CN116814589A - Thermokalite-stable ester bond hydrolase and application of mixed enzyme preparation containing same in degradation of PET (polyethylene terephthalate) products - Google Patents
Thermokalite-stable ester bond hydrolase and application of mixed enzyme preparation containing same in degradation of PET (polyethylene terephthalate) products Download PDFInfo
- Publication number
- CN116814589A CN116814589A CN202310716177.0A CN202310716177A CN116814589A CN 116814589 A CN116814589 A CN 116814589A CN 202310716177 A CN202310716177 A CN 202310716177A CN 116814589 A CN116814589 A CN 116814589A
- Authority
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- China
- Prior art keywords
- enzyme
- pet
- ester bond
- bond hydrolase
- stable ester
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 168
- 229920000139 polyethylene terephthalate Polymers 0.000 title claims abstract description 168
- 239000005020 polyethylene terephthalate Substances 0.000 title claims abstract description 168
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 166
- 238000002360 preparation method Methods 0.000 title claims abstract description 66
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 41
- -1 polyethylene terephthalate Polymers 0.000 title claims abstract description 31
- 230000015556 catabolic process Effects 0.000 title abstract description 38
- 108090000604 Hydrolases Proteins 0.000 claims abstract description 114
- 230000000593 degrading effect Effects 0.000 claims abstract description 54
- 108010005400 cutinase Proteins 0.000 claims abstract description 42
- QPKOBORKPHRBPS-UHFFFAOYSA-N bis(2-hydroxyethyl) terephthalate Chemical compound OCCOC(=O)C1=CC=C(C(=O)OCCO)C=C1 QPKOBORKPHRBPS-UHFFFAOYSA-N 0.000 claims abstract description 40
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 229920003023 plastic Polymers 0.000 claims abstract description 18
- 239000004033 plastic Substances 0.000 claims abstract description 18
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 7
- 239000000758 substrate Substances 0.000 claims description 44
- 238000006243 chemical reaction Methods 0.000 claims description 42
- 229920002799 BoPET Polymers 0.000 claims description 32
- 102000004169 proteins and genes Human genes 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 18
- 239000000543 intermediate Substances 0.000 claims description 16
- 238000009472 formulation Methods 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 14
- JFFQXJXLNZADOF-UHFFFAOYSA-N 4-ethoxycarbonylbenzenecarboperoxoic acid Chemical compound CCOC(=O)C1=CC=C(C(=O)OO)C=C1 JFFQXJXLNZADOF-UHFFFAOYSA-N 0.000 claims description 5
- XRBXGZZMKCBTFP-UHFFFAOYSA-N 4-(2,2-dihydroxyethoxycarbonyl)benzoic acid Chemical compound OC(O)COC(=O)C1=CC=C(C(O)=O)C=C1 XRBXGZZMKCBTFP-UHFFFAOYSA-N 0.000 claims description 3
- 239000003513 alkali Substances 0.000 abstract description 15
- BCBHDSLDGBIFIX-UHFFFAOYSA-M 4-[(2-hydroxyethoxy)carbonyl]benzoate Chemical compound OCCOC(=O)C1=CC=C(C([O-])=O)C=C1 BCBHDSLDGBIFIX-UHFFFAOYSA-M 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 142
- 102000004157 Hydrolases Human genes 0.000 description 94
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 70
- 230000000694 effects Effects 0.000 description 56
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- 108090000371 Esterases Proteins 0.000 description 25
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- 239000000243 solution Substances 0.000 description 23
- 235000013361 beverage Nutrition 0.000 description 22
- BCBHDSLDGBIFIX-UHFFFAOYSA-N 4-[(2-hydroxyethoxy)carbonyl]benzoic acid Chemical compound OCCOC(=O)C1=CC=C(C(O)=O)C=C1 BCBHDSLDGBIFIX-UHFFFAOYSA-N 0.000 description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 13
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 238000011534 incubation Methods 0.000 description 11
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 10
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- 238000001514 detection method Methods 0.000 description 8
- 102000004882 Lipase Human genes 0.000 description 7
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- 230000003301 hydrolyzing effect Effects 0.000 description 7
- 235000019421 lipase Nutrition 0.000 description 7
- 239000000178 monomer Substances 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
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- 229910021645 metal ion Inorganic materials 0.000 description 4
- 239000004094 surface-active agent Substances 0.000 description 4
- YBADLXQNJCMBKR-UHFFFAOYSA-M (4-nitrophenyl)acetate Chemical compound [O-]C(=O)CC1=CC=C([N+]([O-])=O)C=C1 YBADLXQNJCMBKR-UHFFFAOYSA-M 0.000 description 3
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229920004890 Triton X-100 Polymers 0.000 description 3
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- 229920000728 polyester Polymers 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- OLRXUEYZKCCEKK-UHFFFAOYSA-N (4-nitrophenyl) hexanoate Chemical compound CCCCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 OLRXUEYZKCCEKK-UHFFFAOYSA-N 0.000 description 2
- GGIDEJQGAZSTES-UHFFFAOYSA-N (4-nitrophenyl) octanoate Chemical compound CCCCCCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 GGIDEJQGAZSTES-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 108010051152 Carboxylesterase Proteins 0.000 description 2
- 102000013392 Carboxylesterase Human genes 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 240000008415 Lactuca sativa Species 0.000 description 2
- 241001661345 Moesziomyces antarcticus Species 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
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- 238000004519 manufacturing process Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 125000000636 p-nitrophenyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1*)[N+]([O-])=O 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
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- 239000008223 sterile water Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- OTLNPYWUJOZPPA-UHFFFAOYSA-M 4-nitrobenzoate Chemical compound [O-]C(=O)C1=CC=C([N+]([O-])=O)C=C1 OTLNPYWUJOZPPA-UHFFFAOYSA-M 0.000 description 1
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- 102000004308 Carboxylic Ester Hydrolases Human genes 0.000 description 1
- 108090000863 Carboxylic Ester Hydrolases Proteins 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241001480714 Humicola insolens Species 0.000 description 1
- 241001509283 Ideonella Species 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
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- 241000203780 Thermobifida fusca Species 0.000 description 1
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- 210000004899 c-terminal region Anatomy 0.000 description 1
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- 229940112822 chewing gum Drugs 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
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- 238000010353 genetic engineering Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-M hexanoate Chemical compound CCCCCC([O-])=O FUZZWVXGSFPDMH-UHFFFAOYSA-M 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
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- 239000003112 inhibitor Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
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- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 229940070765 laurate Drugs 0.000 description 1
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- 229910052749 magnesium Inorganic materials 0.000 description 1
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- 239000002609 medium Substances 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- WWZKQHOCKIZLMA-UHFFFAOYSA-M octanoate Chemical compound CCCCCCCC([O-])=O WWZKQHOCKIZLMA-UHFFFAOYSA-M 0.000 description 1
- DVDUMIQZEUTAGK-UHFFFAOYSA-N p-nitrophenyl butyrate Chemical compound CCCC(=O)OC1=CC=C([N+]([O-])=O)C=C1 DVDUMIQZEUTAGK-UHFFFAOYSA-N 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 235000012045 salad Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- OESFSXYRSCBAQJ-UHFFFAOYSA-M sodium;3-carboxy-3,5-dihydroxy-5-oxopentanoate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.OC(=O)CC(O)(C(O)=O)CC([O-])=O OESFSXYRSCBAQJ-UHFFFAOYSA-M 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000004901 spalling Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000012289 standard assay Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- KKEYFWRCBNTPAC-UHFFFAOYSA-L terephthalate(2-) Chemical compound [O-]C(=O)C1=CC=C(C([O-])=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-L 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000701 toxic element Toxicity 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B3/00—Destroying solid waste or transforming solid waste into something useful or harmless
- B09B3/60—Biochemical treatment, e.g. by using enzymes
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J11/00—Recovery or working-up of waste materials
- C08J11/04—Recovery or working-up of waste materials of polymers
- C08J11/10—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation
- C08J11/105—Recovery or working-up of waste materials of polymers by chemically breaking down the molecular chains of polymers or breaking of crosslinks, e.g. devulcanisation by treatment with enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01001—Carboxylesterase (3.1.1.1)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09B—DISPOSAL OF SOLID WASTE NOT OTHERWISE PROVIDED FOR
- B09B2101/00—Type of solid waste
- B09B2101/75—Plastic waste
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
- C08J2367/02—Polyesters derived from dicarboxylic acids and dihydroxy compounds
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Environmental & Geological Engineering (AREA)
- Sustainable Development (AREA)
- Polymers & Plastics (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a thermokalite-stable ester bond hydrolase and application of a mixed enzyme preparation containing the enzyme in degrading PET plastics, wherein the amino acid sequence of the thermokalite-stable ester bond hydrolase is shown as SEQ ID No.1, and the coding gene sequence of the thermokalite-stable ester bond hydrolase is shown as SEQ ID No.2. The thermal alkali stability ester bond hydrolase can resist higher temperature and pH, is firstly applied to the degradation of polyethylene terephthalate and polyethylene terephthalate intermediate BHET, and further forms a synthase mixed preparation with PET degrading enzyme cutinase or a mutant thereof, so that the high-efficiency degradation of polyethylene terephthalate and polyethylene terephthalate intermediate under high temperature condition can be realized, the degradation capability is obviously superior to that of the single enzyme treatment, the complete degradation of PET products and PET intermediate BHET, MHET and the like can be realized, pure TPA is produced, and the thermal alkali stability ester bond hydrolase has popularization and application value in the field of plastic degradation.
Description
Technical Field
The invention relates to the technical field of biology, and relates to a thermokalite-stable ester bond hydrolase and application of a mixed enzyme preparation containing the enzyme in the aspect of degrading polyethylene glycol terephthalate.
Background
Plastic waste presents an ecological challenge. Plastic pollution spreads worldwide from deserts to farms, from mountain tops to deep ocean depths, from tropical landfills to arctic snowlands. Reports on plastic waste in marine environments can be traced back to more than half a century ago. Over 60 years, plastic debris has accumulated in the ocean. Eriksen et al evaluate that there are over 5 trillion pieces of plastic in the world's ocean, indicating that there is an urgent need to find innovative solutions to this problem.
The standard method of disposing of waste plastics is landfill and incineration, but toxic elements of the plastics themselves, such as chlorine and some additives, are released during landfill to contaminate the soil and water source. Toxic and harmful gases and particles generated by incineration pollute the atmosphere, generate a large amount of carbon dioxide, and exacerbate global warming. Since enzymes can act on certain composite materials, such as polyesters, biodegradation technology has become a cleaner way of recycling plastics. The biodegradation technique has many advantages such as green pollution-free, simple processing conditions, better selectivity, etc. It has been reported that a number of enzymes can act in the depolymerization of polyethylene terephthalate, some of which are lipases, carboxylesterases and cutinases. Recently, yoshida et al isolated a bacterium Ideonella sakaiensis-F6 from plastic waste that produced a specific enzyme, PET enzyme, that promoted hydrolysis of PET. Sulaiman et al successfully screened leaf branch compost cutinase (LCC) through a metagenomic study based on functional screening lipolytic enzyme encoding genes, and the LCC has good thermal stability and good degradation capability on PET. In addition, it has recently been reported that the PET degradation enzymes cutinase LCC and PET enzyme engineered by genetic engineering are used to completely hydrolyze PET, but the degradation process is still time-consuming. Inhibition of product by the accumulation of monohydroxyethyl terephthalate (MHET) in the reaction medium has proven to be one of the fundamental limitations of polyester hydrolase efficiency. Previous studies have shown that efficiency is improved when the biocatalytic PET membrane hydrolysis process is carried out in an ultrafiltration membrane reaction vessel to continuously remove MHET. However, this requires a large amount of medium buffer to maintain a sufficient dilution ratio in the membrane reactor.
In recent years, the ability of many lipases, esterases and bacterial species to convert bis-hydroxyethyl terephthalate (BHET) to MHET (hydroxyethyl terephthalate) and terephthalic acid (TPA) has been widely studied because they are very important in depolymerizing PET to monomers for reuse. Yellow and et al screen a strain of acinetobacter lettuce, which can effectively degrade PET plastic and polyethylene plastic (CN 114958645A) and a microbial inoculum containing the strain. Yan Zhengfei et al, by mixing the Kehnella JQ-3 with the PET degrading enzyme cutinase ICCG via a bacterial enzyme complex, were able to completely degrade 2mg of PET (CN 115044510A) within 144 hours. Barth et al reported that carboxylesterase TfCa from Thermobifida fusca KW3 was effective in catalyzing the hydrolysis of intermediate BHET and MHET to monomeric TPA in the PET hydrolysis process. Carniel et al found that lipase CalB from Candida Antarctica was able to completely hydrolyze 31.5mM BHET to monomeric TPA in a short period of time. While the addition of CalB during the hydrolysis of PET at Humicola Insolens Cutinase (HiC) increased the yield of TPA by a factor of 7.7. Mrigwani et al characterized the thermophilic carboxylesterase TTCE from thermophilic bacteria and found that TTCE was more catalytic to BHET and MHET. In addition, TTCE was added during the hydrolysis of PET by the enzyme cutinase LCC, which increased TPA production by 30-100%.
Lequan et al isolated and identified a mesophilic esterase EseB from E.coli HY1, which has a good activity on BHET and has a moderate catalytic activity in reported BHET hydrolases. Ion et al screen a lipase DMC from Aspergillus niger Aspergillus niger, which can efficiently catalyze and hydrolyze BHET, and can assist PET degrading enzyme cutinase to degrade better to recover PET. Recently, there have been studies reporting a comparison of two esterases from the group consisting of licheniformis thermophilic, bacillus subtilis, aspergillus chewing gum, candida antarctica and four lipases, as they are more prone to hydrolyse PET plastic subunits. However, few reports have been made on the activity of esterases or lipases from thermophilic bacteria, and it is still highly desirable to find better esterases or lipases than currently tested and to be able to degrade BHET well, together with the PET degrading enzyme cutinase, to degrade PET.
Disclosure of Invention
The invention aims to: aiming at the problems existing in the prior art, the invention provides a thermokalite-stable ester bond hydrolase, which is synthesized and characterized by the invention, and the obtained thermokalite-stable ester bond hydrolase has good thermostability, pH stability, alkali resistance and high catalytic efficiency on artificial substrates p-nitrobenzoate (pN-C4) and BHET. In addition, the enzyme is combined with PET degrading enzyme cutinase LCC, bhrPETase and mutants thereof to degrade PET, so that the degradation rate of PET and the purity and yield of the final product terephthalic acid (TPA) are remarkably improved.
The invention also provides a mixed enzyme preparation containing the thermokalite-stable ester bond hydrolase and application thereof.
The technical scheme is as follows: in order to achieve the above purpose, the amino acid sequence of the thermobase stable ester bond hydrolase is shown as SEQ ID NO. 1.
Wherein, the coding gene sequence of the thermal alkali stability ester bond hydrolase is shown as SEQ ID NO.2.
The mixed enzyme preparation comprises the enzyme such as thermokalite-stable ester bond hydrolase and PET degrading enzyme cutinase as defined in claim 1; the PET degrading enzyme cutinase includes PET degrading enzyme cutinase LCC or BhrPETase or mutants thereof; the amino acid sequences of the PET degrading enzyme cutinase LCC, bhrPETase or the mutant thereof are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
Wherein, the ratio of the enzyme protein amount of thermokali stable ester bond hydrolase to PET degrading enzyme cutinase in the mixed enzyme preparation is 1: 2-2: 1.
the invention relates to an application of thermokalite-stable ester bond hydrolase or a mixed enzyme preparation in degrading polyethylene terephthalate products and intermediates thereof.
The application is that the thermal alkali-stability ester bond hydrolase or the mixed enzyme preparation is added into a reaction system containing polyethylene terephthalate, any one or more of intermediate dihydroxyethyl terephthalate and hydroxyethyl terephthalate of the polyethylene terephthalate to carry out degradation reaction.
Wherein the polyethylene terephthalate-containing product comprises an amorphous PET film or a semi-crystalline PET powder or a plastic product.
Further, the polyethylene terephthalate-containing product is one of mineral water bottles, beverage bottles, fruit and salad plastic packages and containers and appearance plastics of various household appliances.
Wherein the amount of the thermobase-stable ester bond hydrolase in the reaction system is not less than 1.0X10 -3 mg protein/mg substrate; or the amount of the thermobase-stable ester bond hydrolase in the reaction system is not less than 1.0X10 -3 mg protein/mg substrate, and the amount of PET degrading enzyme cutinase is not less than 1.0X10 -3 mg protein/mg substrate.
Wherein the reaction temperature is 55-85 ℃, the pH is 5.0-9.0, the addition amount of each enzyme is 10-20 mug/mL, the concentration of substrate PET is 5-80 mg/mL, and the ratio of the enzyme protein mass of thermokalite-stable ester bond hydrolase to the enzyme protein mass of PET degrading enzyme cutinase is 1: 2-2: 1, the conversion time is 0.5-24 h.
Preferably, the thermal base-stable ester bond hydrolase degradation polyethylene terephthalate process comprises the steps of:
(1) Preparing an enzyme solution of a thermokalite-stable ester bond hydrolase and a PET degrading enzyme cutinase;
(2) The substrate PET was washed with 5g/L Triton X100 solution at 37℃and 130rpm for 10min, then 100mM Na 2 CO 3 The solution was washed for 10min and finally double distilled water (ddH 2 O) washing for 10min;
(3) Adding the enzyme solution prepared in the step (1) and 100mM buffer solution into the treated substrate PET for hydrolysis;
wherein the enzyme solution in the step (1) is a thermokalite-stable ester bond hydrolase or PET degrading enzyme cutinase or a mixed enzyme preparation of thermokalite-stable ester bond hydrolase and PET degrading enzyme cutinase.
Wherein the substrate PET of step (2) comprises an amorphous PET film and a semi-crystalline PET powder.
Wherein the temperature of the hydrolysis in the step (3) is 70 ℃, and the speed of the shaking table is 220rpm.
Wherein the amount of the enzyme protein added in the step (3) is 10 mug/mL.
Preferably, the amount of the substrate PET in the step (3) is 5-80 mg/mL.
Preferably, the pH of the buffer solution for the hydrolysis in the step (3) is 5.0-9.0, and the hydrolysis time is 0.5-24 h.
Preferably, the ratio of the amount of enzyme protein of the thermokali stable ester bond hydrolase to the amount of enzyme protein of the PET degrading enzyme cutinase in the mixed enzyme preparation of step (1) is 1: 2-2: 1.
the enzyme preparation product for degrading the polyethylene terephthalate product and the intermediate thereof can be used for degrading any one or more of polyethylene terephthalate, and the intermediate of polyethylene terephthalate, namely dihydroxyethyl terephthalate and hydroxyethyl terephthalate; the enzyme preparation product contains the thermokalite-stable ester bond hydrolase or the mixed enzyme preparation.
The amino acid sequence of the thermokalite-stable ester bond hydrolase provided by the invention is shown as SEQ ID No. 1; the coding gene sequence is shown as SEQ ID No.2. The engineering bacterium of the escherichia coli constructed by utilizing the coding gene sequence can efficiently express the ester bond hydrolase with the thermal alkali stability. The thermokalite-stable ester bond hydrolase can tolerate higher temperature and pH, and the optimal hydrolysis conditions are pH 8.0 and 80 ℃ to hydrolyze K of p-nitrobenzoic acid ester m ，k cat And k cat /K m The values were 0.42.+ -. 0.01mM, 254.42.+ -. 6.37s, respectively -1 And 610.80 + -20.37 s -1 mM -1 The method comprises the steps of carrying out a first treatment on the surface of the The thermal alkali-stability ester bond hydrolase is applied to the degradation of the polyethylene terephthalate and the polyethylene terephthalate intermediate for the first time; specific activity, K of hydrolyzed polyethylene terephthalate intermediate BHET m ，k cat And k cat /K m The values were 89.87U/. Mu.mol, 0.42.+ -. 0.01mM, 254.42.+ -. 6.37s, respectively -1 And 610.80 + -20.37 s -1 mM -1 The method comprises the steps of carrying out a first treatment on the surface of the And further, the thermokaline stable ester bond hydrolase, the PET degrading enzyme cutinase LCC, bhrPETase and the mutant form a high-temperature double-enzyme mixed preparation, so that the polyethylene terephthalate and the polyethylene terephthalate intermediate can be efficiently degraded under the high-temperature condition, the PET degrading capability of the double-enzyme mixed preparation is far better than that of the single use of the thermokaline stable ester bond hydrolase or the PET degrading enzyme cutinase for degrading PET, and the yield of pure terephthalic acid monomer TPA generated by degrading the polyethylene terephthalate is improved by 100 percent compared with the single treatment of the PET degrading enzyme cutinase in 24 hours at the pH of 7.0 and 70 ℃, and the complete degradation of PET products, PET intermediate BHET, MHET and the like and the pure TPA can be realized. The thermokalite-stable ester bond hydrolase and the mixed preparation containing the enzyme can be effectively applied to biological treatment and monomer recycling of polyester plastics. The invention has simple treatment process and low treatment cost, and has better popularization and application values in the technical field of plastic degradation.
The invention uses the thermal alkali-stability ester bond hydrolase or the thermal alkali-stability ester bond hydrolase and the PET degrading enzyme cutinase to degrade PET together, so that the degradation rate of PET is improved, and the purity and the yield of the final product terephthalic acid (TPA) are improved.
The invention provides a brand-new thermokalite-stable ester bond hydrolase for expression purification and characterization of enzymatic properties, and discovers that the enzyme can efficiently degrade an intermediate product of PET hydrolysis, namely, bis (2-hydroxyethyl) terephthalate (BHET). The thermokalite-stable ester bond hydrolase and the mixed preparation containing the enzyme can effectively degrade PET products at high temperature (70 ℃).
The beneficial effects are that: compared with the prior art, the invention has the following advantages:
1. the invention provides a thermokali-stable ester bond hydrolase for degrading PET and intermediate BHET and MHET thereof, which has good thermostability, pH stability, alkali resistance and high catalytic efficiency for artificial substrates p-nitrobutyrate (pN-C4) and PET degradation monomer BHET, and is applied to degradation of PET intermediate for the first time.
2. The invention combines the thermal alkali-stability ester bond hydrolase with PET degrading enzyme cutinase LCC, bhrPETase and the mutant thereof to form a high-temperature mixed enzyme preparation, can realize the efficient degradation of PET and intermediates thereof under the high-temperature condition, has the degradation capability obviously superior to that of the PET and the intermediates thereof treated by single enzyme, and the mixed enzyme preparation shows excellent synergistic degradation effect, thereby improving the yield of directly recyclable monomer terephthalic acid (TPA) by 100 percent in the degradation of PET.
(3) The invention utilizes brand-new thermokalite-stable ester bond hydrolase or a mixed enzyme preparation thereof to degrade PET, has simple treatment process, low treatment cost and environment protection, and has extremely high application prospect in the aspect of degrading PET.
Drawings
FIG. 1 shows SDS-PAGE analysis of thermobase stable ester bond hydrolase. Wherein M: molecular mass standard; 1: cell crude extract of thermokali-stable ester bond hydrolase; 2: thermal base-stable ester bond hydrolase after nickel affinity chromatography;
FIG. 2 is the effect of pH and temperature on thermobase stable ester bond hydrolase, a: an optimum pH value; b: an optimum temperature; c: pH stability; d: thermal stability; e: alkali resistance;
FIG. 3 is substrate specificity of a thermostable ester bond hydrolase;
FIG. 4 shows the hydrolysis of various initial concentrations of BHET by a thermostable ester bond hydrolase, a: BHET concentration; b: concentration of MHET; c: concentration of TPA; d: conversion of BHET;
FIG. 5 is a graph showing the 24-hour time profile of thermokalite-stable ester bond hydrolase degradation of an amorphous PET film;
FIG. 6 is a comparison of the effects of thermobase stable ester bond hydrolase, LCC and mixed enzyme formulations to degrade amorphous PET films;
FIG. 7 is a comparison of the effect of a thermobase stable ester bond hydrolase, LCC and mixed enzyme formulation to degrade semi-crystalline PET powder;
FIG. 8 shows that the addition amount of the substrate of the amorphous PET film degraded by the mixed enzyme preparation is optimized, a: LCCs treat substrates alone; b: treating the substrate with a mixed enzyme preparation (thermobase stable ester bond hydrolase+lcc);
fig. 9 is a pH optimization of mixed enzyme formulation degradation amorphous PET film, a: LCCs treat substrates alone; b: treating the substrate with a mixed enzyme preparation (thermobase stable ester bond hydrolase+lcc);
FIG. 10 is an enzyme proportion optimization of the degradation of amorphous PET film by the mixed enzyme preparation;
FIG. 11 comparison of the effects of thermokalite-stable ester bond hydrolase, bhrPETase, bhrPETase mutant and mixed enzyme formulations on amorphous PET film degradation;
fig. 12 scanning electron microscope map, a: control (amorphous PET film without enzyme) (7500×); b: control (semi-crystalline PET powder without enzyme) (7500×); c: amorphous PET film surface treated with LCC (7500×); d: amorphous PET film surface treated with a thermokalite-stable ester bond hydrolase+lcc mixed enzyme formulation (7500×); e: semi-crystalline PET powder surface treated with LCC (7500X); f: semi-crystalline PET powder surface treated with a thermobase stable ester bond hydrolase+LCC mixed enzyme formulation (7500X); g: amorphous PET film surface treated with BhrPETase (7500×); h: amorphous PET film surface treated with a thermobase stable ester bond hydrolase+bhrpetase mixed enzyme formulation (7500×); i: amorphous PET film surface treated with BhrPETase mutant (7500×); j: amorphous PET film surface treated with a thermokalite-stable ester bond hydrolase+bhrpetase mutant cocktail enzyme preparation (7500×);
fig. 13 is a comparison of the effects of BhrPETase, bhrPETase mutant and mixed enzyme formulation on degrading PET beverage bottles, a: PET bottle mouth of beverage bottle; b: PET at the bottom of the beverage bottle; c: PET beverage bottle body; d: PET shoulder of beverage bottle; e: PET at the bottom of the beverage bottle; f: PET at the bottom of the beverage bottle;
fig. 14 is a scanning electron microscope map, a: control group (PET surface of beverage bottle neck without enzyme treatment) (7500×); b: amorphous PET film surface treated with BhrPETase (7500×); c: beverage bottle neck PET surface treated with a thermo-base stable ester bond hydrolase+bhrpetase mixed enzyme formulation (7500×); d: beverage bottle neck PET surface treated with BhrPETase mutant (7500×); e: beverage bottle neck PET surface treated with a thermobase stable ester bond hydrolase+bhrpetase mutant cocktail enzyme formulation (7500×).
Detailed Description
The invention will be better understood from the following examples. However, it will be readily appreciated by those skilled in the art that the description of the embodiments is provided for illustration only and should not limit the invention as described in detail in the claims.
The experimental methods described in the examples, unless otherwise specified, are all conventional; the reagents and materials, unless otherwise specified, may be obtained simply by commercial route or by the following procedure.
Example 1
Thermobase-stable ester bond hydrolase expression and purification
Amplification primers were designed based on the thermostable ester bond hydrolase gene (wp_ 004080508.1) and PCR amplification was performed: upstream primer F1 5' -CGCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAATTTTCCACGATGTAAAACTGTG-3 'containing Xba I cleavage site (marked with a dash) and protecting base, downstream primer R1 5' -CCGGCGG CCGCTTCCCCCTTCAGCCACTCGATAAC-3', a NotI cleavage site (underlined) and a protecting base, and a thermokalite-stable ester bond hydrolase gene (WP_ 004080508.1) is used as a template for amplification to obtain the thermokalite-stable ester bond hydrolase gene. The obtained thermal alkali stable ester bond hydrolase gene is subjected to double digestion by Xba I and Not I and then inserted into an expression vector pET-20b (+) subjected to the same double digestion, so as to obtain a recombinant plasmid of the thermal alkali stable ester bond hydrolase, the recombinant plasmid is further transformed into a host E.coli BL21 (DE 3), and the recombinant plasmid is grown to OD at 37 ℃ in LB culture medium containing kanamycin Amp resistance 600 When the pH value is 0.6-0.8, adding IPTG with the final concentration of 0.5mM for induction culture for 8 hours, centrifuging at 4 ℃ and 6000rpm to remove supernatant, washing the precipitate with water for 3 times, re-suspending with three times of pH 7.0 buffer solution, and performing ultrasonic disruption in an ice-water bath or usingCrushing under high pressure to obtain crushed liquid, centrifuging at 0-4 deg.c and 12000rpm for 20min, collecting supernatant to obtain coarse cell extractive liquid of heat alkali stable ester bond hydrolase, and further nickel affinity purifying. The recombinant protein of the invention is added with 6 histidine tags at the C-terminal, can be purified by nickel affinity chromatography, and the protein purity is identified by SDS-PAGE. 1mL of Ni was taken 2+ NTA resin packing column, washing the column with 5-10mL of sterile water, adding 5mL of 1 XStrip buffer (Ni2+ wash), adding 5mL of 1 XCharge buffer (Ni2+) and adding 5mL of 1 Xbinding buffer (equilibrium elution environment) when the sterile water runs out. The target protein to be purified (repeated twice), 5mL of 1 Xbinding buffer (eluting unbound hetero protein), 5mL of 1 Xwashing buffer (eluting weakly bound hetero protein), 5mL of 1 XElute buffer (eluting target protein) and the eluate were collected. The obtained eluent is purified enzyme solution (used in the subsequent experiment of the embodiment) of the thermal alkali-stability ester bond hydrolase, and the purity of the eluent is detected by SDS-PAGE, as shown in figure 1, the thermal alkali-stability ester bond hydrolase has reached electrophoretic purity and is basically consistent with the theoretical value of 29.89kDa. The amino acid sequence of the purified protein is shown as SEQ ID NO.1, and the DNA sequence of the thermobase stable ester bond hydrolase is shown as SEQ ID NO.2.
Example 2
Enzymatic Properties of thermobase-stable ester bond hydrolase
1. The Bradford method measures the total protein mass in the purified enzyme solution of example 1 using BSA as a standard (Bradford 1976) for subsequent enzyme solution addition.
2. Esterase activity assays were quantified by assaying p-nitrophenol (pNP) release using p-nitrophenyl ester as substrate. The total reaction volume was 200. Mu.L, including 50mM Tris-HCl buffer (pH 8.0), 0.27ug/mL enzyme (calculated as protein content in the purified solution prepared in example 1), 1mM p-nitrobutyrate (pN-C4), and the reaction was incubated in 50mM Tris-HCl buffer (pH 8.0) at 70℃for 1min. To the mixture was added 200. Mu.L of 0.5M trichloroacetic acid to stop the reaction, followed by 200. Mu.L of 0.5M sodium carbonate for color development. The enzyme activity was determined by UV absorbance at 405 nm. One unit of esterase activity was defined as the amount of enzyme that released 1. Mu. Mol of p-nitrophenol per minute.
The hydrolysis activity was measured by HPLC using bis (2-hydroxyethyl) terephthalate (BHET) as a substrate. One unit of enzyme activity is defined as the amount of enzyme that cleaves 1. Mu. Mol BHET per minute. The standard reaction system was 1mL, including 50mM Tris-HCl buffer (pH 8.0), 5.4. Mu.g/mL enzyme (calculated as protein content in the purified solution prepared in example 1), and 1mM substrate BHET. The reaction was carried out at 70℃in 50mM Tris-HCl buffer (pH 8.0) for 1 hour. At the end of the experiment, the reaction was stopped with an ice-water bath. The amount of substrate BHET reacted was confirmed and analyzed by HPLC.
HPLC conditions: ZORBOX-C18 column (5 μm; 4.6X1250 mM) equipped with Refractive Index (RI) detector, column temperature 30 ℃, mobile phase 76:24 in water/acetonitrile at a flow rate of 0.5mL/min. TPA, MHET and BHET can be detected at 241 nm. BHET and TPA were measured according to calibration curves generated using commercially available TPA and BHET standards (aladine, usa).
Influence of pH and temperature on esterase Activity
The pH value and temperature curve graph of esterase activity is established by taking pNP-C4 as a substrate. The effect of pH values of 3.0 to 10.0 on esterase activity was investigated. The buffer used was 50mM citrate-sodium citrate (pH 3.0-6.0), naH 2 PO 4 -Na 2 HPO 4 (pH 6.0-7.5) and Tris-HCl buffer (pH 7.5-10.0). According to the above method for measuring esterase activity, the activity at the optimum pH is measured at 70℃and the highest enzyme activity is 100%. The effect of temperature on esterase activity was studied in 50mM Tris-HCl buffer (pH 8.0) from 30 to 100 ℃. According to the method, the highest enzyme activity is 100%. The enzyme pH stability was determined by incubating the enzyme in buffers of different pH values (3.0-10.0) at 37℃for 1 hour. The enzyme was incubated at 37℃for 0 to 8 hours at different pH values (7.0 to 10.0) and examined for alkali resistance. The enzyme was incubated in 50mM Tris-HCl buffer (pH 8.0) at 70, 80 and 90℃for 0-8 hours to investigate the thermostability of the esterase. The residual activity was calculated by measuring the absorbance at 405nm according to the method described above, taking the enzyme activity of the untreated enzyme as 100%. Knot(s)As shown in FIGS. 2a-e, the optimum temperature and pH of the thermostable ester bond hydrolase were 85℃and 8.0, respectively. And the heat alkali stable ester bond hydrolase retains 53% of activity after 6 hours of incubation in a buffer solution at pH 10.0, and retains 57% of enzyme activity after 8 hours of incubation at 90 ℃, which both show good alkali tolerance and heat stability of the heat alkali stable ester bond hydrolase.
2. Influence of organic solvents, metal ions and surfactants on esterase Activity
Using pNP-C4 as a substrate, various metal salts (Fe) were used in 50mM Tris-HCl buffer (pH 8.0) at 70℃according to the above-described esterase activity assay 3+ 、Cu 2+ 、Fe 2+ 、Mg 2+ 、Ca 2+ 、Mn 2+ 、Ni 2+ 、Co 2+ 、Zn 2+ 、Na + 、K + 1 and 10 mM) to determine the effect of metal ions on enzyme activity. The enzyme activity of the esterase to which no metal ion was added was determined to be 100%. Simultaneously, using pNP-C4 as a substrate, PMSF (1 and 10 mM), EDTA (1 and 10 mM) and a surfactant (1% and 5%, v/v) were added to the above-mentioned 50mM Tris-HCl buffer (pH 8.0) assay solution according to the above-mentioned esterase activity assay method, and the effect of PMSF, EDTA and surfactants (SDS, triton X-100 and Tween-80) on esterase activity was investigated. The activity of the residual enzyme was measured by adding the substrate pNP-C4 under standard assay conditions and the reaction mixture was incubated at 37℃for 1 hour. The esterase activity of the enzyme without additives was taken as control and defined as 100% activity. The stability of esterases to organic solvents (DMSO, methanol, ethanol, acetone and chloroform) was determined by storage at 37℃for 1 hour in the presence of organic solvents (50% and 90%, v/v) in 50mM Tris-HCl buffer (pH 8.0), the residual enzymatic activity being determined according to the method described above using pNP-C4 as substrate. The activity of the untreated enzyme was defined as 100% activity. All reactions were in triplicate, relative standard deviation<5%. The results are shown in Table 1, and when the concentration is 1mM, mg 2+ 、Na + And K + Can obviously improve esterase activity and Ni 2+ 、Cu 2+ 、Fe 2+ 、Zn 2+ And Fe (Fe) 3+ The esterase activity is reduced. Na at a concentration of 10mM + 、Li + 、K + And Mg (magnesium) 2+ Has an effect of promoting esterase activity, while Ca 2+ 、Mn 2+ 、Co 2+ 、Ni 2+ 、Cu 2+ 、Fe 2+ And Fe (Fe) 3+ Has inhibiting effect on the activity of ester bond hydrolase with heat alkali stability. As shown in Table 2, PMSF greatly inhibited the activity of the thermostable ester bond hydrolase, whereas EDTA, SDS, tween 80 and Triton-X100 increased the activity of the thermostable ester bond hydrolase at a concentration of 1mM, and slightly inhibited the activity of the thermostable ester bond hydrolase at a concentration of 10 mM. As shown in Table 3, the heat base stable ester bond hydrolase remained largely active after 1 hour of storage in each organic solvent.
TABLE 1 influence of various metal ions on enzyme Activity
TABLE 2 influence of various inhibitors and surfactants on enzyme Activity
TABLE 3 Effect of organic solvents on enzyme stability
3. Substrate specificity and kinetic parameters
According to the above method for measuring esterase activity, p-nitrophenylacetate (pN-C2), p-nitrobutyrate (pN-C4), p-nitrophenylcaproate (pN-C6), p-nitrophenyloctanoate (pN-C8), p-nitrophenylaurate (pN-C12) and p-nitrophenylacetonate were usedPalmitate (pN-C16) and BHET were used as substrates and the substrate specificity was determined in 50mM Tris-HCl buffer (pH 8.0) at 70 ℃. The kinetic parameters K of the thermobase-stable ester bond hydrolases for the hydrolysis of p-nitrophenylacetate, butyrate, caproate and caprylate were determined using the corresponding substrates at concentrations of 0.01mM to 2mM m And V max (wherein all p-nitrophenyl esters are used in accordance with the method for determining p-nitrobutyrate). The esterase activity of the different p-nitrophenyl esters was determined under optimal conditions at pH 8.0 and 85 ℃. K (K) m And V max Values were calculated from the Lineweaver-Burk plot. To calculate the catalytic constant k cat The number of proteins divided by the subunit molecular mass of the thermostable ester bond hydrolase 29.89kDa. After 15min of reaction at a concentration of 0.01mM to 5mM, the K of BHET hydrolysis by the thermostable ester bond hydrolase was determined by quantification of the hydrolysate isolated by HPLC m And V max Values. Each experiment was performed in triplicate, relative standard deviation<5%. The results are shown in FIG. 3 and Table 4, and the specific activity of the thermostable ester bond hydrolase was determined according to pNP-butyrate (C4)>pNP-acetate (C2)>pNP-caproic acid salt (C6)>pNP-octanoate (C8)>pNP-laurate (C12)>pNP-palmitate (C16)>BHET sequence.
TABLE 4 kinetic parameters of thermobase-stable ester bond hydrolase
Example 3
Application of thermokalite-stable ester bond hydrolase in hydrolysis of BHET with different concentrations
Different concentrations of substrate BHET (2, 5, 10, 20 and 40 mM) were used to determine the ability of the thermostable ester bond hydrolase to catalyze the hydrolysis of BHET. The reaction volume was 5mL and the thermobase stable ester bond hydrolase prepared in example 1 (calculated as protein content in the purified solution) was added to 50mM Tris-HCl buffer (pH 8.0) containing different concentrations of substrate BHET (2, 5, 10, 20 and 40 mM) at a load of 4mg protein/g BHET at 85℃to start the reaction for 24 hours. The reacted samples were diluted, filtered through a 0.22 micron filter plate, and stored at-20 ℃ for subsequent HPLC detection. All reactions were performed in duplicate. As a result, as shown in FIG. 4, the heat-stable ester bond hydrolase was effective in catalyzing the hydrolysis of BHET and MHET (MHET is first produced and then degraded) to produce TPA, i.e., the heat-stable ester bond hydrolase was able to completely hydrolyze 40mM of BHET within 24 hours to produce 3.4mM of MHET and 36.8mM of TPA.
Example 4
Thermal alkali-stable ester bond hydrolase degradation amorphous PET film
The reaction mixture consisted of an amorphous PET film (available from Goodfelt Co.) at a final concentration of 10g/L and a thermobase stable ester bond hydrolase (calculated as protein content in the purified solution prepared in example 1) at a final concentration of 10. Mu.g/mL in 100mM Tris-HCl buffer (pH 8.0) and incubated at 70℃for 24 hours at 220rpm. Reaction samples were taken at 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% by mass acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. The control group was incubated with buffer instead of enzyme. All reactions were performed in duplicate. As a result, as shown in FIG. 5, the thermokali-stable ester bond hydrolase had a certain degradation ability for amorphous PET, and after 24 hours of reaction, 0.39mM of TPA,0.15mM of MHET and 0.03mM of BHET could be obtained.
Example 5
Mixed enzyme preparation degradation amorphous PET film
Wherein, cutinase LCC (SEQ ID NO. 3) was synthesized by Shanghai and purchased, and 10. Mu.g/mL means that 1mL of the reaction solution contains an enzyme in an amount of 10. Mu.g of protein, the amount of protein being determined according to the Briand Folder method in example 2.
The same procedure as in example 4 was followed except that 10. Mu.g/mL of the PET degrading enzyme cutinase LCC alone was used to degrade the amorphous PET film as a control. As an experimental group, a mixed enzyme preparation having a ratio of the amounts of protein of thermokali-stable ester bond hydrolase and cutinase LCC of PET (the ratio of the amounts of protein measured by the Briand Folder method in example 2) of 1:1 was used, wherein the concentration of each enzyme was 10. Mu.g/mL. Incubate at pH 7.0, 70℃and 220rpm for 24 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. All reactions were performed in duplicate. As shown in FIG. 6, the total amount of the product produced by hydrolysis of PET by the mixed enzyme preparation was 15.77mM, and the amount of TPA produced was 5.28mM. The total amount of product produced by hydrolysis of PET by LCC alone was 9.68mM, with TPA produced in an amount of 2.91mM. As can be seen, the total amount of product from hydrolysis of PET by the thermostable ester bond hydrolase alone was only 0.57mM, with TPA being produced in an amount of only 0.39mM. The total product amount generated by hydrolyzing PET by the mixed enzyme preparation is 1.63 times and 27.67 times that of PET by independently hydrolyzing LCC and thermokalite-stable ester bond hydrolase respectively, namely the total product amount is increased, and the product amount of double enzyme action is obviously more than the sum of the two single enzyme hydrolysis products. The amount of TPA product produced by hydrolyzing PET with the mixed enzyme preparation is 1.81 times and 13.54 times that of PET by independently hydrolyzing the LCC and the thermokalite-stable ester bond hydrolase. As shown in fig. 12c and d, the degradation rate of the PET film treated with the mixed enzyme preparation was 38.87% 1.53 times that of the single enzyme LCC (25.37%). Compared with the film PET surface (FIG. 12 a) which has not been subjected to the enzyme treatment, the film PET surface (FIGS. 12c and d) which has been subjected to the enzyme treatment exhibits roughness, voids and the like. The surface of the PET film treated with the mixed enzyme preparation (FIG. 12 d) showed a phenomenon that the pores were enlarged and the texture was coarser as compared with the PET film treated with the single enzyme preparation (FIG. 12 c), which is consistent with the result of the product analysis. The experiment effectively proves that although the thermokalite-stable ester bond hydrolase has a general effect on degrading PET, the mixed enzyme preparation added with the thermokalite-stable ester bond hydrolase can obviously improve the degradation capability of the cutinase on PET and can also improve the amount of a monomer product TPA.
Example 6
Mixed enzyme preparation for degrading semi-crystalline PET powder
The procedure of example 5 was followed except that the substrate was a semi-crystalline PET powder (available from Goodfelt, inc. with a crystallinity of 60.3%) and incubated at pH 7.0, 70℃and 220rpm. After the incubation was completed, the total amount of product hydrolyzed semi-crystalline powder PET by the mixed enzyme preparation with the addition of the thermostable ester bond hydrolase was 0.4mM, which is 1.60 times the total amount of single enzyme LCC hydrolysis product (0.25 mM), as shown in FIG. 11. Wherein the amount of TPA produced by hydrolysis of the mixed enzyme preparation was 0.17mM, which is 1.89 times that of the single enzyme LCC (0.09 mM). As shown in fig. 12b, e and f, the mixed enzyme formulation treated semi-crystalline powder PET surface (fig. 12 f) showed significant deep erosion and a large number of pores compared to the non-enzyme treated surface (fig. 12 b), whereas the single enzyme LCC treatment showed spalling of the semi-crystalline powder PET surface layer. More traces of erosion were observed for the mixed enzyme formulation treated semi-crystalline powder PET (fig. 12 f) than for the LCC-degraded semi-crystalline powder PET surface alone (fig. 12 e).
Example 7
Mixed enzyme preparation degradation amorphous PET film substrate addition amount optimization
The same procedure as in example 5 was followed except that different amounts of substrate (5, 10, 20, 30, 40, 50 g/L) were added and incubated at pH 7.0, 70℃and 220rpm for 24 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% by mass acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. The control group was incubated with buffer instead of enzyme. All reactions were performed in duplicate. As shown in FIG. 7, the optimal substrate amount for degrading an amorphous PET film by a mixed enzyme preparation was 20g/L, at which time the total amount of the hydrolyzed PET-derived product was 16.76mM, and the TPA-derived product was 5.82mM. The optimal substrate amount for single PET degrading enzyme cutinase LCC to degrade amorphous PET film is 10g/L, the total amount of the produced product is 9.68mM, and the production amount of TPA is 2.91mM.
Example 8
Mixed enzyme preparation degradation amorphous PET film pH optimization
The same procedure as in example 5 was followed except that the reaction pH for hydrolyzing the amorphous PET film was 5.0, 6.0, 7.0, 8.0 and 9.0, respectively, the substrate addition amount was 20g/L, and the incubation was carried out at 70℃and 220rpm for 24 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% by mass acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. The control group was incubated with buffer instead of enzyme. All reactions were performed in duplicate. As shown in FIG. 8, the pH optimum for degrading an amorphous PET film by a mixed enzyme preparation was 7.0, at which time the total amount of the hydrolyzed PET-derived product was 16.76mM, and the amount of TPA-derived product was 5.82mM.
Example 9
Enzyme proportion optimization for degrading amorphous PET film by mixed enzyme preparation
The same procedure as in example 5 was followed except that the ratio of thermokalite-stable ester bond hydrolase to PET degrading enzyme cutinase LCC protein in the mixed enzyme formulation was 1:2, 2:3, 3:4, 1:1, 4:3, 3:2 and 2:1, respectively, the amount of substrate added was 20g/L and incubated at pH 7.0, 70℃and 220rpm for 24 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. The control group was incubated with buffer instead of enzyme. All reactions were performed in duplicate. As shown in FIG. 9, the optimal ratio of the amounts of the thermokali-stable ester bond hydrolase and the PET degrading enzyme cutinase LCC protein in the mixed enzyme preparation was 3:4 (the ratio of the amounts of the proteins measured by the Brillar method in example 2), at which time the total amount of the hydrolyzed PET-derived product was 18.18mM, wherein the amount of TPA produced was 5.93mM.
Example 10
Degradation of amorphous PET film by different enzyme mixtures
Wherein, PET degrading enzyme cutinase BhrPETase (SEQ ID NO. 4) and its mutant EQK (SEQ ID NO. 5) are synthesized and purchased by Shanghai, and 10 mug/mL means that 1mL of the reaction solution contains 10ug of protein, and the protein is measured according to the Briand Fund method in example 2.
The same procedure as in example 5 was followed except that the PET degrading enzyme cutinase LCC in the mixed enzyme preparation was replaced with the PET degrading enzyme cutinase BhrPETase and its mutants. Incubate at pH 7.0, 70℃and 220rpm for 24 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. All reactions were performed in duplicate. As shown in FIG. 10, the total concentrations of the products obtained by hydrolyzing PET with BhrPETase mixed enzyme preparation and BhrPETase mutant mixed enzyme preparation to which the thermostable ester bond hydrolase was added were 14.67 and 19.35mM, respectively, 1.49 times and 1.64 times that of the single enzymes BhrPETase (9.84 mM) and BhrPETase mutant (11.82 mM). Wherein the mixed enzyme preparation hydrolyzed PET to generate TPA concentrations of 5.35 and 7.8mM, respectively, which are 2-fold and 1.89-fold higher than the single enzyme BhrPETase (2.67 mM) and BhrPETase mutant EQK (4.13 mM), respectively. As shown in fig. 12, the amorphous film PET degradation rates of the BhrPETase mixed enzyme preparation and the BhrPETase mutant mixed enzyme preparation were 46.8% and 51.8%, respectively, which were 1.86 times and 1.48 times that of the single enzymes BhrPETase (25.2%) and BhrPETase mutant (35.1%), respectively. And the amorphous film PET surface treated with the mixed enzyme formulation (fig. 12h and j) exhibited more voids, larger holes, and a deeper degree of corrosion than the amorphous film PET surface treated with the single enzyme (fig. 12g and i). This is consistent with the results of examples 5 and 6, showing that the use of a thermostable ester bond hydrolase in combination with a cutinase significantly improves the degradation of PET.
Example 11
PET beverage bottle degraded by different mixed enzyme preparations
The procedure was as in example 10, except that the substrate used was a waste PET beverage bottle, and different parts (bottle mouth, bottle shoulder, bottle body, bottle bottom and bottle bottom center) of the PET beverage bottle after use (500 mL) were cut out to 10mg of the substrate and 1mL of the mixture of BhrPETase, bhrPETase enzyme preparation, bhrPETase mutant and BhrPETase mutant enzyme preparation, and 100mM phosphate buffer (pH 7.0) were added to a 2mL centrifuge tube together, and the enzyme amounts were the same as in example 5 and example 10. Incubate at 70℃at 220rpm for 72 hours. At the end of incubation, the reaction supernatant was diluted 10-fold in a solution containing 24% acetonitrile. The diluted sample was filtered through a 0.22 micron filter plate and stored at-20 ℃ for subsequent HPLC detection. All reactions were performed in duplicate. As shown in FIG. 13, the BhrPETase mixed enzyme preparation and BhrPETase mutant enzyme preparation hydrolyzed beverage bottle neck finish PET released TPA yields of 16.49 and 24.27mM, which were 166% and 158% higher, respectively, than the single enzymes BhrPETase (6.20 mM) and BhrPETase mutant (9.39 mM). The total yield of PET released from the bottles of the beverage bottles was 23.16 and 31.58mM, which was 22.73% and 23.50% higher, respectively, than the single enzymes BhrPETase (18.87 mM) and BhrPETase mutant (25.57 mM). The degradation rate of beverage bottle PET was analyzed by comparing the weight difference before and after enzymatic degradation by a weighing method, and the result is shown in fig. 14, and compared with the untreated control group (fig. 14 a), the degradation rate of PET at the mouth of the beverage bottle treated with the BhrPETase mixed enzyme preparation and the BhrPETase mutant enzyme preparation was 51.8% and 70.0%, respectively, and improved by 10.66% and 10.92% compared with the BhrPETase (46.8%) and the BhrPETase mutant (62.7%), respectively, which indicates that the ability of double enzyme to degrade PET is superior to that of single enzyme. As shown in fig. 14, the surface of the beverage bottle neck finish PET (fig. 14c and e) treated with the mixed enzyme preparation exhibited more voids, larger holes, and a deeper degree of corrosion than the surface of the beverage bottle neck finish PET (fig. 14b and d) treated with the single enzyme. The results are consistent with the results of product analysis and weight difference analysis, and the degradation conditions of other parts of the PET beverage bottle are similar to those of the bottle mouth, so that the hydrolysis capability and TPA yield of PET can be obviously improved by adding the thermokalite-stable ester bond hydrolase in the process of hydrolyzing the PET plastic product by the cutinase.
Claims (10)
1. The thermokalite-stable ester bond hydrolase is characterized in that the amino acid sequence of the thermokalite-stable ester bond hydrolase is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the gene sequence encoding the thermostable ester bond hydrolase is shown in SEQ ID No.2.
3. A mixed enzyme preparation, characterized in that the enzyme preparation contains the enzyme of the thermokalite-stable ester bond hydrolase and the enzyme of PET degrading enzyme cutinase; the PET degrading enzyme cutinase includes PET degrading enzyme cutinase LCC or BhrPETase or mutants thereof; the amino acid sequences of the PET degrading enzyme cutinase LCC, bhrPETase or the mutant thereof are respectively shown as SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5.
4. A mixed enzyme preparation according to claim 3, characterized in that the ratio of the amount of enzyme protein of thermokali stable ester bond hydrolase to the amount of enzyme protein of PET degrading enzyme cutinase is 1: 2-2: 1.
5. use of a thermokali stable ester bond hydrolase according to claim 1 or a mixed enzyme formulation according to claim 4 for degrading polyethylene terephthalate products or intermediates thereof.
6. The use according to claim 5, wherein the use is to add the thermokalite-stable ester bond hydrolase or the mixed enzyme preparation to a reaction system containing any one or more of polyethylene terephthalate, dihydroxyethyl terephthalate and hydroxyethyl terephthalate for degradation reaction.
7. Use according to claim 5, characterized in that the polyethylene terephthalate containing product preferably comprises an amorphous PET film or a semi-crystalline PET powder or a plastic product.
8. The use according to claim 5, wherein the amount of the thermobase-stable ester bond hydrolase in the reaction system is not less than 1.0X10 -3 mg protein/mg substrate; or the amount of the thermobase-stable ester bond hydrolase in the reaction system is not less than 1.0X10 -3 mg protein/mg substrate, and the amount of PET degrading enzyme cutinase is not less than 1.0X10 -3 mg protein/mg substrate.
9. The use according to claim 5, wherein the reaction temperature is 55 to 85 ℃, the pH is 5.0 to 9.0, the addition amount of each enzyme is 10 to 20 μg/mL, the concentration of the substrate PET is 5 to 80mg/mL, and the ratio of the amount of the enzyme protein of the thermokali stable ester bond hydrolase to the amount of the enzyme protein of the PET degrading enzyme cutinase is 1: 2-2: 1, the conversion time is 0.5-24 h.
10. An enzyme preparation product for degrading polyethylene terephthalate or an intermediate thereof, wherein the enzyme preparation product is useful for degrading any one or more of polyethylene terephthalate, an intermediate of polyethylene terephthalate, bis-hydroxyethyl terephthalate and hydroxyethyl terephthalate; the enzyme preparation product contains the thermokalite-stable ester bond hydrolase according to claim 1 or the mixed enzyme preparation according to claim 3.
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