CN111662851B - Bacterial strain for oxidizing aromatic compounds and application thereof - Google Patents
Bacterial strain for oxidizing aromatic compounds and application thereof Download PDFInfo
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- 150000001491 aromatic compounds Chemical class 0.000 title abstract description 19
- 230000001590 oxidative effect Effects 0.000 title abstract description 10
- 230000001580 bacterial effect Effects 0.000 title description 7
- 241000589516 Pseudomonas Species 0.000 claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 6
- 238000000855 fermentation Methods 0.000 claims description 44
- 230000004151 fermentation Effects 0.000 claims description 44
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 38
- 239000000843 powder Substances 0.000 claims description 25
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 claims description 24
- 239000002609 medium Substances 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- 239000001888 Peptone Substances 0.000 claims description 19
- 108010080698 Peptones Proteins 0.000 claims description 19
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 19
- 239000001963 growth medium Substances 0.000 claims description 19
- 235000019319 peptone Nutrition 0.000 claims description 19
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 239000002904 solvent Substances 0.000 claims description 19
- 238000011218 seed culture Methods 0.000 claims description 18
- 239000007787 solid Substances 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 13
- 238000007254 oxidation reaction Methods 0.000 claims description 12
- 239000000758 substrate Substances 0.000 claims description 12
- 230000003647 oxidation Effects 0.000 claims description 11
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 239000011942 biocatalyst Substances 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 238000004321 preservation Methods 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 2
- 238000012216 screening Methods 0.000 abstract description 8
- 239000002689 soil Substances 0.000 abstract description 6
- 230000007613 environmental effect Effects 0.000 abstract description 5
- 238000003786 synthesis reaction Methods 0.000 abstract description 5
- 230000015572 biosynthetic process Effects 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 3
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- 230000000813 microbial effect Effects 0.000 abstract description 2
- 150000004053 quinones Chemical class 0.000 abstract description 2
- 238000005067 remediation Methods 0.000 abstract description 2
- 239000012450 pharmaceutical intermediate Substances 0.000 abstract 1
- 239000000047 product Substances 0.000 description 18
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 14
- 238000001291 vacuum drying Methods 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 11
- 239000006228 supernatant Substances 0.000 description 11
- FRASJONUBLZVQX-UHFFFAOYSA-N 1,4-naphthoquinone Chemical compound C1=CC=C2C(=O)C=CC(=O)C2=C1 FRASJONUBLZVQX-UHFFFAOYSA-N 0.000 description 10
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 10
- 239000011521 glass Substances 0.000 description 10
- 238000000605 extraction Methods 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000009210 therapy by ultrasound Methods 0.000 description 6
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 239000000543 intermediate Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- QIXDHVDGPXBRRD-UHFFFAOYSA-N 2,3,5-trimethylcyclohexa-2,5-diene-1,4-dione Chemical compound CC1=CC(=O)C(C)=C(C)C1=O QIXDHVDGPXBRRD-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 150000004057 1,4-benzoquinones Chemical class 0.000 description 1
- 229930192627 Naphthoquinone Natural products 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical group O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000008204 material by function Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- -1 naphthoquinone compounds Chemical class 0.000 description 1
- 238000010525 oxidative degradation reaction Methods 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/66—Preparation of oxygen-containing organic compounds containing the quinoid structure
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a strain for oxidizing aromatic compounds and application thereof. The strain is used for screening a strain of pseudomonas from soil near a chemical plant and is named asPseudomonas spLk 08. The strain can grow and ferment by taking aromatic compounds as a unique carbon source, and the aromatic compounds are oxidized into quinone compounds. The invention provides a microbial strain capable of oxidizing aromatic compounds, has the characteristics of mild reaction, environmental friendliness and the like, and has wide application prospects in the aspects of environmental remediation and pharmaceutical intermediate synthesis.
Description
Technical Field
The invention relates to a method for oxidizing aromatic compounds, in particular to pseudomonas and application thereof in oxidizing aromatic compounds.
Background
The oxidation reaction of aromatic compounds relates to the synthesis of medical intermediates and functional materials, the metabolism of toxic substances, the biodegradation of pollutants and the like. However, the oxidation process of aromatic compounds still has the problems of complicated steps, low reaction selectivity, difficult post-treatment of the catalyst, low product yield, environmental pollution, and the like. Quinone is one of oxidation products of aromatic compounds, is widely applied to chemical product production and is an important medical intermediate. For example, 2, 3, 6-trimethyl-p-benzoquinone is one of the main intermediates for vitamin E synthesis, 2, 6-dimethyl-1, 4-p-benzoquinone is used as an anti-aging high-activity antioxidant, p-benzoquinone derivatives can inhibit the migration ability of HepG2 liver cancer cells, naphthoquinone compounds have broad-spectrum antibacterial effect and the like. Therefore, the development of the aromatic compound oxidation process with high purity, high yield, low pollution and low cost has great significance.
At present, 1, 4-naphthoquinone substances are industrially synthesized mainly by adopting a gas-phase oxidation method, a liquid-phase oxidation method and other chemical synthesis methods, and the problems of non-orientation, various byproducts and the like exist. The currently reported microbial strains for oxidative degradation of aromatic compounds have the main purpose of degrading substrates, and no report of a specified target synthetic product exists.
Disclosure of Invention
The invention aims to provide pseudomonas capable of oxidizing aromatic compoundsPseudomonas spLk08, the growth and fermentation conditions of the strain and the application in oxidizing aromatic compounds, and has the characteristics of mild reaction, environmental protection and the like.
The technical scheme adopted by the invention is as follows:
a strain for oxidizing aromatic compounds, which is classified and named as pseudomonasPseudomonas spLk08, deposited in the center of chinese type culture collection, with the address of the collection unit: the preservation number of the strain is CCTCC NO: M20191048 and the preservation date is 12 months and 16 days in 2019 at Wuhan university in Wuhan, China.
The pseudomonas isPseudomonas spScreening and characterization of lk 08:
strain screening
1) The strain source is as follows: separating and screening soil near a Nanjing Yangzi petrochemical plant;
2) strain screening: collecting soil from the neighborhood of Nanjing Yangzi petrochemical plant, placing in sterile sample bottle, taking to laboratory, mixing soil sample with sterile water to obtain bacterial suspension, centrifuging the mixture, and diluting the upper suspension to bacterial concentration of 10 6 Individual celland/mL, coating on the surface of an enrichment medium, and taking 1-naphthol as the only carbon source in the enrichment medium. The enrichment culture time is 48h, the culture temperature is 30 ℃, and a single colony is obtained. Single colonies were inoculated into the fermentation medium. These single colonies were cultured at 25-35 ℃ for 24-48 hours, and the culture broth was centrifuged to extract the supernatant. Measuring the content of quinone substances in the culture solution by high performance liquid chromatography; finally, the strain pseudomonas with the highest quinone content in the culture solution is screened outPseudomonas sp. lk08。
The enrichment medium is an inorganic salt medium and comprises 0.5 g/L1-naphthol and 4g/L Na 2 HPO 4 、1.5g/L KH 2 PO 4 、1 g/L NH 4 Cl、0.2g/L MgSO 4 •7H 2 O、0.02g/L CaCl 2 、0.03g/L FeSO 4 •7H 2 O、1.0g/L NaNO 3 1mL of trace element/L of medium (per 100mL of solution comprising 0.25 mg/L of CoCl) 2 •6H 2 O、4.0mg/L CuSO 4 •5H 2 O、4.3mg/L MnSO 4 •5H 2 O、4.3mg/L ZnSO 4 •7H 2 O）。
The strain morphological characteristics and physiological and biochemical characteristics are as follows: the pseudomonas isPseudomonas spLk08 colonies were yellow, round, moist and smooth on the surface, convex in the center, opaque at the edges, growth temperature of the strain 25-35 ℃, pH 7.0-8.0, gram negative.
The pseudomonas isPseudomonas spUse of lk08 in the oxidation of aromatic compounds.
By pseudomonasPseudomonas spUsing the cell fermented and cultured by lk08 as a biocatalyst, using phenol and 1-naphthol as substrates, and oxidizing the substrates to obtain the required product, wherein the reaction temperature is 25-37 ℃, the pH value is 7.0-8.0, and the reaction time is 10-12 h.
The biocatalyst is pseudomonasPseudomonas spThe fermentation culture solution obtained after lk08 fermentation culture, wherein the concentration of the substrate is 0.5-1.5 g/L, and the substrate comprises phenol, 1-naphthol and the like.
The biocatalyst is prepared by the following steps:
(1) plate culture: pseudomonas spPseudomonas spLk08 was inoculated in solid medium, cultured at 30-37 ℃ for 12-24h, to obtain single colonies on the plate: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder, pH7-8 and water as a solvent;
(2) seed culture: selecting a single colony from a solid plate, inoculating the single colony to a seed culture medium, and culturing at 30-37 ℃ and 200rpm for 8 hours to obtain a seed solution, wherein the concentration of the seed culture medium consists of: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent;
(3) fermentation culture: inoculating the seed liquid to a fermentation culture medium with the inoculation amount of 1%, culturing at 30-37 ℃ and 200rpm for 12h to obtain a fermentation liquid, extracting, performing ultrasonic treatment and 6000rpm centrifugation on the fermentation liquid for 10min, taking out the supernatant, and performing vacuum drying to obtain the required product. The concentration of the fermentation medium is as follows: 0.5-1.5 g/L of aromatic compound, 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as solvent.
Furthermore, the pseudomonas isPseudomonas spThe use of lk08 in the oxidation of aromatic compounds is: by pseudomonasPseudomonas spLk08 cells cultured by fermentation are used as biocatalyst, phenol and 1-naphthol are used as substrate, the temperature is 30 ℃, the pH value is 7.0, the fermentation culture is carried out for 12h, the product yield is the highest, and the hydroxylation of the substrate is determined by extraction, centrifugation, vacuum drying and liquid phase detection.
The extraction method comprises the following steps: absorbing 25mL of fermentation liquid into a 50mL shaking tube, adding 5mL of methyl tert-butyl ether into the tube, concentrating according to the ratio of 5:1, shaking, extracting for three minutes, centrifuging: after the extraction is finished, the ultrasonic treatment is carried out for 30min, then the centrifugation is carried out for 10min at 6000rpm, 2mL of supernatant is sucked into a clean glass tube,
the vacuum drying method comprises the following steps: putting the glass tube into a vacuum drying oven, vacuum-pumping for 2h, and volatilizing ether in the tube to obtain the desired product
The liquid phase detection and analysis: 500 mL of methanol was added to the tube and after the product had dissolved, the tube was tested for analysis by membrane loading. The mobile phase is acetonitrile-water solution, the concentration of acetonitrile is 50%, the column temperature is 30 ℃, the flow rate of the mobile phase is 1 mL/min, and the chromatographic column is C18.
Has the advantages that:
compared with the prior art, the invention has the following beneficial effects: the strain can oxidize 1-naphthol to generate quinone compounds, has the characteristics of mild reaction, environmental friendliness and the like, and has wide application prospects in the aspects of environmental remediation, medical intermediate synthesis and the like.
Detailed Description
The invention is described in one step with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1 Strain screening
Collecting soil from the neighborhood of Nanjing Yangzi petrochemical plant, placing in a sterile sample bottle to bring to the laboratory, mixing the soil sample with sterile water to obtain a bacterial suspension, centrifuging the mixture, and diluting the upper suspension in gradient to a bacterial concentration of 10 6 cells/mL and coated on the surface of enrichment medium (containing 0.5 g/L of 1-naphthol each), 1-naphthol is used as carbon source in the enrichment medium. The enrichment culture time is 48h, and the culture temperature is 30 ℃. The single colony obtained above was inoculated in a primary screening medium (containing 1-naphthol 0.5 g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 3 g/L, magnesium sulfate 1 g/L, ferrous sulfate 1 g/L, manganese sulfate 0.5 g/L, sodium dihydrogen phosphate 2.5 g/L, calcium carbonate 1.7 g/L, pH 7.0-8.0). These single colonies were cultured at 25-35 ℃ for 24-48 hours, and single colonies were collected. The single colony obtained above is inoculated into a fermentation medium (aromatic compound 0.5-1.5 g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 5g/L, pH7-8, solvent is water) and cultured for 24-48 h at 25-35 ℃. And (3) determining the content of the 1, 4-naphthoquinone in the culture solution by adopting a high performance liquid chromatography, and screening out the bacterial colony with the highest yield. Finally, the strain pseudomonas with the highest content of 1, 4-naphthoquinone in the culture solution is screened outPseudomonas sp. lk08。
EXAMPLE 2 Oxidation of 1-Naphthol to prepare 1, 4-naphthoquinone
1) Plate culture: pseudomonas spPseudomonas spLk08 was inoculated in solid medium and incubated at 30 ℃ for 24h to obtain single colonies on the plate, said solid culture beingThe base is as follows: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder, pH7-8 and water as a solvent;
2) seed culture: selecting a single colony from a solid plate, inoculating the single colony to a seed culture medium, and culturing at 30 ℃ and 200rpm for 8h to obtain a seed solution, wherein the concentration of the seed culture medium is as follows: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent;
3) fermentation culture: inoculating the seed liquid to a fermentation culture medium with the inoculation amount of 1%, culturing at 30 ℃ and 200rpm for 12h to obtain a fermentation liquid, extracting, performing ultrasonic treatment and 6000rpm centrifugation on the fermentation liquid for 10min, taking out the supernatant, and performing vacuum drying to obtain the required product. The concentration of the fermentation medium is as follows: 0.5 g/L of 1-naphthol, 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent.
4) Sucking 25mL of fermentation liquid into a 50mL shaking tube, adding 5mL of methyl tert-butyl ether into the tube, concentrating according to a ratio of 5:1, shaking, extracting for three minutes, and centrifuging: after the extraction was completed, sonication was performed for 30min, followed by centrifugation at 6000rpm for 10min, and 2mL of the supernatant was aspirated into a clean glass tube.
5) And (3) putting the glass tube into a vacuum drying oven, extracting for 2 hours in vacuum, and volatilizing the ether in the tube to obtain the required product. The yield of 1, 4-naphthoquinone is 0.11mg/L by liquid chromatography analysis.
EXAMPLE 3 Oxidation of the substrate
1) Plate culture: pseudomonas spPseudomonas spLk08 was inoculated in a solid medium, which was: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder, pH7-8 and water as a solvent;
2) seed culture: selecting a single colony from a solid plate, inoculating the single colony to a seed culture medium, and culturing at 30 ℃ and 200rpm for 8 hours to obtain a seed solution, wherein the seed culture medium comprises the following components in concentration: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent;
3) fermentation culture: inoculating the seed liquid to a fermentation culture medium with the inoculation amount of 1%, culturing at 30 ℃ and 200rpm for 12h to obtain a fermentation liquid, extracting, performing ultrasonic treatment and 6000rpm centrifugation on the fermentation liquid for 10min, taking out the supernatant, and performing vacuum drying to obtain the required product. The concentration of the fermentation medium is as follows: 1.5 g/L of phenol, 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent.
4) Absorbing 25mL of fermentation liquid into a 50mL shaking tube, adding 5mL of methyl tert-butyl ether into the tube, concentrating according to the ratio of 5:1, shaking, extracting for three minutes, centrifuging: after the extraction was completed for 30min, followed by centrifugation at 6000rpm for 10min, 2mL of the supernatant was pipetted into a clean glass tube.
5) And (3) putting the glass tube into a vacuum drying oven, extracting for 2 hours in vacuum, and volatilizing the ether in the tube to obtain the required product. The liquid chromatography analysis shows that the output of the p-benzoquinone is 0.40 mg/L.
EXAMPLE 4 Oxidation of the substrate
1) Plate culture: pseudomonas spPseudomonas spLk08 was inoculated in a solid medium, which was: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder, pH7-8 and water as a solvent;
2) seed culture: selecting a single colony from a solid plate, inoculating the single colony to a seed culture medium, and culturing at 30 ℃ and 200rpm for 8h to obtain a seed solution, wherein the concentration of the seed culture medium is as follows: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent;
3) fermentation culture: inoculating the seed liquid to a fermentation culture medium with the inoculation amount of 1%, culturing at 30 ℃ and 200rpm for 12h to obtain a fermentation liquid, extracting, performing ultrasonic treatment and 6000rpm centrifugation on the fermentation liquid for 10min, taking out the supernatant, and performing vacuum drying to obtain the required product. The concentration of the fermentation medium is as follows: 1.0 g/L of phenol, 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent.
4) Sucking 25mL of fermentation liquid into a 50mL shaking tube, adding 5mL of methyl tert-butyl ether into the tube, concentrating according to a ratio of 5:1, shaking, extracting for three minutes, and centrifuging: after the extraction was completed, sonication was performed for 30min, followed by centrifugation at 6000rpm for 10min, and 2mL of the supernatant was aspirated into a clean glass tube.
5) And (3) putting the glass tube into a vacuum drying oven, vacuumizing for 2h, and volatilizing ether in the tube to obtain the required product. The liquid chromatography analysis shows that the output of the p-benzoquinone is 0.52 mg/L.
Example 5 genetic stability of the Strain
And selecting single colonies from the solid culture medium, streaking the single colonies onto a new solid plate for culture, and carrying out first passage when new single colonies grow out, so that the single colonies are continuously subjected to 6 times. And selecting a single colony from each generation of separation plate to perform seed culture and fermentation culture, performing product pretreatment, and analyzing the output of p-benzoquinone by liquid chromatography to determine the stability of the strain. The culture conditions of each generation are as follows:
1) plate culture: pseudomonas spPseudomonas spLk08 was inoculated in solid medium, which was: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder, pH7-8 and water as a solvent;
2) seed culture: selecting a single colony from a solid plate, inoculating the single colony to a seed culture medium, and culturing at 30 ℃ and 200rpm for 8h to obtain a seed solution, wherein the concentration of the seed culture medium is as follows: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent;
3) fermentation culture: inoculating the seed liquid to a fermentation culture medium with the inoculation amount of 1%, culturing at 30 ℃ and 200rpm for 12h to obtain a fermentation liquid, extracting, performing ultrasonic treatment and 6000rpm centrifugation on the fermentation liquid for 10min, taking out the supernatant, and performing vacuum drying to obtain the required product. The concentration of the fermentation medium is as follows: 1.0 g/L of phenol, 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent.
4) Sucking 25mL of fermentation liquid into a 50mL shaking tube, adding 5mL of methyl tert-butyl ether into the tube, concentrating according to a ratio of 5:1, shaking, extracting for three minutes, and centrifuging: after the extraction was completed for 30min, followed by centrifugation at 6000rpm for 10min, 2mL of the supernatant was pipetted into a clean glass tube.
5) And (3) putting the glass tube into a vacuum drying oven, extracting for 2 hours in vacuum, and volatilizing the ether in the tube to obtain the required product. The results of the liquid chromatography analysis are shown in Table 1.
TABLE 1
| Passage of culture | P-benzoquinone yield (mg/L) |
| F1 | 0.52 |
| F2 | 0.51 |
| F3 | 0.48 |
| F4 | 0.54 |
| F5 | 0.55 |
| F6 | 0.53 |
Claims (8)
1. Pseudomonas bacteria (Pseudomonas spLk08, preserved in China center for type culture Collection with the preservation number of CCTCC NO: M20191048, and the preservation date is 2019, 12 months and 16 days.
2. Use of pseudomonas lk08 of claim 1 in the oxidation of naphthol or phenol.
3. The use of claim 2, wherein the cell obtained by fermentation culture of pseudomonas lk08 is used as a biocatalyst, the concentration of the naphthol or phenol substrate is 0.5-1.5 g/L, and the substrate is oxidized by adopting a one-step fermentation method.
4. The application of claim 3, wherein pseudomonad lk08 cell is used as biocatalyst, naphthol and phenol are used as substrates, the reaction temperature is 25-37 ℃, the pH value is 7-8, and the reaction time is 10-12 h.
5. The use of claim 4, wherein said Pseudomonas lk08 cells are prepared by the following steps:
(1) plate culture: inoculating pseudomonas lk08 in a solid culture medium, and culturing at 30-37 ℃ for 12-24h to obtain a single colony on a plate;
(2) seed culture: selecting a single colony from the solid plate, inoculating the single colony to a seed culture medium, and culturing at 30-37 ℃ and 200rpm for 8h to obtain a seed solution;
(3) fermentation culture: inoculating the seed liquid to a fermentation culture medium by an inoculation amount of 1%, and culturing at 30-37 ℃ and 200rpm for 12h to obtain a fermentation liquid.
6. Use according to claim 5, wherein the solid medium is: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, 20g/L of agar powder, pH7-8 and water as a solvent.
7. Use according to claim 5, wherein the seed medium consists of: 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent.
8. Use according to claim 5, wherein the fermentation medium consists of: 0.5-1.5 g/L of naphthol or phenol, 10g/L of peptone, 5g/L of yeast powder, 5g/L of sodium chloride, pH7-8 and water as a solvent.
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