericides and herbicides in the agricultural production process and generated by degrading certain organic matters.
The technical scheme of the invention is summarized as follows:
the amino and carboxyl groups of the artificial enzyme benzophenone alanine with laccase-like activity and chloride salt composed of transition metal are coordinated and combined to form a supermolecular material with laccase-like activity, which is denoted as BpA-M, wherein BpA is benzophenone alanine, and M represents transition metal copper and zinc.
The preparation method of the laccase-like active artificial enzyme comprises the following steps:
1) Adding chloride aqueous solution of transition metal copper or zinc into alkaline aqueous solution of benzophenone alanine, stirring to uniformly mix the two to complete coordination chelation reaction, and standing in a water bath kettle at 30-80 ℃ for 0-60h to complete self-assembly;
2) Centrifuging at 8000-10000rpm for 10-20min, discarding supernatant, collecting precipitate, cleaning with ultrapure water, and vacuum drying to obtain BpA-Cu and BpA-Zn solid.
The alkaline solution of the benzophenone alanine in the step 1) adopts 10mM sodium hydroxide to dissolve 5-50mM benzophenone alanine; preparing NaOH and transition metal salt solution, wherein the water specification of the NaOH and transition metal salt solution is at least ultrapure water; the concentration of the transition metal chloride salt solution is 10-20mM.
The step 1) is that the benzophenone alanine alkaline aqueous solution and the transition metal chloride salt solution are incubated for 30-60min in a water bath kettle at 40-80 ℃ before being mixed; the process of adding the transition metal chloride salt solution into the alkaline aqueous solution of the benzophenone alanine is required to be carried out under the water bath condition of 40-80 ℃.
The molar ratio of the transition metal chloride salt solution to the benzophenone alanine in the step 1) is 1:1-1:10.
The temperature of the vacuum drying oven in the step 2) is set to be 40-80 ℃, and the drying time is 24-72 hours until the precipitate containing a small amount of water is dried to powder BpA-Cu and BpA-Zn.
The invention discloses application of an artificial enzyme with laccase-like activity in the field of simulating laccase activity and solving phenolic pollutants.
The invention uses benzophenone alanine and transition metal such as copper, zinc plasma to self-assemble to form a laccase-like active mimic by coordination, and utilizes the self-assembled nano material prepared by the method to simulate part of laccase functions. The activity and stability of the mimetic laccase and the natural laccase were determined using 2, 4-dichlorophenol (2.4-DP) as substrate and 4-aminoantipyrine (4-AP) as a chromogenic agent. This is mainly because laccase oxidizes 2,4-DP in the presence of oxygen to free radicals, which can couple with 4-AP to a substance with a color change, which has a distinct absorption peak at 510 nm. Compared with the natural laccase, the mimic laccase provided by the invention has higher substrate affinity compared with the natural laccase. The Km values of the preparation method are 0.07 and 0.27), and the preparation method has higher catalytic efficiency under extreme conditions (high temperature, strong acid and strong alkali) of 89.9 percent, 39.5 percent and 50.2 percent respectively, and good biocompatibility, wherein 800 mug/mL mimic enzyme BpA-Cu has lower harm to the microglial cells BV-2 of mice, and the survival rate of the cells can reach 83.84 percent. The mimic enzyme with laccase-like activity is applied to the field of catalytic nano materials, and can solve the problems of poor reusability and disappearance or reduction of the activity of the natural laccase under extreme conditions (strong acid, strong alkali, high temperature and high salt).
In addition, to study the degradation ability of the mimic enzymes BpA-Cu and laccase to environmental phenolic contaminants, various phenolic contaminants such as hydroquinone, 2-aminophenol, 2,4, 6-trichlorophenol, catechol, phenol, 2-naphthol, 2-nitrophenol, 2,4-DP, and the like were selected as model substrates for the phenolic contaminants. The catalytic activities of BpA-Cu and laccase on various substrates are calculated in a standardized way by taking the activity of BpA-Cu on 2,4-DP as 100%, and the relative catalytic capacities of mimic enzyme BpA-Cu on 7 phenolic pollutants serving as substrates are higher than those of natural laccase except for 2, 4-DP. In particular, the oxidation capacities of the para-catechol, the phenol and the 2-nitrophenol can reach 97.1 percent, 70.1 percent and 81.2 percent of the catalytic capacity of the 2,4-DP substrate respectively. However, the catalytic capacity of natural laccase on these substrates is only 23.3%, 0.1% and 1.7%. Although the relative catalytic capacities of the mimic enzyme and laccase are lower when hydroquinone, 2,4, 6-trichlorophenol are used as substrates, the activity of the mimic enzyme is still higher than that of laccase. The oxidizing ability of the mimic enzyme BpA-Cu to a variety of environmental phenolic contaminants suggests substrate versatility.
The invention has the advantages of simple preparation condition, mature synthesis condition of the unnatural amino acid, low price and wide source. The catalytic process of the simulated enzyme can be monitored by an ultraviolet-visible spectrophotometer and an enzyme-labeled instrument. Compared with the materials of the natural laccase and the simulated laccase reported in the literature, the invention has the advantages that:
1) The prepared mimic enzyme has higher capability of catalyzing the oxidation of a substrate and lower Km, and the value corresponding to the Km of the natural enzyme is 0.07mM VS 0.27mM;
2) The catalytic capability of the prepared mimic enzyme is enhanced along with the increase of the salt concentration, so that the mimic enzyme can play a role in the fields of treating lignin, organic phenol pollution in seawater and the like in the future;
3) The activity of the prepared mimic enzyme under alkaline conditions can be kept at 50.2% of the original activity and at 90 ℃ at high temperature to be kept at 89.9%, so that the mimic enzyme can be used for removing phenolic organic pollutants in water bodies in the fields of printing and dyeing and the like.
Drawings
FIG. 1 is a scanning electron microscope image (b) of the self-assembled artificial enzyme BpA-Cu of example 2, which is an enlarged view of the image (a).
FIG. 2 is a graph showing the relative catalytic activity of the self-assembled artificial enzyme of example 7 and a natural laccase.
FIG. 3 is a graph showing the relative catalytic activity of the self-assembled artificial enzyme of example 8 in buffers containing different salt concentrations.
FIG. 4 is a graph showing the relative catalytic activity of the self-assembled artificial enzyme and laccase of example 9 after incubation for 30min at different temperatures.
FIG. 5 is a graph showing the relative catalytic activities of the self-assembled artificial enzyme and laccase of example 10 in buffers of different pH values.
FIG. 6 shows the catalytic activity of the nano-enzyme BpA-Cu of example 11 after multiple cycles.
FIG. 7 is a graph showing cytotoxicity of the self-assembled artificial enzyme BpA-Cu and the starting material of example 12.
FIG. 8 is a graph showing the relative catalytic activity of the self-assembled artificial enzymes BpA-Cu and laccase of example 13 on various phenolic contaminants.
Detailed Description
The method of the present invention is further illustrated by the following description in conjunction with the specific embodiments and the accompanying drawings, but the specific embodiments described herein are intended to be illustrative only and not limiting in any way.
The preparation method of the nano enzyme with laccase activity formed by self-assembling the unnatural amino acid and the transition metal ion through coordination action comprises the following steps:
1) Dissolving BpA with 10-50mM sodium hydroxide solution prepared from ultrapure water to obtain BpA alkaline aqueous solution with concentration of 10-50mM, and preparing CuCl with concentration of 10-20mM with ultrapure water 2 、ZnCl 2 Solution, placing the obtained two solutions inThe temperature is kept for 30-60min in a water bath kettle with the temperature of 40-80 ℃, then the heat-preserving transition metal salt solution is slowly added into BpA alkaline solution, the magnetic stirring is needed in the process to fully and uniformly mix the transition metal chloride solution until the transition metal chloride solution is fully added, and the ratio of BpA alkaline solution to transition metal chloride solution is needed to be controlled to be 1:1-1:10 in the process. After fully mixing, self-assembling in water bath at 40-80 ℃ for 0-60h to obtain the final self-assembled supermolecule aggregate suspension.
2) Centrifuging the suspension obtained after the reaction at high speed at 4-25deg.C at 8000-10000rpm for 10-20min, discarding supernatant to obtain precipitate, and washing the precipitate with ultrapure water for 3-5 times; placing the cleaned precipitate in a vacuum drying oven at 40-80deg.C for 24-72 hr to obtain self-assembled nanometer enzyme powder with laccase-like activity. The activity, stability and biocompatibility of the nano enzyme powder are measured.
Example 1 BpA and CuCl at 40 ℃ 2 The molar ratio of laccase-like active nano-enzyme to the laccase is 1:1, 1:5 and 1:10.
40mg of sodium hydroxide solid NaOH is dissolved in 100mL of ultrapure water to obtain a NaOH solution with the concentration of 10mM for standby, 59.264mg of benzophenone alanine BpA is dissolved in the NaOH solution to obtain 20mL of BpA alkaline solution with the concentration of 10 mM; 170.5mg of copper chloride dihydrate was dissolved in 100mL of ultrapure water to give CuCl at a concentration of 10mM 2 The solution is ready for use, and the dosage cylinder is used for measuring 20mL, 4 mL and 2mL of CuCl respectively 2 The solutions are respectively placed in a water bath kettle at 40 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated CuCl 2 Slowly adding the solution into BpA alkaline solution, magnetically stirring until all the solution is uniformly mixed, standing in a water bath kettle at 40 ℃ for 60 hours after the solution is uniformly mixed so as to meet the requirement of the self-assembly process, and obtaining BpA-Cu suspension.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 8000rpm for 20min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 40 ℃ for 72h to obtain the nano enzyme solid powder BpA-Cu synthesized under different conditions.
EXAMPLE 2 BpA and CuCl at 60 ℃ 2 The molar ratio of laccase-like active nano-enzyme is 1:1, 1:2 and 1:10.
40mg of sodium hydroxide solid (NaOH) was dissolved in 100mL of ultrapure water to obtain a 10mM NaOH solution for use, and 118.528mg of benzophenone alanine (BpA) was dissolved in the above NaOH solution to obtain 20mL of BpA alkaline solution having a concentration of 20 mM; 341mg copper chloride dihydrate (CuCl) 2 ·2H 2 O) was dissolved in 100mL of ultrapure water to give CuCl at a concentration of 20mM 2 The solution is ready for use, and the dosage cylinder is used for measuring 20, 10 and 2mL of CuCl 2 The solutions are respectively placed in a water bath kettle at 60 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated CuCl 2 Slowly adding the solution into BpA alkaline solution, magnetically stirring until all the solution is uniformly mixed, standing in a water bath kettle at 60 ℃ for 48 hours after the solution is uniformly mixed so as to meet the requirement of the self-assembly process, and obtaining BpA-Cu suspension.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 9000rpm for 18min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 60 ℃ for 48h to obtain nano enzyme solid powder BpA-Cu.
The morphology of the synthesized nano-enzyme is observed by using a scanning electron microscope, and the structure formed when the standing time is 0h is found to have no obvious structure, and as can be seen from fig. 1, the structure of the nano-enzyme is gradually complete with the extension of the self-assembly time.
Example 3 BpA and CuCl at 80 ℃ 2 The molar ratio of laccase-like active nano-enzyme is 1:1, 1:2 and 1:10.
40mg of sodium hydroxide solid (NaOH) was dissolved in 100mL of ultrapure water to give a 10mM NaOH solution for use, and 296.32mg of benzophenone alanine (BpA) was dissolved in the above NaOH solution to give a 50mM BpA base20mL of sexual solution; 852.5mg of copper chloride dihydrate was dissolved in 100mL of ultrapure water to give CuCl at a concentration of 50mM 2 The solution is ready for use, and the dosage cylinder is used for measuring 20, 4 and 2mL of CuCl 2 The solutions are respectively placed in a water bath kettle at 80 ℃ for incubation for 30min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated CuCl 2 Slowly adding the solution into BpA alkaline solution, magnetically stirring until all the solutions are uniformly mixed, and standing in a water bath kettle at 80 ℃ for 24 hours after the solutions are uniformly mixed so as to meet the requirement of the self-assembly process to obtain BpA-Cu.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 10000rpm for 20min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 80 ℃ for 24h to obtain the nano enzyme solid powder BpA-Cu synthesized under different conditions.
EXAMPLE 4 BpA and ZnCl at 40℃ 2 The molar ratio of laccase-like active nano-enzyme to the laccase is 1:1, 1:5 and 1:10.
40mg of sodium hydroxide solid NaOH is dissolved in 100mL of ultrapure water to obtain a NaOH solution with the concentration of 10mM for standby, 59.264mg of benzophenone alanine BpA is dissolved in the NaOH solution to obtain 20mL of BpA alkaline solution with the concentration of 10 mM; 136.315mg of zinc chloride was dissolved in 100mL of ultrapure water to give ZnCl having a concentration of 10mM 2 The solution is ready for use, and the dosage cylinder is used for respectively measuring 20mL, 4 mL and 2mL ZnCl 2 The solutions are respectively placed in a water bath kettle at 40 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated ZnCl 2 Slowly adding the solution into BpA alkaline solution, magnetically stirring until all the solution is uniformly mixed, standing in a water bath kettle at 40 ℃ for 60 hours after the solution is uniformly mixed so as to meet the requirement of the self-assembly process, and obtaining BpA-Zn suspension.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 8000rpm for 20min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 40 ℃ for 72h to obtain the nano enzyme solid powder BpA-Zn synthesized under different conditions.
Example 5 BpA and ZnCl at 60℃ 2 The molar ratio of laccase-like active nano enzyme to laccase is 1:1, 1:2 and 1:10
40mg of sodium hydroxide solid (NaOH) was dissolved in 100mL of ultrapure water to obtain a 10mM NaOH solution for use, and 118.528mg of benzophenone alanine (BpA) was dissolved in the above NaOH solution to obtain 20mL of BpA alkaline solution having a concentration of 20 mM; 272.63mg of zinc chloride ZnCl 2 Dissolved in 100mL of ultrapure water to give ZnCl having a concentration of 20mM 2 The solution is ready for use, and the dosage cylinder is used for measuring 20, 10 and 2mL mL of ZnCl 2 The solutions are respectively placed in a water bath kettle at 60 ℃ for incubation for 60min until the reactants are completely dissolved; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated ZnCl 2 Slowly adding the solution into BpA alkaline solution, magnetically stirring until all the solution is uniformly mixed, standing in a water bath kettle at 60 ℃ for 48 hours after the solution is uniformly mixed so as to meet the requirement of the self-assembly process, and obtaining BpA-Zn suspension.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 9000rpm for 18min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 60 ℃ for 48h to obtain nano enzyme solid powder BpA-Zn.
EXAMPLE 6 BpA and CuCl at 80 ℃ 2 The molar ratio of laccase-like active nano-enzyme to the laccase is 1:1, 1:5 and 1:10.
40mg of sodium hydroxide solid (NaOH) was dissolved in 100mL of ultrapure water to obtain a 10mM NaOH solution for use, and 296.32mg of benzophenone alanine (BpA) was dissolved in the above NaOH solution to obtain 20mL of BpA alkaline solution having a concentration of 50 mM; 681.575mg of zinc chloride was dissolved in 100mL of ultrapure water to give ZnCl having a concentration of 50mM 2 The solution is ready for use, and the dosage cylinder is used for measuring 20, 4 and 2mL ZnCl 2 The solutions are respectively placed in a water bath kettle at 80 ℃ for incubation for 30min until the reactants are completeDissolving; after the two reactants are completely dissolved in the solution and the temperature reaches a set value, the incubated ZnCl 2 Slowly adding the solution into BpA alkaline solution, magnetically stirring until all the solutions are uniformly mixed, and standing in a water bath kettle at 80 ℃ for 24 hours after the solutions are uniformly mixed so as to meet the requirement of the self-assembly process to obtain BpA-Zn.
Centrifuging the suspension obtained by the reaction in a low-temperature refrigerated centrifuge at 4 ℃ and 10000rpm for 15min, discarding the supernatant, namely the reactant which does not participate in the reaction, washing the precipitate with ultrapure water for 5 times to further remove the reactant solution, and drying the precipitate obtained by the last centrifugation in a vacuum drying oven at 80 ℃ for 24h to obtain the nano enzyme solid powder BpA-Zn synthesized under different conditions.
Example 7 laccase-like Activity catalytic Performance of nanoenzyme studies.
Weighing BpA-Cu and BpA-Zn of the invention, respectively dissolving in PB (10 mM, pH 7.0) buffer solution, and performing intense ultrasound for 20min to obtain a suspension with the concentration of 1mg/mL for later use; 163.0014mg of 2, 4-dichlorophenol (2, 4-DP) was weighed and dissolved in 100mL of absolute ethanol to give a 10mM2,4-DP solution for use; 203.24mg of 4-aminoantipyrine (4-AP) is weighed and dissolved in 100mL of absolute ethyl alcohol to obtain a 4-AP solution with the concentration of 10mM for later use; mu.L of 2,4-DP, 100. Mu.L of 4-AP, 100. Mu.L of pA-Cu or BpA-Zn, 700. Mu.L of PB buffer were mixed uniformly in a 1.5mL centrifuge tube (total reaction volume: 1 mL), reacted at room temperature for 1 hour, centrifuged at 10000rpm for 5 minutes to leave a supernatant, and the absorbance of the solution at 510nm (indoxyl aminoantipyrine dye) was monitored by using a microplate reader to determine the oxidation potential of 2,4-DP by using the molar extinction coefficient of indoxyl aminoantipyrine dye.
As can be seen from FIG. 2, bpA and BpA-Zn have a lower laccase-like activity, whereas BpA-Cu of the present invention can oxidize 2,4-DP and bind to 4-AP to darken the solution. The results show that the catalytic reaction rates are different when different concentrations of 2,4-DP are added, and the dynamic parameters of the catalyst of BpA-Cu are calculated according to the different reaction rates to be Km of 0.07mM and Vmax of 2.1X10 -5 mM·S -1 The lower Km value for BpA-Cu compared to other studies suggests a higher affinity of BpA-Cu for the substrate 2, 4-DP.
Example 8 study of the active salt tolerance of a nano-enzyme laccase.
To examine the catalytic performance of the BpA-Cu suspension of the invention after incubation in buffers with different ionic strengths, 100. Mu.L of a 1mg/mL BpA-Cu suspension was mixed with 700. Mu.L of 10mM PB (pH 7.0) buffer, incubated in 50, 100, 200, 300, 400, 500, 600mM NaCl for 30min, 100. Mu.L of 10mM2,4-DP and 100. Mu.L of 10mM 4-AP were added and mixed uniformly (total reaction volume: 1 mL), the supernatant was retained by centrifugation at 10000rpm for 5min after reaction at room temperature for 1h, and the absorbance at 510nm of the solution was monitored using a microplate reader to determine the oxidation capacity of 2,4-DP using the molar extinction coefficient of indophenol aminoantipyrine dye.
As can be seen from FIG. 3, the catalytic activity of BpA-Cu of the present invention increases with increasing salt ion concentration, and the activity thereof can reach 3.62 times of the NaCl-free condition in the presence of 600mM NaCl, whereas the activity of the native laccase decreases with increasing salt concentration. The nano-enzyme can be applied to the treatment of the seawater phenolic pollution field.
Example 9 catalytic performance of laccase-like Activity of nanoenzymes under high temperature conditions.
To examine the catalytic performance of BpA-Cu prepared in the invention after incubation at high temperature, 100. Mu.L of a 1mg/mL BpA-Cu suspension was mixed with 700. Mu.L of 10mM PB (pH 7.0) buffer, incubated at 0, 30, 40, 50, 60, 70, 80, 90℃for 30min, after the mixture was allowed to return to room temperature, 100. Mu.L of 10mM2,4-DP and 100. Mu.L of 10mM 4-AP were added and mixed uniformly (total reaction volume: 1 mL), the mixture was reacted at room temperature for 1 hour and centrifuged at 10000rpm for 5min to retain the supernatant, the absorbance at 510nm of the solution was monitored by using a microplate reader, and the oxidation capacity of 2,4-DP was determined by using the molar extinction coefficient of indophenol aminoantipyrine dye, so that the catalytic activity after incubation at 0℃for 30min was 100%, and the relative activity under other conditions was calculated.
From FIG. 4 it can be seen that the temperature change has little effect on the catalytic activity of BpA-Cu, and the activity can be maintained to 89.98% after incubation for 30min at 90℃whereas the catalytic activity of laccase decreases with increasing temperature, decreasing to 36.85% of the original catalytic activity when the incubation temperature is 50℃and substantially completely losing its catalytic activity when the incubation temperature is 80 ℃. Compared with laccase, bpA-Cu still has good catalytic performance in a higher temperature range (60-90 ℃). Therefore, the BpA-Cu catalyst shows higher laccase-like catalytic capability at higher reaction temperature, and can be used for removing phenolic organic pollutants in water bodies in the fields of printing and dyeing and the like.
Example 10 catalytic performance of laccase-like activity of nanoenzymes under extreme pH conditions.
To examine the catalytic performance of BpA-Cu prepared according to the present invention under different pH conditions, 100. Mu.L of a 1mg/mL BpA-Cu suspension was incubated with 700. Mu.L of buffers of pH 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 for 30min, 100. Mu.L of 2,4-DP, 100. Mu.L of 4-AP were mixed uniformly (total reaction volume: 1 mL), and after 1h of reaction at room temperature, the supernatant was retained by centrifugation at 10000rpm for 5min, and the absorbance of the solution at 510nm was monitored using an enzyme-labeled instrument to determine the oxidation ability of 2,4-DP using the molar extinction coefficient of indophenol aminoantipyrine dye. Wherein the conditions of pH 3.0, 4.0 and 5.0 are formulated with 50mM glycine-hydrochloric acid buffer, the conditions of pH 6.0, 7.0 and 8.0 are formulated with 10mM PBS buffer, and the conditions of pH 9.0 and 10.0 are formulated with 50mM glycine-sodium hydroxide buffer.
It can be seen from FIG. 5 that BpA-Cu exhibits a strong catalytic activity in the pH range of 5.0 to 8.0. In addition, laccase does not show catalytic activity at pH 3.0-4.0, which shows that natural enzyme is easy to inactivate at acid extreme pH, while BpA-Cu can keep its highest activity 39.46% and 54.37% at the pH, laccase does not show catalytic activity at pH 9.0-10.0, while BpA-Cu still has a certain activity at the pH, thereby proving that natural laccase is easy to inactivate at extreme pH, while nano-enzyme BpA-Cu of the invention still has better catalytic ability at severe pH, so that the nano-enzyme can be applied to removing phenolic organic pollutants in water body in the fields of printing and dyeing and the like.
Example 11 reusability of laccase-like Activity of nanoenzymes.
1mg of BpA-Cu of the invention is weighed and placed in a 1.5mL centrifuge tube, 800 mu L of 10mM PB (pH 7.0) buffer solution, 100 mu L of 10mM2,4-DP and 100 mu L of 10mM 4-AP are added, the mixture is uniformly mixed and then reacted for 1h at room temperature, the mixture is centrifuged at 10000rpm for 5min at 4 ℃ after the reaction is finished, the supernatant is taken to measure the light absorption value at 510nm, the precipitate is washed 3 times by ultrapure water, and the reactants are added for repeated circulation for 10 times, so as to calculate the relative activity of BpA-Cu.
Natural laccase, by nature, is readily soluble in water and therefore cannot be recycled after a single use, which has limited industrial applications. BpA-Cu can be recovered and reused by centrifugation, and as can be seen from FIG. 6, bpA-Cu still maintains 83.96% activity after 10 cycles. This indicates that BpA-Cu has good recycling properties. The activity loss is caused by the fact that the cleaning process inevitably causes the mass loss of the catalyst, thereby causing the activity to be reduced, and the recycling advantage of BpA-Cu can be utilized to treat the environmental sewage.
Example 12 cytotoxicity of laccase-like active nanoenzymes.
Colorimetric method for artificial enzyme BpA-Cu, bpA, cu by thiazole blue 2+ Is characterized by cytotoxicity. Sterilized 96-well plates were taken and 80. Mu.L of mouse microglial BV-2 (8X 10) was added to each well 3 And culturing for 24h to adhere the cells. Then 20. Mu.L of samples to be tested with different concentrations are added and the culture is continued for 24 hours. A5.5 mg/mL MTT solution was prepared with a sterilized PBS solution (containing 10mM PB and 10mM NaCl, pH 7.0). mu.L of MTT solution was added to each well, and the culture was continued for 3-4 hours. The cell culture plates were then centrifuged at 1500rpm for 10min and the supernatant discarded. 100 μl of dimethyl sulfoxide (DMSO) was added to each well, and the 96-well plate was placed in an air shaker at 37 ℃ and 150rpm and shaken until the blue-violet formazan particles in the plate were completely dissolved. Finally, absorbance at 570nm was measured for each sample using a microplate reader. The cell group added with PBS buffer was used as a control group, and the sample containing only the medium and no cells was used as a blank group. Cell activity was calculated using equation 1.
As can be seen from FIG. 7, the in vitro cytotoxicity of artificial enzyme BpA-Cu was evaluated by MTT assaySex, studies have found that various catalytic materials show enhanced cytotoxicity with increasing material concentration after 24h incubation with BV-2 cells. From the study, it was found that the toxicity of BpA-Cu to cells was gradually increased with increasing concentration, but when the concentration in the mixed solution reached 800. Mu.g/mL, the cell activity was maintained at 83.84%. After BpA addition, toxicity increased gradually with increasing concentration, and when the concentration reached 100 μg/mL, cell activity remained 58.58% of the control group, which was more toxic than BpA-Cu cytotoxicity BpA. And when Cu of 12.5. Mu.g/mL was added 2+ The cell activity was reduced to 39.28% of the original one, indicating Cu 2+ Has stronger toxicity to cells. Therefore, we will have a slightly toxic BpA and a more toxic Cu 2+ The toxic phenol pollutants in the environmental sewage are oxidized into non-toxic oxides through coordination interaction to form the environment-friendly and oxidation-effect environment-friendly sewage.
Example 13 catalytic ability of laccase-like active nanoenzymes to various phenolic contaminants.
To investigate the catalytic ability of BpA-Cu and natural laccase on various phenolic substrates, the content of free radicals of the generated product was calculated by using the beer-lambert law and molar extinction coefficient to obtain the catalytic ability. The solid powder BpA-Cu obtained was ultrasonically dispersed in 10mM phosphate buffer (pH 7.0) to give BpA-Cu suspension at a concentration of 1 mg/mL. mu.L of BpA-Cu suspension (1 mg/mL) was added to 700. Mu.L of phosphate buffer (10 mM, pH 7.0), and 100. Mu.L of hydroquinone, 2-aminophenol, 2,4, 6-trichlorophenol, catechol, phenol, 2-naphthol, 2-nitrophenol and 2, 4-dichlorophenol in ethanol (10 mM) and 100. Mu.L of 4-AP (10 mM) aqueous solution were further added and mixed uniformly to give a total reaction volume of 1mL. The reaction was carried out at 25℃for 1 hour, and then centrifuged at 12000rpm for 5 minutes, bpA-Cu was separated and the supernatant after the reaction was collected, and the absorbance of the solution after the reaction at 510nm was measured by using a microplate reader. Its molar extinction coefficient is 13.6mM -1 ·cm -1 (2, 4-dichlorophenol), 9.2mM -1 ·cm -1 (hydroquinone), 7.8mM -1 ·cm -1 (phenol). Other relative catalytic capacities were calculated as 100% of the catalytic capacity of the mimic enzyme BpA-Cu to catalyze 2, 4-dichlorophenol. Living 2,4-DP with BpA-CuThe activity was 100%, and the catalytic activities of BpA-Cu and laccase on various substrates were calculated as standardized.
As can be seen from FIG. 8, the relative catalytic capacity of the mimic enzyme BpA-Cu for 7 phenolic contaminants was higher than that of the native laccase, except for 2, 4-DP. In particular, the oxidation capacities of the para-catechol, the phenol and the 2-nitrophenol can reach 97.1 percent, 70.1 percent and 81.2 percent of the catalytic capacity by taking the 2, 4-dichlorophenol as a substrate respectively. However, the catalytic capacity of natural laccase on these substrates is only 23.3%, 0.1% and 1.7%. Although the relative catalytic capacities of the mimic enzyme and laccase are lower when hydroquinone, 2,4, 6-trichlorophenol are used as substrates, the activity of the mimic enzyme is still higher than that of laccase. Thus, the mimic enzyme BpA-Cu has oxidizing capability on various environmental phenolic pollutants, which indicates that the mimic enzyme has substrate universality. It is possible that the natural laccase catalytic center has a steric hindrance effect besides the substrate binding site, so that the laccase only has an oxidation effect on the adaptive substrate, and the mimic enzyme BpA-Cu provides an effective catalytic site for various substrate molecules due to the simple supramolecular structure of the mimic enzyme BpA-Cu, thereby having the universality of the substrate.
The technical scheme disclosed and proposed by the invention can be realized by a person skilled in the art by appropriately changing the condition route and other links in consideration of the content of the present invention, although the method and the preparation technology of the invention have been described by the preferred embodiment examples, the related person can obviously modify or recombine the method and the technical route described herein to realize the final preparation technology without departing from the content, spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be included within the spirit, scope and content of the invention.
Claims (3)
1. An artificial enzyme with laccase-like activity is characterized in that a supramolecular material with laccase-like activity is formed by coordination combination of an amino group and a carboxyl group of benzophenone alanine and chloride salt composed of transition metal, wherein BpA is benzophenone alanine, and M represents transition metal copper or zinc.
2. The method for preparing the artificial enzyme with laccase-like activity as claimed in claim 1, comprising the following steps:
1) Adding chloride aqueous solution of transition metal copper or zinc into alkaline aqueous solution of benzophenone alanine, stirring to uniformly mix the two to complete coordination chelation reaction, standing in a water bath kettle at 30-80 ℃ for 0-60h which is not 0, and completing self-assembly;
2) Centrifuging at 8000-10000rpm for 10-20min, discarding supernatant, collecting precipitate, cleaning with ultrapure water, and vacuum drying the precipitate to obtain BpA-Cu and BpA-Zn solids;
the alkaline solution of the benzophenone alanine in the step 1) is prepared by dissolving 5-50mM of benzophenone alanine in 10mM sodium hydroxide; preparing NaOH and transition metal salt solution, wherein the water specification of the NaOH and transition metal salt solution is at least ultrapure water; the concentration of the transition metal chloride salt solution is 10-20 mM;
the step 1) is that the benzophenone alanine alkaline aqueous solution and the transition metal chloride salt solution are incubated for 30-60min in a water bath kettle at 40-80 ℃ before being mixed; the process of adding the transition metal chloride solution into the alkaline aqueous solution of the benzophenone alanine is required to be carried out under the water bath condition of 40-80 ℃;
the molar ratio of the transition metal chloride salt to the benzophenone alanine in the step 1) is 1:1-1:10;
the temperature setting of the vacuum drying in the step 2) is 40-80 ℃, and the drying time is 24-72h until the precipitate containing a small amount of water is dried to powder BpA-Cu and BpA-Zn.
3. The use of an artificial enzyme having laccase-like activity as claimed in claim 1 for mimicking laccase activity to address phenolic contaminants, wherein the phenolic contaminants are hydroquinone, 2-aminophenol, 2,4, 6-trichlorophenol, catechol, phenol, 2-naphthol, 2-nitrophenol and 2, 4-dichlorophenol.
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