CN109536392B - Aspergillus versicolor ZJB16085 and its application in the synthesis of R-2-(4-hydroxyphenoxy)propionic acid - Google Patents
Aspergillus versicolor ZJB16085 and its application in the synthesis of R-2-(4-hydroxyphenoxy)propionic acid Download PDFInfo
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- CN109536392B CN109536392B CN201811635789.2A CN201811635789A CN109536392B CN 109536392 B CN109536392 B CN 109536392B CN 201811635789 A CN201811635789 A CN 201811635789A CN 109536392 B CN109536392 B CN 109536392B
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- aspergillus versicolor
- hydroxyphenoxy
- propionic acid
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- 241000203233 Aspergillus versicolor Species 0.000 title claims abstract description 38
- AQIHDXGKQHFBNW-ZCFIWIBFSA-N (2r)-2-(4-hydroxyphenoxy)propanoic acid Chemical compound OC(=O)[C@@H](C)OC1=CC=C(O)C=C1 AQIHDXGKQHFBNW-ZCFIWIBFSA-N 0.000 title claims abstract description 37
- 230000015572 biosynthetic process Effects 0.000 title abstract description 8
- 238000003786 synthesis reaction Methods 0.000 title abstract description 8
- SXERGJJQSKIUIC-SSDOTTSWSA-N (2r)-2-phenoxypropanoic acid Chemical compound OC(=O)[C@@H](C)OC1=CC=CC=C1 SXERGJJQSKIUIC-SSDOTTSWSA-N 0.000 claims abstract description 27
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 13
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- 238000000855 fermentation Methods 0.000 claims description 37
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- 239000001963 growth medium Substances 0.000 claims description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 16
- 239000008367 deionised water Substances 0.000 claims description 14
- 229910021641 deionized water Inorganic materials 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 12
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 5
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 5
- 229940041514 candida albicans extract Drugs 0.000 claims description 5
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 claims description 5
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 5
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 5
- ISPYRSDWRDQNSW-UHFFFAOYSA-L manganese(II) sulfate monohydrate Chemical compound O.[Mn+2].[O-]S([O-])(=O)=O ISPYRSDWRDQNSW-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
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- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- 239000004327 boric acid Substances 0.000 claims description 3
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 claims description 3
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 claims description 3
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- MPTQRFCYZCXJFQ-UHFFFAOYSA-L copper(II) chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Cu+2] MPTQRFCYZCXJFQ-UHFFFAOYSA-L 0.000 claims 1
- 239000002054 inoculum Substances 0.000 claims 1
- LAIZPRYFQUWUBN-UHFFFAOYSA-L nickel chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Ni+2] LAIZPRYFQUWUBN-UHFFFAOYSA-L 0.000 claims 1
- KSLUMEQTEAUMJZ-UHFFFAOYSA-L zinc;sulfate;tetrahydrate Chemical compound O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O KSLUMEQTEAUMJZ-UHFFFAOYSA-L 0.000 claims 1
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- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 14
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 description 10
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- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 description 2
- TXFPEBPIARQUIG-UHFFFAOYSA-N 4'-hydroxyacetophenone Chemical compound CC(=O)C1=CC=C(O)C=C1 TXFPEBPIARQUIG-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 2
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- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- 229960003767 alanine Drugs 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- -1 aryloxy phenoxy propionic acid Chemical compound 0.000 description 2
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- 238000010170 biological method Methods 0.000 description 2
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- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 150000004683 dihydrates Chemical class 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
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- NESLWCLHZZISNB-UHFFFAOYSA-M sodium phenolate Chemical compound [Na+].[O-]C1=CC=CC=C1 NESLWCLHZZISNB-UHFFFAOYSA-M 0.000 description 2
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- 229960001763 zinc sulfate Drugs 0.000 description 2
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- XLHUBROMZOAQMV-UHFFFAOYSA-N 1,4-benzosemiquinone Chemical group [O]C1=CC=C(O)C=C1 XLHUBROMZOAQMV-UHFFFAOYSA-N 0.000 description 1
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- AQIHDXGKQHFBNW-UHFFFAOYSA-N 2-(4-hydroxyphenoxy)propanoic acid Chemical compound OC(=O)C(C)OC1=CC=C(O)C=C1 AQIHDXGKQHFBNW-UHFFFAOYSA-N 0.000 description 1
- SXERGJJQSKIUIC-UHFFFAOYSA-N 2-Phenoxypropionic acid Chemical compound OC(=O)C(C)OC1=CC=CC=C1 SXERGJJQSKIUIC-UHFFFAOYSA-N 0.000 description 1
- MPPOHAUSNPTFAJ-UHFFFAOYSA-N 2-[4-[(6-chloro-1,3-benzoxazol-2-yl)oxy]phenoxy]propanoic acid Chemical compound C1=CC(OC(C)C(O)=O)=CC=C1OC1=NC2=CC=C(Cl)C=C2O1 MPPOHAUSNPTFAJ-UHFFFAOYSA-N 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/66—Aspergillus
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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- 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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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/42—Hydroxy-carboxylic acids
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Abstract
The invention discloses Aspergillus versicolor (Aspergillus versicolor) ZJB16085 and application thereof in synthesis of R-2- (4-hydroxyphenoxy) propionic acid, wherein the preservation number is CCTCC NO: M2018408. The invention separates Aspergillus versicolor (Aspergillus versicolor) CCTCC NO: M2018408 which can effectively convert R-2-phenoxypropionic acid into R-2- (4-hydroxyphenoxy) propionic acid from humus soil, and can synthesize 4.5g/L of R-2- (4-hydroxyphenoxy) propionic acid under the condition of 10g/L of substrate, and the conversion rate is 45%.
Description
Technical Field
The invention relates to a microorganism for synthesizing R-2- (4-hydroxyphenoxy) propionic acid by converting R-2 phenoxypropionic acid and a screening method thereof, belongs to the technical field of bioengineering, and particularly relates to aspergillus versicolor for synthesizing R-2- (4-hydroxyphenoxy) propionic acid by converting R-2 phenoxypropionic acid.
Background
R-2- (4-hydroxyphenoxy) propionic acid is a key intermediate in the synthesis process of phenoxy carboxylic acid and aryloxy phenoxy propionic acid herbicides, and can be used for synthesizing herbicides such as fenoxaprop (soybean, rice), fluazifop-p-butyl (rape, potato), clodinafop-propargyl (grain) and the like. As phenoxy carboxylic acid herbicides are receiving increased attention, their advantages as herbicides are also increasingly recognized. As chiral pesticide with enantiomer, compared with racemic pesticide, the chiral pesticide has low efficiency and high environmental pollution, and the optically pure phenoxy carboxylic acid herbicide has the advantages of high weeding efficiency, low harm to crops, low environmental pollution and the like. The synthesis of phenoxy carboxylic acid herbicides uses chiral R-2- (4-hydroxyphenoxy) propionic acid as an intermediate, so the development of efficient synthesis methods of R-2- (4-hydroxyphenoxy) propionic acid is attracting much attention.
Elango V et al (U.S. Pat. No. 3, 5008439A) reported that p-hydroxyacetophenone as a starting material was reacted with an α -halopropionate, oxidized and hydrolyzed to give a racemic modification of 2- (4-hydroxyphenoxy) propionic acid, which was further resolved to give R-2- (4-hydroxyphenoxy) propionic acid of high optical purity. The technical route is systematically improved by Shenhengxi et al (synthetic chemistry, 2006, 04: 398-. Although the product is easy to separate and purify, the method needs multi-step reaction to complete, so that the yield of the product is not high, and meanwhile, reaction raw materials and reagents are not suitable for large-scale production.
Umbelliferae et al (proceedings of Zhejiang university of Industrial science 2009, 04: 362-365) reported that high-optical-purity R-2- (4-hydroxyphenoxy) propionic acid was produced by esterification, sulfonylation, etherification and the like using L-lactic acid as a starting compound. Wherein the configuration is reversed in the reaction of S-2- (4-tosyloxy) propionic acid to generate R-2- (4-hydroxyphenoxy) propionic acid, and the reaction mechanism is as follows: under the condition of sodium hydroxide, hydroquinone is firstly generated into sodium phenolate, ethyl S-2- (4-tosyloxy) propionate is changed into sodium salt, sodium ions are positively charged, oxygen of the sodium phenolate is negatively charged, chiral carbon in the sodium S-2- (4-tosyloxy) propionate is attacked from the surface with small steric hindrance, and after an intermediate is formed, the configuration is inverted and is changed into an R type. Wuchunlei et al (Guangzhou chemical 2015, 08:92-93) reported that L-alanine was used as a starting compound, and the product R-2- (4-hydroxyphenoxy) propionic acid was produced by steps of chlorination, etherification and acidification, in which the configuration was inverted during the etherification reaction. The process route of the L-alanine and the L-lactic acid is simpler, the reaction steps are fewer, but the conditions are difficult to control. Because hydroquinone participates in the reaction process, the hydroquinone is easy to be oxidized to generate quinone in an alkaline environment, so that the disubstituted product of the hydroquinone and the hydroquinone which are not completely reacted is difficult to remove in the later purification treatment, and the purity of the product is influenced. Therefore, in the synthesis process, the reaction conditions and the material ratio need to be strictly controlled to prevent side reactions from occurring.
At present, few studies have been made in foreign countries on the biological production of R-2- (4-hydroxyphenoxy) propionic acid, and among them, BASF company Dingler et al (Pest Management Science,2015,46(1):33-35) have reported that R-2- (4-hydroxyphenoxy) propionic acid is synthesized by hydroxylating the substrate R-2-phenoxypropionic acid by Beauveria bassiana. Kinne M et al (Tetrahedron Letters,2008,49(41):5950-2O2Under the condition, the racemic 2-phenoxypropionic acid is selectively hydroxylated to generate R-2- (4-hydroxyphenoxy) propionic acid. With the attention of people on environmental protection, the original chemical industry is impacted to a certain extent, and meanwhile, the rapid development of the green biological manufacturing industry is promoted, so that the biological method for preparing the R-2- (4-hydroxyphenoxy) propionic acid has a great application prospect.
Disclosure of Invention
The invention aims to provide Aspergillus versicolor (Aspergillus versicolor) ZJB16085 and a method for synthesizing R-2- (4-hydroxyphenoxy) propionic acid by converting R-2 phenoxypropionic acid.
The technical scheme adopted by the invention is as follows:
the invention provides Aspergillus versicolor (Aspergillus versicolor) ZJB16085 which is preserved in China center for type culture collection with the preservation number of CCTCC NO: M2018408, the preservation date of 2018, 6 months and 28 days, the preservation address of Wuhan university, China, and the postal code of 430072.
M2018408 is subjected to enrichment culture twice from humus soil, strains which can grow in a culture medium containing a substrate R-2-phenoxypropionic acid are selected to be inoculated into a 96-pore plate fermentation primary sieve, a 4-aminoantipyrine color development method is adopted to screen out primary sieve strains which can convert the substrate R-2-phenoxypropionic acid into R-2- (4-hydroxyphenoxy) propionic acid, and then the primary sieve strains are transferred into a shake flask filled with the fermentation culture medium for fermentation and secondary sieve.
The invention also provides application of the aspergillus versicolor ZJB16085 in catalyzing R-2 phenoxypropionic acid to synthesize R-2- (4-hydroxyphenoxy) propionic acid, wherein fermentation liquor obtained by fermentation culture of the aspergillus versicolor ZJB16085 is used as a catalyst and a reaction medium, a substrate R-2 phenoxypropionic acid is added, conversion reaction is carried out at the temperature of 25-30 ℃ and the shaking table rotation speed of 150-. The final concentration of the substrate in the fermentation liquor is 10-50g/L, preferably 10 g/L; the content of wet bacteria in the fermentation liquor is 15-20 g/L, and preferably 20 g/L.
Further, a fermentation liquid obtained by fermentation culture of the aspergillus versicolor ZJB16085 is prepared according to the following method: inoculating Aspergillus versicolor ZJB16085 into a fermentation culture medium, and performing fermentation culture (preferably culture until the wet thallus content in the fermentation broth is 15-20 g/L) at the temperature of 25-30 ℃ and the shaking table rotation speed of 150-; the fermentation medium comprises the following components: 5-20g/L glucose, 5-10g/L yeast extract, 5-10g/L ammonium sulfate, 0.5-1.0g/L magnesium sulfate heptahydrate, 0.05-0.12g/L manganese sulfate monohydrate, 1.5-2.5g/L potassium dihydrogen phosphate, 3.6-6.0g/L dipotassium hydrogen phosphate trihydrate, 1-5mL/L trace element liquid, deionized water as a solvent, 5M sodium hydroxide solution for adjusting the pH value to 6.8, and sterilizing at 115 ℃ for 20 min; the trace element liquid comprises the following components: 2g/L of ferrous sulfate heptahydrate, 100mg/L of zinc sulfate (II) tetrahydrate, 300mg/L of boric acid, 200mg/L of cobalt chloride (II) hexahydrate, 10mg/L of copper chloride (II) dihydrate, 20mg/L of nickel chloride (II) hexahydrate and 30mg/L of sodium molybdate dihydrate, and deionized water is used as a solvent.
Further, the fermentation medium composition is: 5g/L of glucose, 5g/L of yeast extract, 5g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate monohydrate, 1.5g/L of potassium dihydrogen phosphate, 3.6g/L of dipotassium hydrogen phosphate trihydrate, 1mL/L of trace element liquid and deionized water as a solvent, adjusting the pH value to 6.8 by using 5M sodium hydroxide solution, and sterilizing at 115 ℃ for 20 min.
Further, before inoculation, aspergillus versicolor ZJB16085 is subjected to slant activation and seed culture, and then the seed solution is inoculated to a fermentation medium in an inoculation amount with the volume concentration of 3-10% (preferably 3%), specifically: inoculating Aspergillus versicolor ZJB16085 to slant culture medium, and standing at 28 deg.C for 6-7 days to obtain slant strain; selecting thalli from the inclined plane, inoculating the thalli into a 250mL shaking flask filled with 50mL seed culture medium, and culturing at 28 ℃ and 150-; the slant culture medium (PDA medium): 200g/L of potato, 20g/L of glucose, 20g/L of agar and deionized water as a solvent, wherein the pH is natural, and the potato is sterilized at 115 ℃ for 20 min; the seed medium (PDA medium): 200g/L of potato, 20g/L of glucose and deionized water as a solvent, wherein the pH is natural, and the potato is sterilized at 115 ℃ for 20 min.
The invention establishes a method for quickly and effectively screening a microorganism strain for synthesizing R-2- (4-hydroxyphenoxy) propionic acid by a biological method, namely, designs an experimental scheme aiming at screening microorganisms which can grow in a culture medium containing a substrate R-2-phenoxypropionic acid and can convert the R-2-phenoxypropionic acid to synthesize the R-2- (4-hydroxyphenoxy) propionic acid: firstly, enriching microorganisms in humus soil in a culture medium containing a substrate R-2-phenoxypropionic acid, and primarily screening strains capable of growing on an enrichment culture medium plate. Then the ability of converting R-2-phenoxypropionic acid into R-2- (4-hydroxyphenoxy) propionic acid is detected, namely, a single colony which grows out is selected and inoculated into a 96 deep-hole plate containing a fermentation culture medium (the formula is the same as that of an enrichment culture medium) for culture, and the ability of each strain to convert a substrate into a product is detected by a 4-aminoantipyrine color development method. And preserving the screened strains to obtain primary screened strains. And respectively inoculating the strains obtained by primary screening into 250mL triangular flasks filled with 50mL fermentation medium (the formula of the enrichment medium is the same as that of the enrichment medium) for secondary screening, culturing for about 7 days, and detecting the concentration of the R-2- (4-hydroxyphenoxy) propionic acid in the fermentation liquor by HPLC. Through the screening work of a large number of strains of a plurality of soil samples, the ZJB16085 strain with higher transformation capacity is screened from more than 1000 separated soil microorganisms.
The microorganism ZJB16085 which is obtained by screening and can effectively convert R-2-phenoxypropionic acid to synthesize R-2- (4-hydroxyphenoxy) propionic acid is cultured on a solid flat plate for about 7 days, the diameter of a bacterial colony is about 1.8cm, the bacterial colony is loose, the hypha is short, the texture is villous, the middle part is convex, the bacterial colony is concentric, the color is gradually changed from dark green to cyan, and the edge part is white. The edge part of the back of the colony is white, and the middle part of the colony gradually becomes light yellow. Observed under an optical microscope, hyphae are long and sparse and have branch shapes, spores are formed at the top of the hyphae, and the number of the spores is small. Observing under electron microscope, the mycelium is smooth and branched, and the diameter of the mycelium is 2.0-3.0 μm; the top sac is enlarged, the single round of conidiophore looks like wheat spike, and no separation exists. Conidiophores are oval or nearly spherical, single spores, 4.0-5.0 μm in diameter, and according to morphology, the strain was preliminarily determined to be Aspergillus (Aspergillus). In combination with the morphological analysis of microorganisms and the identification of molecular biology, Aspergillus versicolor (Aspergillus versicolor) is considered to belong to the genus Aspergillus of the family Hyphomycetes.
Compared with the prior art, the invention has the following beneficial effects: the invention separates Aspergillus versicolor (Aspergillus versicolor) CCTCC NO: M2018408 which can effectively convert R-2-phenoxypropionic acid into R-2- (4-hydroxyphenoxy) propionic acid from humus soil, and can synthesize 4.5g/L of R-2- (4-hydroxyphenoxy) propionic acid under the condition of 10g/L of substrate, and the conversion rate is 45%.
Drawings
The results of the preliminary screening of the strains of FIG. 1 are shown in the letter symbols for the column number and the number symbols for the row number.
FIG. 2R-2- (4-hydroxyphenoxy) propionic acid standard (A), R-2-phenoxypropionic acid standard (B) and strains.
HPLC chromatogram of ZJB16085 fermentation conversion liquid (C).
FIG. 3 is a front (A) and back (B) morphogram of the ZJB16085 strain.
FIG. 4 shows the mycelium morphology of strain ZJB16085 under an optical microscope at 10X 10 times.
FIG. 5 SEM electron micrograph of strain ZJB 16085.
FIG. 6 phylogenetic tree of strain ZJB 16085.
The specific implementation mode is as follows:
the following is an example of R-2- (4-hydroxyphenoxy) propionic acid synthesis by screening, identifying and fermenting R-2-phenoxypropionic acid with Aspergillus versicolor (CCTCC NO: M2018408).
Example 1: screening of Strain ZJB16085
(1) Sampling soil from multiple plant growing areas in campus of Zhejiang industry university, weighing 1g of soil sample (wet weight), preparing suspension with 9mL of 0.9% physiological saline, sucking 1mL of the suspension, inoculating the suspension into a 250mL triangular flask containing 50mL of enrichment medium containing 10g/L R-2-phenoxypropionic acid, culturing on a shaking table at 28 ℃ and 150rpm for 1-2d, and performing first enrichment culture; taking 1mL of the bacterial liquid of the first enrichment culture to perform the second enrichment culture in a fresh enrichment culture medium containing 10g/L R-2-phenoxypropionic acid under the same conditions. Carrying out gradient dilution on the bacterial liquid subjected to the second enrichment culture, and respectively taking 100 mu L of diluted 103And 105Spreading the diluted solution on a screening medium plate, and performing static culture in a constant temperature incubator at 28 ℃ for 3-4 d. A single colony grown on the screening plate is picked by using a sterile toothpick and inoculated in a 96-deep-well plate (1 mL of fermentation medium containing 10g/L R-2-phenoxypropionic acid is added in each well), after the single colony is cultured for 6-7d at 28 ℃ and 150rpm, the 96-deep-well plate is centrifuged for 15min at 1000rpm, 100 mu L of supernatant is absorbed in the 96-deep-well plate, and the capacity of each strain for converting a substrate into a product is detected by using a 4-aminoantipyrine color development method (figure 1). 64 primary screening strains with transformation ability were preserved.
(2) Inoculating the primary screened strain into a 250mL shaking flask containing 50mL of seed culture medium, culturing at 28 ℃ and 150rpm for 2-3d, inoculating the primary screened strain into a 250mL shaking flask containing 50mL of fermentation culture medium containing 10g/L R-2-phenoxypropionic acid at a volume concentration of 3%, performing fermentation and re-screening at 28 ℃ and 150rpm for 7d, centrifuging at 10000rpm for 10min, filtering the supernatant through a 0.22 μm microporous membrane, and detecting the concentration of R-2-phenoxypropionic acid and R-2- (4-hydroxyphenoxy) propionic acid by HPLC (an Erite C18 reverse phase column of 250mm x 4.6mm, a mobile phase V (a phosphoric acid aqueous solution with pH of 2): V (acetonitrile): 3: 2, a flow rate of 1mL/min, a detector DAD, a detection wavelength of 210nm, and a column temperature of 30 ℃). The retention times of R-2-phenoxypropionic acid and R-2- (4-hydroxyphenoxy) were 8.1min and 3.9min, respectively, as shown in FIG. 2. And (3) screening out strains with higher transformation capacity from the 64 strains to obtain a re-screened strain ZJB16085, and performing slant preservation.
Among them, the enrichment medium of this example consists of: 5g/L of glucose, 5g/L of yeast extract, 5g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate monohydrate, 1.5g/L of potassium dihydrogen phosphate, 3.6g/L of dipotassium hydrogen phosphate trihydrate, 1mL/L of trace element liquid and deionized water as a solvent, adjusting the pH value to 6.8 by using 5M sodium hydroxide solution, and sterilizing at 115 ℃ for 20 min.
The trace element liquid comprises the following components: 2g/L of ferrous sulfate heptahydrate, 100mg/L of zinc sulfate (II) tetrahydrate, 300mg/L of boric acid, 200mg/L of cobalt chloride (II) hexahydrate, 10mg/L of copper chloride (II) dihydrate, 20mg/L of nickel chloride (II) hexahydrate and 30mg/L of sodium molybdate dihydrate, and deionized water is used as a solvent.
Screening a culture medium: 20g/L agar is added in the enrichment medium formula.
Slant medium (PDA medium): 200g/L of potato, 20g/L of glucose, 20g/L of agar and deionized water as a solvent, wherein the pH is natural, and the potato is sterilized at 115 ℃ for 20 min.
Seed medium (PDA medium): 200g/L of potato, 20g/L of glucose and deionized water as a solvent, wherein the pH is natural, and the potato is sterilized at 115 ℃ for 20 min.
The fermentation medium is composed of the same enrichment medium.
4-aminoantipyrine color development: 100 mu L of sample to be tested is added into a 96 micro-porous plate, and 0.06g/L potassium ferricyanide is added into the 4-aminoantipyrine reagent to prepare the color developing agent. Adding 100 mu L of color developing agent into each micropore, reacting for 20min at 30 ℃, and detecting the light absorption value of the sample at 550nm by using an enzyme-labeling instrument.
The preparation of the 4-aminoantipyrine reagent comprises the following steps: 0.6g of 4-aminoantipyrine, 2mL of 1mol/L NaOH, 10mL of 20% Na2CO3The volume is 100 mL. Stored in brown bottle and protected from light.
Example 2: identification of Strain ZJB16085
(1) Observation of colony morphology
A small amount of spores of the strain are picked by an inoculating needle, a PDA plate is streaked, the strain is cultured for about 7 days at the temperature of 28 ℃, and the single colony morphology is shown in figure 3. The diameter of the colony is about 1.8cm, the colony is loose, the hypha is short, the texture is villiform, the middle is raised, the concentric circles are shaped, the color is gradually changed from dark green to cyan, and the most edge part is white. The edge part of the back of the colony is white, and the middle part of the colony gradually becomes light yellow.
(2) Observation of mycelium morphology
Spreading a round filter paper on the bottom of a culture dish with a diameter of 9.0cm, placing a U-shaped glass rod on the round filter paper, placing a clean glass slide and two cover slips on the glass rod, covering the dish with a cover, sterilizing, inoculating, culturing at 28 deg.C for about 7d, and observing the hypha shape under an optical microscope at a power of 10 × 10 times, as shown in FIG. 4. The hyphae are long and sparse and have branch shapes, spores are formed on the top of the hyphae, and the number of the spores is small.
(3) SEM electron microscope observation
The SEM results are shown in FIG. 5. The strain has smooth hypha with branches, and the diameter of the hypha is 2.0-3.0 μm; the top sac is enlarged, the single round of conidiophore looks like wheat spike, and no separation exists. Conidiophores are oval or nearly spherical, single spores, 4.0-5.0 μm in diameter, and according to morphology, the strain was preliminarily determined to be Aspergillus (Aspergillus).
(4) Molecular identification
Chromosomal DNA was extracted according to the molecular kit instructions, and 18S rDNA sequences were PCR-amplified using total DNA as template and ITS1 (5'-TC CGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') as forward and reverse primers. The PCR amplification system was (50 mL): 10 × Taq Buffer (Mg)2+)mu.L of 5. mu.L, 1. mu.L of 10mM dNTPs, 0.5. mu.L of Taq DNA polymerase, 0.5. mu.L of each of primers ITS1 and ITS4, 1.5. mu.L of template, ddH2O41. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 30s, annealing at 50 deg.C for 30s, extension at 72 deg.C for 1min, 35 cycles, preservation at 72 deg.C for 10min, and storage at 4 deg.C. PCR products were sequenced by Higzhou Zhikexi Biotechnology Limited (SEQ ID NO:1) and then placed on NCBI websiteThe rDNA ITS sequences with higher similarity to this sequence were selected by BLAST search, and the neighbor junction tree (NJ phylogenetic tree) was generated by 1000 repeats using MEGA5 software Align by Clustalw automated analysis (FIG. 6). The results showed 99% similarity of ZJB16085 to Aspergillus versicolor strain ATCC 9677. The strain is considered to be a variant of Aspergillus versicolor by combining morphological identification and growth characteristic research of the strain, is named as Aspergillus versicolor (Aspergillus versicolor) ZJB16085, is preserved in China center for type culture collection, has a preservation number of CCTCC NO: M2018408, and has a preservation date of 2018, 6-28 days, and a preservation address of university of Wuhan, China, and a postal code of 430072.
Any nucleotide sequence obtained by carrying out one or more nucleotide substitutions, deletions or insertions on the nucleotide sequence shown in SEQ NO. 1 is within the protection scope of the present invention as long as the nucleotide sequence has more than 90% of similarity with the nucleotide sequence.
Example 3: aspergillus versicolor CCTCC No. M2018408 fermentation conversion of R-2-phenoxy propionic acid to produce R-2- (4-hydroxy phenoxy) propionic acid
Inoculating Aspergillus versicolor CCTCC NO: M2018408 to slant culture medium, and standing at 28 deg.C for 6-7d to obtain slant strain. The thalli is picked from the inclined plane and inoculated into a 250mL shaking flask filled with 50mL seed culture medium, and cultured for 2-3d at 28 ℃ and 200rpm to obtain seed liquid. Inoculating the seed solution into a 250mL shaking flask containing 50mL fermentation transformation medium by the inoculation amount with the volume concentration of 3%, culturing at 28 ℃ and 150-. The reaction mixture was centrifuged at 10000rpm for 10min, and the supernatant was filtered through a 0.22 μm microfiltration membrane and then subjected to HPLC (Ulipret C18 reverse phase column 250 mm. times.4.6 mm, mobile phase V (aqueous phosphoric acid solution having pH 2): V (acetonitrile): 3: 2, flow rate 1mL/min, detector DAD, detection wavelength 210nm, column temperature 30 ℃ C.) to determine the concentrations of R-2-phenoxypropionic acid and R-2- (4-hydroxyphenoxy) propionic acid. The results show that the concentration of R-2- (4-hydroxyphenoxy) propionic acid at the end of the reaction is 4.5 g/L; the mass conversion was 45%.
The composition of the slant medium, seed medium and fermentation medium in this example was the same as that in example 1.
Sequence listing
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<120> Aspergillus versicolor ZJB16085 and application in synthesis of R-2- (4-hydroxyphenoxy) propionic acid
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acctgcggaa ggatcattac tgagtgcggg ctgcctccgg gcgcccaacc tcccacccgt 60
gaatacctaa cactgttgct tcggcgggga accccctcgg gggcgagccg ccggggacta 120
ctgaacttca tgcctgagag tgatgcagtc tgagtctgaa tataaaatca gtcaaaactt 180
tcaacaatgg atctcttggt tccggcatcg atgaagaacg cagcgaactg cgataagtaa 240
tgtgaattgc agaattcagt gaatcatcga gtctttgaac gcacattgcg ccccctggca 300
ttccgggggg catgcctgtc cgagcgtcat tgctgcccat caagcccggc ttgtgtgttg 360
ggtcgtcgtc ccccccgggg gacgggcccg aaaggcagcg gcggcaccgt gtccggtcct 420
cgagcgtatg gggctttgtc acccgctcga ctagggccgg ccgggcgcca gccgacgtct 480
ccaaccattt ttcttcaggt tgacctcgga tcaggtaggg atacccgctg aacttaagca 540
tatcaataag gcggagga 558
Claims (7)
1. Aspergillus versicolor (Aspergillus versicolor) ZJB16085, preserved in China center for type culture Collection with preservation number CCTCC NO: M2018408, preservation date of 2018, 6 months and 28 days, and preservation address of Wuhan university, China, Zip code 430072.
2. An application of aspergillus versicolor ZJB16085 in catalyzing R-2-phenoxypropionic acid to synthesize R-2- (4-hydroxyphenoxy) propionic acid according to claim 1.
3. The application as claimed in claim 2, wherein the application is to use fermentation liquid obtained by fermentation culture of aspergillus versicolor ZJB16085 as a catalyst and a reaction medium, add substrate R-2-phenoxypropionic acid, perform conversion reaction at the temperature of 25-30 ℃ and the table rotation speed of 150-200rpm to obtain conversion liquid containing R-2- (4-hydroxyphenoxy) propionic acid, and separate and purify the conversion liquid to obtain R-2- (4-hydroxyphenoxy) propionic acid.
4. Use according to claim 3, characterized in that the substrate is present in the fermentation broth to a final concentration of 10-50 g/L; the content of wet bacteria in the fermentation liquor is 15-20 g/L.
5. The use according to claim 3, wherein the fermentation broth obtained by fermentation culture of Aspergillus versicolor ZJB16085 is prepared by the following method: inoculating Aspergillus versicolor ZJB16085 into the fermentation culture medium, and performing fermentation culture at 25-30 deg.C and shaking table rotation speed of 150-; the fermentation medium comprises the following components: 5-20g/L of glucose, 5-10g/L of yeast extract, 5-10g/L of ammonium sulfate, 0.5-1.0g/L of magnesium sulfate heptahydrate, 0.05-0.12g/L of manganese sulfate monohydrate, 1.5-2.5g/L of potassium dihydrogen phosphate, 3.6-6.0g/L of dipotassium hydrogen phosphate trihydrate, 1-5mL/L of trace element liquid, deionized water as a solvent and the pH value of 6.8; the trace element liquid comprises the following components: 2g/L of ferrous sulfate heptahydrate, 100mg/L of zinc sulfate tetrahydrate, 300mg/L of boric acid, 200mg/L of cobalt chloride hexahydrate, 10mg/L of copper chloride dihydrate, 20mg/L of nickel chloride hexahydrate and 30mg/L of sodium molybdate dihydrate, and deionized water is used as a solvent.
6. The use according to claim 5, characterized in that the fermentation medium consists of: 5g/L of glucose, 5g/L of yeast extract, 5g/L of ammonium sulfate, 0.5g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate monohydrate, 1.5g/L of potassium dihydrogen phosphate, 3.6g/L of dipotassium hydrogen phosphate trihydrate, 1mL/L of trace element liquid, deionized water as a solvent, and 5M sodium hydroxide solution is used for adjusting the pH value to be 6.8.
7. The use according to claim 5, wherein the Aspergillus versicolor ZJB16085 is subjected to slant activation and seed culture before inoculation, and then the seed solution is inoculated into the fermentation medium at an inoculum size of 3-10% by volume: inoculating Aspergillus versicolor ZJB16085 to slant culture medium, and standing at 28 deg.C for 6-7 days to obtain slant strain; selecting thalli from the inclined plane, inoculating the thalli into a 250mL shaking flask filled with 50mL seed culture medium, and culturing at 28 ℃ and 150-; the slant culture medium comprises the following components: 200g/L of potato, 20g/L of glucose, 20g/L of agar and deionized water as a solvent, wherein the pH value is natural; the seed culture medium comprises the following components: potato 200g/L, glucose 20g/L, deionized water as solvent, and natural pH.
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