CN110038250A - A method of the phthalate of degradation metal ion or organic solvent - Google Patents
A method of the phthalate of degradation metal ion or organic solvent Download PDFInfo
- Publication number
- CN110038250A CN110038250A CN201910360374.7A CN201910360374A CN110038250A CN 110038250 A CN110038250 A CN 110038250A CN 201910360374 A CN201910360374 A CN 201910360374A CN 110038250 A CN110038250 A CN 110038250A
- Authority
- CN
- China
- Prior art keywords
- phthalate
- lesterase
- carboxy
- baces01
- degradation
- 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.)
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Links
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 24
- 229910021645 metal ion Inorganic materials 0.000 title claims abstract description 17
- 239000003960 organic solvent Substances 0.000 title claims abstract description 16
- 230000015556 catabolic process Effects 0.000 title abstract description 40
- 238000006731 degradation reaction Methods 0.000 title abstract description 40
- 108010051152 Carboxylesterase Proteins 0.000 claims abstract description 62
- 102000013392 Carboxylesterase Human genes 0.000 claims abstract description 62
- MGWAVDBGNNKXQV-UHFFFAOYSA-N diisobutyl phthalate Chemical compound CC(C)COC(=O)C1=CC=CC=C1C(=O)OCC(C)C MGWAVDBGNNKXQV-UHFFFAOYSA-N 0.000 claims abstract description 22
- FLKPEMZONWLCSK-UHFFFAOYSA-N diethyl phthalate Chemical compound CCOC(=O)C1=CC=CC=C1C(=O)OCC FLKPEMZONWLCSK-UHFFFAOYSA-N 0.000 claims abstract description 20
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 claims abstract description 18
- 241000588724 Escherichia coli Species 0.000 claims abstract description 12
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims abstract description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 238000006243 chemical reaction Methods 0.000 claims description 21
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 10
- UHOVQNZJYSORNB-UHFFFAOYSA-N monobenzene Natural products C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 150000002148 esters Chemical class 0.000 claims description 7
- 235000019441 ethanol Nutrition 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 3
- 239000003054 catalyst Substances 0.000 claims description 2
- 230000000593 degrading effect Effects 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 50
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- 230000002255 enzymatic effect Effects 0.000 abstract description 4
- 229940088598 enzyme Drugs 0.000 description 49
- 239000000243 solution Substances 0.000 description 30
- RJNPPEUAJCEUPV-UHFFFAOYSA-N naphthalen-2-yl acetate Chemical compound C1=CC=CC2=CC(OC(=O)C)=CC=C21 RJNPPEUAJCEUPV-UHFFFAOYSA-N 0.000 description 23
- 230000000694 effects Effects 0.000 description 22
- 239000000758 substrate Substances 0.000 description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 14
- 239000008057 potassium phosphate buffer Substances 0.000 description 11
- -1 acyl glycerine Chemical compound 0.000 description 9
- BUCIWTBCUUHRHZ-UHFFFAOYSA-K potassium;disodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[K+].OP(O)([O-])=O.OP([O-])([O-])=O BUCIWTBCUUHRHZ-UHFFFAOYSA-K 0.000 description 8
- PRPINYUDVPFIRX-UHFFFAOYSA-M 1-naphthaleneacetate Chemical compound C1=CC=C2C(CC(=O)[O-])=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-M 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 238000000746 purification Methods 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- XNGIFLGASWRNHJ-UHFFFAOYSA-N o-dicarboxybenzene Natural products OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 6
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241001062009 Indigofera Species 0.000 description 3
- 230000007613 environmental effect Effects 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- VGKONPUVOVVNSU-UHFFFAOYSA-N naphthalen-1-yl acetate Chemical compound C1=CC=C2C(OC(=O)C)=CC=CC2=C1 VGKONPUVOVVNSU-UHFFFAOYSA-N 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
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- 238000005215 recombination Methods 0.000 description 3
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- 150000003839 salts Chemical class 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 108010093581 aspartyl-proline Proteins 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 150000001733 carboxylic acid esters Chemical class 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 125000005498 phthalate group Chemical class 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 150000004782 1-naphthols Chemical class 0.000 description 1
- PJKVFARRVXDXAD-UHFFFAOYSA-N 2-naphthaldehyde Chemical compound C1=CC=CC2=CC(C=O)=CC=C21 PJKVFARRVXDXAD-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 1
- STACJSVFHSEZJV-GHCJXIJMSA-N Ala-Asn-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STACJSVFHSEZJV-GHCJXIJMSA-N 0.000 description 1
- CRWFEKLFPVRPBV-CIUDSAMLSA-N Ala-Gln-Met Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(O)=O CRWFEKLFPVRPBV-CIUDSAMLSA-N 0.000 description 1
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 1
- QDGMZAOSMNGBLP-MRFFXTKBSA-N Ala-Trp-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=C(C=C3)O)C(=O)O)N QDGMZAOSMNGBLP-MRFFXTKBSA-N 0.000 description 1
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- PVDTYLHUWAEYGY-CIUDSAMLSA-N Ser-Glu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PVDTYLHUWAEYGY-CIUDSAMLSA-N 0.000 description 1
- SQBLRDDJTUJDMV-ACZMJKKPSA-N Ser-Glu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SQBLRDDJTUJDMV-ACZMJKKPSA-N 0.000 description 1
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- SCSVNSNWUTYSFO-WDCWCFNPSA-N Thr-Lys-Glu Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O SCSVNSNWUTYSFO-WDCWCFNPSA-N 0.000 description 1
- NOOMDULIORCDNF-IRXDYDNUSA-N Tyr-Gly-Phe Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O NOOMDULIORCDNF-IRXDYDNUSA-N 0.000 description 1
- HSBZWINKRYZCSQ-KKUMJFAQSA-N Tyr-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O HSBZWINKRYZCSQ-KKUMJFAQSA-N 0.000 description 1
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 description 1
- AEMPCGRFEZTWIF-IHRRRGAJSA-N Val-Leu-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O AEMPCGRFEZTWIF-IHRRRGAJSA-N 0.000 description 1
- ZRSZTKTVPNSUNA-IHRRRGAJSA-N Val-Lys-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)C(C)C)C(O)=O ZRSZTKTVPNSUNA-IHRRRGAJSA-N 0.000 description 1
- YLRAFVVWZRSZQC-DZKIICNBSA-N Val-Phe-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YLRAFVVWZRSZQC-DZKIICNBSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 102000005922 amidase Human genes 0.000 description 1
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
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- 230000007547 defect Effects 0.000 description 1
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- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 230000036267 drug metabolism Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 108010006664 gamma-glutamyl-glycyl-glycine Proteins 0.000 description 1
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 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
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108010056582 methionylglutamic acid Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- ASHGTUMKRVIOLH-UHFFFAOYSA-L potassium;sodium;hydrogen phosphate Chemical compound [Na+].[K+].OP([O-])([O-])=O ASHGTUMKRVIOLH-UHFFFAOYSA-L 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000005067 remediation Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- 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
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B09B5/00—Operations not covered by a single other subclass or by a single other group in this subclass
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- 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)
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- 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)
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/40—Inorganic substances
- A62D2101/47—Inorganic substances containing oxygen, sulfur, selenium or tellurium, i.e. chalcogen
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Abstract
The invention discloses a kind of degradation metal ion or the methods of the phthalate of organic solvent, belong to enzyme engineering field.The present invention is by amino acid sequence carboxy-lesterase BaCEs01 heterogenous expression in Escherichia coli as shown in SEQ ID NO.1, obtained carboxy-lesterase BaCEs01 optimal reactive temperature is 40 DEG C, after placing 1 hour at 40 DEG C, enzymatic activity is maintained at 40% or more, has good temperature stability.It purifies obtained carboxy-lesterase BaCEs01 and 84.2%, 89.6%, 81.3% is up to respectively to the degradation rate of phthalate (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate), provide a kind of method of efficient degradation plasticiser phthalate.
Description
Technical field
The present invention relates to a kind of degradation metal ion or the methods of the phthalate of organic solvent, belong to enzyme work
Journey field.
Background technique
Carboxy-lesterase (carboxylesterase, EC 3.1.1.1) be refer to catalyzing hydrolysis carboxylate generate carboxylic acid and
The nonspecific esterase of alcohol belongs to B esterase with single acyl glycerine esterase, fragrant amidase and cholinesterase etc., in structure with
Lipase belongs to α/β hydrolase family.
Carboxy-lesterase is widely present in animal, plant and microorganism, and current research, which is concentrated mainly in animal's liver, to be joined
With in the carboxy-lesterase and insect bodies of drug metabolism with the carboxy-lesterase of drug resistance, to grinding for the carboxy-lesterase of microbial source
Study carefully relatively fewer.But it since the carboxy-lesterase yield from microbial source is high, stability is good, high catalytic efficiency, therefore obtains
The concern of more and more people.
Phthalic acid ester (PAEs) is widely used in production and living as plasticiser and enters environment, seriously threatens life
State Environmental security and human health.In environment the remaining efficient degradation of plasticiser have become present environmental protection there is an urgent need to
Research hotspot.Biological prosthetic is one of the important means of environmental improvement, and current main method is carried out using microbial strains
Degradation under the conditions of temperature is 25-30 DEG C, is cultivated 0.5-10 days from the point of view of the phthalic acid ester degradation bacteria isolated
The phthalic acid ester degradation rate of various concentration is reached 50% or so.Therefore using microbial strains degradation, there is degradations to imitate
The disadvantages of rate is low, and stability is poor, and there are certain pollutions, to allow researchers that attention to be placed on enzymic degradation.It is micro-
Biology is most wide, most species biology to be distributed in nature, therefore microbe-derived enzyme is also very extensive.Study micro- life
The degradation of the enzyme that object generates to environmental pollutants is with very promising.
It is relatively limited for the research of esterase degradation phthalate at present, and focus mainly on its mechanism of degradation
And the toxicity of catabolite, in the esterase having found, it is low that there is degradation efficiencies, and reaction condition requires the defects of stringenter.
Such as in 2015, the carboxy-lesterase EstZ22 of what Wang Chaofan et al. was cloned from bacillus can degrade phthalic acid ester
Reaction temperature need 70 DEG C or more, be not particularly suited in practical application；2016, Do Kyung Hong et al. clone's
For esterase EstSP1 in 1h, the degradation rate to 1mM dibutyl phthalate is only 30%；2014, Xiao-Yan Zhang
Et al. the most suitable degradation condition of esterase EstS1 of clone be 60 DEG C.But major part carboxy-lesterase for metal ion and has at present
The tolerance of solvent is lower, seriously limits the application of carboxy-lesterase.Therefore, reaction condition bacterium of less demanding is obtained to come
The carboxy-lesterase in source will have highly important value and significance in industrial application.
Summary of the invention
The first purpose of the invention is to provide a kind of can degrade at a lower reaction temperature to contain metal ion or organic
The method of phthalate in the system of solvent is to be with amino acid sequence carboxy-lesterase as shown in SEQ ID NO.1
Catalyst, phthalate of degrading.
In one embodiment, the reaction temperature is 15~50 DEG C.
In one embodiment, the reaction temperature is preferably 30~50 DEG C.
In one embodiment, the reaction temperature is preferably 40 DEG C.
In one embodiment, the degradation condition of the carboxy-lesterase are as follows: pH5.0~8.0.
In one embodiment, the degradation condition of the carboxy-lesterase is preferred are as follows: pH 6.5.
In one embodiment, the degradation time is 1~6h.
In one embodiment, the metal ion is Na+、K+、Zn2+、NH4 +、Mg2+、Ca2+、Cu2+Or Fe3+。
In one embodiment, the concentration of the metal ion is 0.5~2mM.
In one embodiment, the organic solvent is methanol, ethyl alcohol, acetonitrile, acetone, n-hexane or isopropanol.
In one embodiment, the concentration of the organic solvent is 0.5~2% (v/v).
In one embodiment, the phthalate are as follows: diethyl phthalate, two fourth of phthalic acid
Ester or diisobutyl phthalate.
A second object of the present invention is to provide a kind of recombinant bacteriums for producing carboxy-lesterase, are host with Escherichia coli, expression
Amino acid sequence gene as shown in SEQ ID NO.1.
In one embodiment, it is expression vector in expression in escherichia coli that the expression, which is using pColdII,.
Third object of the present invention is to provide a kind of method for producing carboxy-lesterase, the method is using the recombination
Bacterium carries out fermenting and producing.
The present invention also provides the recombinant bacterium or its production carboxy-lesterase field of environment protection application.
Beneficial effects of the present invention:
(1) present invention is by amino acid sequence as carboxy-lesterase BaCEs01 shown in SEQ ID NO.1 is different in Escherichia coli
Source expression, obtained carboxy-lesterase BaCEs01 optimal reactive temperature are 40 DEG C, after placing 1 hour at 40 DEG C, and enzymatic activity is kept
40% or more, there is good temperature stability.
(2) remaining enzyme activity is respectively after carboxy-lesterase BaCEs01 places 1h in the buffer containing ethyl alcohol and acetonitrile
76.9% and 71.6%, and organic solvent (methanol, acetone, n-hexane, isopropanol) does not influence its enzyme activity significantly.
(3) the carboxy-lesterase BaCEs01 that purifying obtains is to phthalate (diethyl phthalate, adjacent benzene two
Formic acid dibutyl ester, diisobutyl phthalate) degradation rate respectively be up to 84.2%, 89.6%, 81.3%, provide one kind
The method of efficient degradation plasticiser phthalate.
Detailed description of the invention
Fig. 1 is optimal reactive temperature.
Fig. 2 is temperature stability.
Fig. 3 is optimal reaction pH.
Fig. 4 is pH stability.
Fig. 5 is 1- naphthyl acetate enzymatic reaction rate figure.
Fig. 6 is 2- naphthyl acetate enzymatic reaction rate figure.
Specific embodiment
(1) method that carboxy-lesterase enzyme activity is measured as substrate using 1- naphthyl acetate or 2- naphthyl acetate:
1% solid indigo plant B salt: the solid indigo plant B salt for weighing 1g, which is dissolved in distilled water, is settled to 100mL, is kept in dark place.
5% SDS: the SDS for weighing 5g is dissolved in distilled water, in 37 DEG C water-bath 1 hour, be settled to after it is completely dissolved
100mL is saved in refrigerator.
The 1- naphthyl acetate or 2- naphthyl acetate of 0.6M: the 1- naphthyl acetate or 2- naphthyl acetate for weighing 11.17g are dissolved in
In 95% ethyl alcohol, it is settled to 100mL, is kept in dark place.
Disodium hydrogen phosphate-potassium phosphate buffer: 1/15M disodium hydrogen phosphate and 1/15M potassium dihydrogen phosphate mixed configuration
To pH7.0.
The substrate 1- naphthyl acetate of 15 μ L or 2- naphthyl acetate are added to 1.5mL disodium hydrogen phosphate-potassium dihydrogen phosphate to delay
(pH 7.0) keeps the temperature 5min in 37 DEG C of water-baths in fliud flushing, and the enzyme solution of 250 μ L after purification is added, reacts 5min, and 0.5mL is added and terminates
Developing solution DBLS (1% solid indigo plant B salt is mixed with 5%SDS with 2:5), shakes up, stands 10min, measure light absorption value under 595/555nm.
Enzyme activity definition: under optimum reaction conditions, in 1min from the 1- naphthyl acetate or 2- naphthyl acetate solution of 0.6M
Enzyme amount needed for the 1- naphthols or beta naphthal of 1 μM of release is an enzyme-activity unit.
(2) measurement of carboxy-lesterase protein concentration: according to Bradford protein quantification kit method, by certain times of dilution
Several enzyme solutions is mixed with G250 dyeing liquor, with the light absorption value at microplate reader measurement 595nm, calculates albumen according to protein concentration mark song
Concentration.Specific enzyme activity (Umg-1)=enzyme activity (UmL-1) × [protein concentration (mgmL-1)]-1。
(3) phthalate high performance liquid chromatography detection condition are as follows: C18 column (4.6 × 250mm of Agilent), wave
Long 254nm, mobile phase ratio are methanol: water=80:20, and detection temperature is 30 DEG C.
(4) phthalate degradation rate calculation formula: degradation rate (%)=residue concentration of substrate/initial substrate concentration
Embodiment 1: the building of engineered strain
The carboxylic of artificial synthesized nucleotide sequence (amino acid sequence is as shown in SEQ ID NO.1) as shown in SEQ ID NO.2
Acid esters enzyme BaCEs01 gene order.By carboxy-lesterase BaCEs01 gene order and plasmid vector pColdII restriction enzyme
Connection conversion obtains recombinant bacterium E.coli into E.coli BL21 (DE3) competent cell after I double digestion of enzyme Sac I and Xba
BL21-pColdII-BaCEs01。
Embodiment 2: the expression and purification of carboxy-lesterase (BaCEs01)
LB culture medium g/L: sodium chloride 10, tryptone 10, Yeast Extract 5, pH 7.
Recombination bacillus coli E.coli BL21-pColdII-BaCEs01 is inoculated in containing 100mgmL-1The LB of ammonia benzyl
In fluid nutrient medium, (it is transferred in E.coli BL21 (DE3) with original strain E.coli BL21 (DE3) and unloaded bacterial strain
PClodII plasmid) as control, 37 DEG C, 200rmp cultivates 12h, and then the 500 above-mentioned seed liquors of μ L are inoculated in containing 50 μ L ammonia
In the 50mL LB culture solution of benzyl, 37 DEG C of culture 2.5h, until OD600It is 0.6, shaking table is cooled to 15 DEG C, stands 30min.Every bottle
The IPTG of the 40 final concentration of 0.4mol/L of μ L is added as inducer, inducer is not added as a control group, in 15 DEG C of 200rmp
Culture is for 24 hours.
Bacterium solution is collected, 4 DEG C, 8000rmp obtains thallus after being centrifuged 10min, and 5mL phosphate buffer (0.02mol/ is added
L, pH7.0) thallus is resuspended, Ultrasonic Cell Disruptor is broken, and supernatant is collected by centrifugation and obtains crude enzyme liquid.Crude enzyme liquid obtained above is adopted
Ni-sepharose purification, which is carried out, with 150 protein purification system of AKTA avant obtains BaCEs01 enzyme solution.
Embodiment 3: the enzyme activity determination of carboxy-lesterase (BaCEs01)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 7), using 2- naphthyl acetate as substrate, 20~50 DEG C of ranges
It is interior, every 5 DEG C, measure carboxy-lesterase BaCEs01 enzyme activity, it is known that the optimum temperature of carboxy-lesterase BaCEs01 is 40 DEG C.Most suitable
Under the conditions of 40 DEG C of reaction temperature, in 5.0~9.0 range of pH, every 0.5, enzyme activity is measured, determines that optimum response pH is 6.5.
Under optimum reaction conditions, i.e., in disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5), at 40 DEG C, respectively
The ratio of the carboxy-lesterase BaCEs01 purified in embodiment 2 is measured using 0.6M1- naphthyl acetate and 2- naphthyl acetate as substrate
Enzyme activity respectively reaches 5.20U/mg and 1.20U/mg.
Temperature stability: by 250 μ L of carboxy-lesterase (BaCEs01) enzyme solution, when pH is 6.5 respectively 10,20,30,40,
Remaining enzyme activity is measured after saving 1h in 50,60 DEG C, is set as 100% so that enzyme activity is highest.As the result is shown: carboxy-lesterase
After BaCEs01 is placed 1 hour at 40 DEG C, enzymatic activity is maintained at 40% or more, has good temperature stability.
PH stability: by 250 μ L carboxy-lesterase BaCEs01 enzyme solutions under the conditions of 40 DEG C of temperature, respectively in pH5.0,5.5,
Remaining enzyme activity is measured after saving 1h in 6.0,6.5,7.0,7.5,8.0, is set as 100% so that enzyme activity is highest.As the result is shown: carboxylic
Acid esters enzyme is maintained at 40% or more in pH5.5-7.5 enzymatic activity, has good pH stability.
Embodiment 4: influence of the metal ion to carboxy-lesterase (BaCEs01)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5) of 1.5mL, at 40 DEG C, 250 μ L are added
BaCEs01 enzyme solution, the 0.6M 2- naphthyl acetate of 15 μ L, 1mM different metal ions (Na+,K+,Zn+,NH4 +,Mg2+,Ca2+,Cu2+,
Fe3+), measure influence of the metal ion to carboxy-lesterase BaCEs01 enzyme activity.The results are shown in Table 1, it is known that K+, NH4 +, Mg2+It is right
Enzyme activity has facilitation slightly, remaining metal ion has different degrees of inhibiting effect to enzyme.
Influence of 1 different metal ions of table to carboxy-lesterase enzyme activity
Embodiment 5: influence of the organic solvent to carboxy-lesterase (BaCEs01)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5) of 1.5mL, at 40 DEG C, 250 μ L are added
BaCEs01 enzyme solution, the 0.6M 2- naphthyl acetate of 15 μ L, 1% (v/v) different organic solvents (methanol, ethyl alcohol, acetonitrile, acetone, just
Hexane, isopropanol), after placing 1h, DBLS solution is added and terminates reaction, shadow of the measurement different organic solvents to Carboxylesterase Activity
It rings.The results are shown in Table 2, it is known that remaining enzyme activity is respectively after BaCEs01 places 1h in the buffer containing ethyl alcohol and acetonitrile
76.9% and 71.6%, remaining organic solvent does not influence its enzyme activity significantly.
Influence of 2 different organic solvents of table to carboxy-lesterase enzyme activity
Embodiment 6: the substrate specificity of carboxy-lesterase (BaCEs01)
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5) of 1.5mL, at 40 DEG C, 250 μ L are added
BaCEs01 enzyme solution, measurement carboxy-lesterase BaCEs01 are catalyzed 0.2-3.4mmolL-11- naphthyl acetate and 0.1-3.2mmol
L-12- naphthyl acetate reaction rate, using Origin software carry out nonlinear fitting curve Vmax and Km value is calculated,
And then Kcat/Km value is calculated (see Fig. 5, Fig. 6).As shown in table 3, substrate 1- naphthyl acetate is compared with 2- naphthyl acetate, carboxylic
Acid esters enzyme (K bigger to the affinity of 1- naphthyl acetatemLower, affinity is bigger), to the catalytic efficiency (K of 1- naphthyl acetatecat/
Km) more preferably, reach 0.042.
The different substrate kinetic parameters of table 3
Embodiment 7: the application of carboxy-lesterase (BaCEs01)
Carboxy-lesterase (BaCEs01) enzyme solution of 50 μ L after purification is taken to be added to the phthalate of final concentration of 1mM
In (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate), in the disodium hydrogen phosphate-of 1.5mL
In potassium phosphate buffer (pH 6.5), carboxy-lesterase (BaCEs01) enzyme solution is not added as a control group, in 40 DEG C of water-bath 1h
Afterwards, it is that 1M HCl solution terminates reaction, then is extracted with isometric ethyl acetate that 100 μ L concentration are added in the reaction system, often
Group three parallel laboratory tests of experimental setup.The amount of remaining substrate is measured, by high performance liquid chromatography to judge its hydrolysis degree.It calculates
Carboxy-lesterase BaCEs01 is obtained to 3 kinds of plasticiser phthalates (diethyl phthalate, two fourth of phthalic acid
Ester, diisobutyl phthalate) degradation rate be respectively 74.5%, 80.3%, 70.2%.Know therefrom, the carboxy-lesterase
70% or more is reached to the degradation rate of three kinds of plasticisers of low concentration, there is very big application value in environment remediation.
Degradation of 4 BaCEs01 of table to low concentration phthalate
Embodiment 8: the application of carboxy-lesterase (BaCEs01)
It is identical as process in embodiment 7, except that in system phthalate final concentration of 10mM.
Carboxy-lesterase (BaCEs01) enzyme solution in 50 μ L embodiments 2 after purification is added to the adjacent benzene of final concentration of 10mM
In diformic ester (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate), 1.5mL's
In disodium hydrogen phosphate-potassium phosphate buffer (pH 6.5), carboxy-lesterase (BaCEs01) enzyme solution is not added as a control group,
After 40 DEG C of water-bath 1h, 100 μ L concentration are added in the reaction system and are that 1M HCl solution terminates reaction, then with isometric acetic acid
Ethyl ester extraction, three parallel laboratory tests of every group of experimental setup.The amount of remaining substrate is measured, by high performance liquid chromatography to judge its water
The carboxy-lesterase is calculated to 3 kinds of plasticiser phthalates (diethyl phthalates, O-phthalic in solution degree
Dibutyl phthalate, diisobutyl phthalate) degradation rate be respectively 44.6%, 50.4%, 40.7%.Thus result it is found that
In the case where not increasing enzyme amount, which has all reached 40% or more to the degradation rate of 3 kinds of plasticisers of high concentration.
Degradation of 5 BaCEs01 of table to high concentration phthalate
Embodiment 9: the application of carboxy-lesterase (BaCEs01)
Identical as process in embodiment 7, different hydrolysis times extend to 6h.
By the enzyme solution of 50 μ L after purification be added to final concentration of 1mM phthalate (diethyl phthalate,
Dibutyl phthalate, diisobutyl phthalate) in, enzyme solution is not added as a control group, in the phosphoric acid hydrogen two of 1.5mL
In sodium-potassium phosphate buffer (pH 6.5), after 40 DEG C of water-bath 6h, it is that 1M HCl solution terminates instead that 100 μ L concentration, which are added,
It answers, then is extracted with isometric ethyl acetate, three parallel laboratory tests of every group of experimental setup.It is measured by high performance liquid chromatography remaining
The amount of substrate, to judge its hydrolysis degree, be calculated the carboxy-lesterase be respectively 75.6% to the degradation rate of 3 kinds of plasticisers,
84.7.%, 72.3%.The result illustrates the extension reaction time, which reaches the degradation rate of 3 kinds of plasticisers
75% or more.
Degradation of 6 BaCEs01 of table to low concentration phthalate
Embodiment 10: the application of carboxy-lesterase (BaCEs01)
It is identical as process in embodiment 7, except that it is 100 μ that the additive amount of carboxy-lesterase (BaCEs01) enzyme solution, which increases,
L。
The enzyme solution of 100 μ L after purification is added to phthalate (the phthalic acid diethyl of final concentration of 1mM
Ester, dibutyl phthalate, diisobutyl phthalate) in, enzyme solution is not added as a control group, in the phosphoric acid of 1.5mL
In disodium hydrogen-potassium phosphate buffer (pH 6.5), after 40 DEG C of water-bath 1h, it is that 1M HCl solution is whole that 100 μ L concentration, which are added,
It only reacts, then is extracted with isometric ethyl acetate, three parallel laboratory tests of every group of experimental setup.It is measured by high performance liquid chromatography
The amount of remaining substrate, to judge its hydrolysis degree.The amount for analyzing remaining substrate is measured through high performance liquid chromatography, and the carboxylic is calculated
Acid esters enzyme is respectively 84.2%, 89.6%, 81.3% to the degradation rate of 3 kinds of plasticisers.The result illustrates that increasing enzyme amount increases drop
Solution rate.
Degradation of 7 BaCEs01 of table to low concentration phthalate
Embodiment 11: recombinant bacterium E.coli BL21-pColdII-BaCEs01 whole-cell catalytic reaction
Disodium hydrogen phosphate-is used after recombinant bacterium E.coli BL21-pColdII-BaCEs01 obtained in embodiment 1 is collected
Potassium phosphate buffer (pH 6.5) resuspension is diluted to OD600It is 1.0, is separately added into 100 μ L bacterium solutions to final concentration 1mM neighbour's benzene
In diformic ester (diethyl phthalate, dibutyl phthalate, diisobutyl phthalate), in 40 DEG C of water-baths
After 1h, it is that 1M HCl solution terminates reaction, then is extracted with isometric ethyl acetate, every group of experimental setup that 100 μ L concentration, which are added,
Three parallel laboratory tests.The recombination is calculated to judge its hydrolysis degree by the amount that high performance liquid chromatography measures remaining substrate
Bacterium is respectively 19.8%, 15.4% and 21.6% to the degradation rate of 3 kinds of plasticisers.
Degradation of 8 recombinant bacterium of table to phthalate
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>method of the phthalate of a kind of degradation metal ion or organic solvent
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 200
<212> PRT
<213>artificial synthesized
<400> 1
Met Lys His Val Phe Glu Gln Gly Thr Ser Glu Asn Val Leu Leu Leu
1 5 10 15
Leu His Gly Thr Gly Gly Asn Glu His Asp Leu Leu Ser Leu Gly Arg
20 25 30
Phe Ile Asp Pro Asn Ala Ser Leu Leu Gly Val Arg Gly Ser Val Ser
35 40 45
Glu Asn Gly Met Pro Arg Phe Phe Lys Arg Leu Lys Glu Gly Val Phe
50 55 60
Asp Glu Lys Asp Leu Ile Glu Arg Thr Glu Glu Leu Lys Asn Phe Ile
65 70 75 80
Asp Glu Ala Ala Gln Met Tyr Gly Phe Ser Arg Glu Asn Val Ile Ala
85 90 95
Ala Gly Tyr Ser Asn Gly Ala Asn Ile Ala Ala Ser Leu Leu Phe His
100 105 110
Tyr Lys Asp Val Leu Lys Gly Ala Val Leu His His Pro Met Val Pro
115 120 125
Ile Arg Gly Val Lys Leu Pro Asp Met Glu Gly Leu Pro Val Phe Ile
130 135 140
Gly Ala Gly Lys Arg Asp Pro Leu Cys Thr Lys Glu Glu Ser Glu Glu
145 150 155 160
Leu Tyr Gln Tyr Leu Thr Asp Ala His Ala Ser Val Ala Leu Glu Trp
165 170 175
Gln Glu Gly Gly His Gln Leu Thr Gln Gln Glu Ala Glu Gln Ala Arg
180 185 190
Ala Trp Tyr Ser Glu Arg Phe Leu
195 200
<210> 2
<211> 603
<212> DNA
<213>artificial synthesized
<400> 2
atgaaacatg tgtttgaaca gggaacgtca gaaaatgttc tgctgctttt gcacggaacg 60
ggaggaaatg aacatgatct tctgtcgctc ggacgtttta tcgatccgaa cgcaagtctg 120
ctgggggtca gaggatcagt gtcggaaaac ggcatgcccc gtttttttaa gcggctgaaa 180
gaaggggtat ttgacgagaa agatttaatt gaacggacgg aagaattaaa gaattttatt 240
gatgaagcgg cgcaaatgta cgggttcagc agagaaaacg tgattgctgc cggctattca 300
aacggcgcca atattgcagc aagtcttttg tttcattata aagatgtact gaaaggagcg 360
gttctgcatc atccgatggt gccgatacgc ggagtgaagc tgcctgatat ggaagggctg 420
cctgtgttta tcggtgcggg aaaacgcgat cccctatgta caaaagaaga atcggaggag 480
ctttatcagt atctgacaga tgctcatgca tccgttgctc ttgaatggca ggagggcggt 540
catcagctca cacagcagga agccgagcag gcacgagcct ggtacagtga gcgattccta 600
taa 603
Claims (10)
1. phthalate in a kind of system containing metal ion or organic solvent that can degrade at a lower reaction temperature
Method, which is characterized in that be the adjacent benzene of degrading using amino acid sequence carboxy-lesterase as shown in SEQ ID NO.1 as catalyst
Diformic ester.
2. the method according to claim 1, wherein the reaction temperature is 15~50 DEG C.
3. the method according to claim 1, wherein the metal ion is Na+、K+、Zn2+、NH4 +、Mg2+、Ca2 +、Cu2+Or Fe3+。
4. the method according to claim 1, wherein the concentration of the metal ion is 0.5~2mM.
5. the method according to claim 1, wherein the organic solvent is methanol, ethyl alcohol, acetonitrile, acetone, just
Hexane or isopropanol.
6. the method according to claim 1, wherein the concentration of the organic solvent is 0.5~2% (v/v).
7. the method according to claim 1, wherein the phthalate are as follows: phthalic acid diethyl
Ester, dibutyl phthalate or diisobutyl phthalate.
8. a kind of recombinant bacterium for producing carboxy-lesterase, which is characterized in that with Escherichia coli be host, express amino acid sequence such as SEQ
Gene shown in ID NO.1.
9. a kind of method for producing carboxy-lesterase, which is characterized in that the method is carried out using recombinant bacterium according to any one of claims 8
Fermenting and producing.
10. recombinant bacterium according to any one of claims 8 or its production carboxy-lesterase field of environment protection application.
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| CN109943515A (en) * | 2019-04-30 | 2019-06-28 | 江南大学 | It is a kind of produce carboxy-lesterase recombinant bacterium and its application |
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| US6291222B1 (en) * | 1998-01-09 | 2001-09-18 | Heska Corporation | Carboxylesterase nucleic acid molecules and uses thereof |
| CN103013944A (en) * | 2013-01-10 | 2013-04-03 | 南京大学 | Hydrolytic enzyme for diethyl phthalate plasticizer |
| CN104109659A (en) * | 2014-07-23 | 2014-10-22 | 云南师范大学 | Carboxylesterase as well as coding gene and application thereof |
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