CN109536392A - Aspergillus versicolor ZJB16085 and the application for synthesizing R-2- (4- hydroxyphenoxy) propionic acid - Google Patents
Aspergillus versicolor ZJB16085 and the application for synthesizing R-2- (4- hydroxyphenoxy) propionic acid Download PDFInfo
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Abstract
The invention discloses the application of a kind of aspergillus versicolor (Aspergillus versicolor) ZJB16085 and synthesis R-2- (4- hydroxyphenoxy) propionic acid, deposit number CCTCC NO:M 2018408.The present invention isolates aspergillus versicolor (Aspergillus versicolor) CCTCC NO:M 2018408 of effectively conversion R-2- phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid from peat, it is under 10g/L substrate existence condition, R-2- (4- hydroxyphenoxy) propionic acid of 4.5g/L, conversion ratio 45% can be synthesized.
Description
Technical field
The present invention relates to it is a kind of conversion R-2 phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid microorganism and its
Screening technique, belongs to technical field of bioengineering, in particular to one plant conversion R-2 phenoxy propionic acid synthesizes R-2- (4- hydroxy benzenes
Oxygroup) propionic acid aspergillus versicolor.
Background technique
R-2- (4- hydroxyphenoxy) propionic acid is in phenoxy carboxylic acid and fragrant phenoxy phenoxy propionic acid herbicide synthesis process
Key intermediate can be used for synthesizing fenoxaprop-P (soybean, rice), fluazifop (rape, potato), clodinafop-propargyl (paddy
Object) etc. herbicides.As phenoxy carboxylic acid herbicides is more and more paid close attention to, the advantages of it is as herbicide, is also more and more
It is recognized.As the Chiral pesticide with enantiomter, relative to the inefficiencies and environment high pollution of racemic pesticide, light
Learning pure phenoxy carboxylic acid herbicides has the advantages that weeding ratio is high, it is low with environmental pollution etc. to injure to crops.Benzene oxycarboxylic acid
The synthesis of class herbicide can all use chiral R-2- (4- hydroxyphenoxy), and propionic acid is as intermediate, therefore, develops R-2- (4-
Hydroxyphenoxy) propionic acid high-efficiency synthesis method is concerned.
Elango V etc. (US Patent 5008439A) was once reported using parahydroxyacet-ophenone as raw material and alpha-halogenate third
Acid esters reaction, is aoxidized and hydrolysis obtains the racemic modification of 2- (4- hydroxyphenoxy) propionic acid, further obtained by fractionation
High optically pure R-2- (4- hydroxyphenoxy) propionic acid.Shen perpetuity etc. (synthesis chemistry, 2006,04:398-401) is to the technique road
Line has carried out the improvement of system, is reacted with parahydroxyacet-ophenone with alpha-chloro propionic ester, and oxidation, hydrolysis and fractionation etc. are passed through
Reaction, has synthesized optically pure R-2- (4- hydroxyphenoxy) propionic acid.It is easier to, needs although this method product isolates and purifies
Multistep reaction could be completed, and cause the yield of product not high, and simultaneous reactions raw materials and reagents are also not suitable for being mass produced.
Father-in-law builds congruent (Zhejiang Polytechnical University's journal, 2009,04:362-365) and once reported using Pfansteihl as initial compounds,
By esterification, sulfonylation and etherificate and etc., generate high optically pure R-2- (4- hydroxyphenoxy) propionic acid.Wherein S-2- (4-
Tosyloxy) propionic acid generate R-2- (4- hydroxyphenoxy) propionic acid reaction in configuration inverted, reaction mechanism:
Under the conditions of sodium hydroxide, hydroquinone generates phenol sodium first, and S-2- (4- tosyloxy) ethyl propionate becomes sodium salt, by
Positively charged in sodium ion, the oxygen of phenol sodium is negatively charged, in one side attack S-2- (4- tosyloxy) sodium propionate small from steric hindrance
Chiral carbon, formed intermediate after, configuration inverts, and is changed into R type.Wu Chunlei etc. (Chemical Industry in Guangzhou, 2015,08:92-93)
Once it reported using l-Alanine as initial compounds, by chlorination, etherificate and acidification and etc., wherein configuration turns over when etherification reaction
Turn, generates product R-2- (4- hydroxyphenoxy) propionic acid.L-Alanine and Pfansteihl process route are fairly simple, reaction step compared with
It is few, but condition is difficult to control.Because there is hydroquinone participation in reaction process, hydroquinone generation easy to oxidize in alkaline environment
Quinone makes so the hydroquinone and the di-substituted of hydroquinone that do not react completely in later-period purification processing are difficult to be removed
The purity for obtaining product is affected.Therefore, in the synthesis process, strict control reaction condition and material ratio are needed, with what is prevented
Side reaction generates.
Currently, the external research report for prepare R-2- (4- hydroxyphenoxy) propionic acid about bioanalysis is seldom, wherein BASF
Company Dingler etc. (Pest Management Science, 2015,46 (1): 33-35) is once reported through beauveria bassiana hydroxyl
Change substrate R-2- phenoxy propionic acid, synthesizes R-2- (4- hydroxyphenoxy) propionic acid.(the Tetrahedron such as Kinne M
Letters, 2008,49 (41): 5950-5953) once report was using agrocybe peroxidase, in addition H2O2Under the conditions of will disappear outside
The 2- phenoxy propionic acid selectivity hydroxylating for revolving body generates R-2- (4- hydroxyphenoxy) propionic acid.As people are to environmental protection
Pay attention to, original chemical industry receives certain impact, while having pushed the rapid development of green bio process industry, therefore
Bioanalysis, which prepares R-2- (4- hydroxyphenoxy) propionic acid, very big application prospect.
Summary of the invention
The object of the present invention is to provide a kind of aspergillus versicolor (Aspergillus versicolor) ZJB16085 and conversions
The method of R-2 phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid.
The technical solution adopted by the present invention:
The present invention provides one plant of aspergillus versicolor (Aspergillus versicolor) ZJB16085, is preserved in Chinese Typical Representative
Culture collection, deposit number CCTCC NO:M 2018408, preservation date on June 28th, 2018, preservation address China
Wuhan Wuhan University, postcode 430072.
For aspergillus versicolor CCTCC NO:M 2018408 of the present invention by enrichment culture twice from peat, picking can be
The bacterial strain that grows in culture medium containing substrate R-2- phenoxy propionic acid accesses 96 orifice plates fermentation primary dcreening operation, using 4- amino peace for than
Woods development process filters out the primary dcreening operation bacterial strain that substrate R-2- phenoxy propionic acid can be converted to R-2- (4- hydroxyphenoxy) propionic acid,
Then it transfers again and carries out what fermentation secondary screening obtained in the shaking flask equipped with fermentation medium.
The present invention also provides a kind of aspergillus versicolor ZJB16085 to synthesize R-2- (4- hydroxy benzenes in catalysis R-2 phenoxy propionic acid
Oxygroup) application in propionic acid, the application is the fermentation liquid that is obtained using the fermented culture of aspergillus versicolor ZJB16085 as catalyst
And reaction medium, substrate R-2 phenoxy propionic acid is added, is turned under the conditions of 25-30 DEG C of temperature, shaking speed 150-200rpm
Change reaction, obtain the conversion fluid for containing R-2- (4- hydroxyphenoxy) propionic acid, conversion fluid is isolated and purified, obtains R-2- (4- hydroxyl
Phenoxy group) propionic acid.The substrate final concentration of 10-50g/L in fermentation liquid, preferably 10g/L；Wet thallus in the fermentation liquid
Content is 15~20g/L, preferably 20g/L.
Further, the fermentation liquid that the fermented culture of the aspergillus versicolor ZJB16085 obtains is prepared as follows: will be miscellaneous
Color aspergillus ZJB16085 is seeded in fermentation medium, training of fermenting under the conditions of 25-30 DEG C of temperature, shaking speed 150-200rpm
(preferably culture 15~20g/L of wet thallus content into fermentation liquid) is supported, fermentation liquid is obtained；The fermentation medium composition: grape
Sugared 5-20g/L, yeast extract 5-10g/L, ammonium sulfate 5-10g/L, bitter salt 0.5-1.0g/L, manganese sulfate monohydrate
0.05-0.12g/L, potassium dihydrogen phosphate 1.5-2.5g/L, dipotassium hydrogen phosphate trihydrate 3.6-6.0g/L, liquid microelement 1-5mL/
L, solvent are deionized water, adjust 6.8,115 DEG C of sterilizing 20min of pH value with the sodium hydroxide solution of 5M；The liquid microelement
Composition: FeSO47H2O 2g/L, zinc sulfate (II) tetrahydrate 100mg/L, boric acid 300mg/L, cobalt chloride (II) six
Hydrate 200mg/L, copper chloride (II) dihydrate 10mg/L, nickel chloride (II) hexahydrate 20mg/L, sodium molybdate two are hydrated
Object 30mg/L, using deionized water as solvent.
Further, the fermentation medium composition: glucose 5g/L, yeast extract 5g/L, ammonium sulfate 5g/L, seven hydrations
Magnesium sulfate 0.5g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate trihydrate 3.6g/L, micro member
Plain liquid 1mL/L, solvent are deionized water, adjust 6.8,115 DEG C of sterilizing 20min of pH value with the sodium hydroxide solution of 5M.
Further, the aspergillus versicolor ZJB16085 first carries out slant activation and seed culture before inoculation, then by seed
Liquid is seeded to fermentation medium with the inoculum concentration of volumetric concentration 3-10% (preferably 3%), specifically: by aspergillus versicolor ZJB16085
It is seeded to slant medium, in 28 DEG C of stationary culture 6-7d, obtains slant strains；Picking thallus, which is seeded to, from inclined-plane is equipped with
In the 250mL shaking flask of 50mL seed culture medium, 28 DEG C, 150-200rpm culture 2-3d obtain seed liquor；The slant medium
(PDA culture medium): potato 200g/L, glucose 20g/L, agar 20g/L, solvent are deionized water, and pH is naturally, 115 DEG C of sterilizings
20min；The seed culture medium (PDA culture medium): potato 200g/L, glucose 20g/L, solvent are deionized water, pH naturally,
115 DEG C of sterilizing 20min.
The present invention establishes quickly and effectively screening bioanalysis synthesis R-2- (4- hydroxyphenoxy) propionic acid microorganism
The method of bacterial strain, that is, devise a kind of can be grown and can with screening in the culture medium containing substrate R-2- phenoxy propionic acid
To convert the microorganism of R-2- phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid as the experimental program of target: first will
Microorganism in peat is enriched in the culture medium containing substrate R-2- phenoxy propionic acid, and preliminary screening goes out can be in richness
The bacterial strain grown on collection culture medium flat plate.Then R-2- phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid is converted to it
Ability detected, i.e., the single colonie that picking is grown is inoculated with into containing fermentation medium (identical as enrichment culture based formulas)
It is cultivated in 96 deep-well plates, the ability that substrate is converted to product by each bacterial strain is detected by 4-AA development process.
Screening strain is subjected to preservation, obtains primary dcreening operation strain.The bacterial strain that primary dcreening operation obtains is inoculated into respectively equipped with 50mL fermentation medium
Secondary screening, culture 7d or so are carried out in the 250mL triangular flask of (identical as enrichment culture based formulas), are detected in fermentation liquid by HPLC
The concentration of R-2- (4- hydroxyphenoxy) propionic acid.By a large amount of bacterial strain screenings work to multiple pedotheques, from separating
More than 1000 plants of edaphons in screening obtain the ZJB16085 bacterial strain with higher conversion capability.
What the present invention screened can effectively convert R-2- phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid
Microorganism ZJB16085 cultivates 7d or so on solid plate, and colony diameter is about 1.8cm, and bacterium colony is loose, and mycelia is shorter, quality
Villiform, intermediate projections, concentric circles, color fade to cyan by blackish green, and marginal portion is white.Bacterium colony dorsal edge portion
It is divided into white, centre gradually becomes faint yellow.Optical microphotograph under the microscope, mycelia is longer, sparse, have it is branched, in mycelia
Body top forms spore, and spore count is less.Observed under electron microscope, mycelia is smooth, there is a branch, and 2.0-3.0 μm of hyphal diameter；
Top capsule is expanded, conidiophore single-wheel is like wheat head shape, no separation.Conidium oval is subsphaeroidal, monospore, diameter 4.0-
5.0 μm, according to morphology, primarily determine that the bacterial strain is Eurotium (Aspergillus).In conjunction with microbial morphology analysis and
Molecular biology identification, it is believed that belong to the aspergillus versicolor (Aspergillus versicolor) of Si Bao section Eurotium.
Compared with prior art, the beneficial effects are mainly reflected as follows: the present invention isolates effectively from peat
Convert the aspergillus versicolor (Aspergillus of R-2- phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid
Versicolor) CCTCC NO:M 2018408 can synthesize R-2- (the 4- hydroxyl of 4.5g/L under 10g/L substrate existence condition
Phenoxyl) propionic acid, conversion ratio 45%.
Detailed description of the invention
For Fig. 1 bacterial strain primary dcreening operation as a result, letter represents columns, number represents line number.
Fig. 2 R-2- (4- hydroxyphenoxy) propionic acid standard items (A), R-2- phenoxy propionic acid standard items (B) and bacterial strain.
The HPLC chromatogram of ZJB16085 microbe conversion liquid (C).
Positive (A) and the back side (B) aspect graph of Fig. 3 bacterial strain ZJB16085 bacterium colony.
The mycelium morphology of 10 × 10 times of Fig. 4 optical microscopy lower bacterial strain ZJB16085.
The SEM electron-microscope scanning figure of Fig. 5 bacterial strain ZJB16085.
The phylogenetic tree of Fig. 6 bacterial strain ZJB16085.
Specific embodiment:
It is aspergillus versicolor (Aspergillus versicolor) CCTCC NO:M 2018408 screening, identification and hair below
Ferment converts the embodiment of R-2- phenoxy propionic acid synthesis R-2- (4- hydroxyphenoxy) propionic acid.
Embodiment 1: the screening of bacterial strain ZJB16085
(1) it is sampled from many places plant species growing area soil in Zhejiang Polytechnical University campus, respectively weighs 1g soil sample (weight in wet base),
Suspension is made with the physiological saline of 9mL0.9%, draws 1mL and is inoculated in equipped with the 50mL phenoxy propionic acid of R-2- containing 10g/L
In the 250mL triangular flask of enriched medium, in cultivating 1-2d on 28 DEG C, the shaking table of 150rpm, first time enrichment culture is carried out；It takes
The bacterium solution of 1mL first time enrichment culture is in the enriched medium of the fresh phenoxy propionic acid of R-2- containing 10g/L in same item
Second of enrichment culture is carried out under part.The bacterium solution of second of enrichment culture is subjected to gradient dilution, 100 μ L is taken to dilute 10 respectively3
With 105Dilution again is coated on screening and culturing medium plate, and the stationary culture 3-4d in 28 DEG C of constant incubators.With sterile
The single colonie grown in toothpick picking screening flat board is inoculated in 96 deep-well plates that (each hole is added with the 1mL benzene oxygen of R-2- containing 10g/L
The fermentation medium of base propionic acid), and at 28 DEG C, after cultivating 6-7d under 150rpm, 96 deep-well plates are centrifuged 15min in 1000rpm,
100 μ L supernatants are drawn in 96 microwell plates, each bacterial strain conversion of substrate is detected with 4-AA development process and generates product
Ability (Fig. 1).64 plants of primary dcreening operation bacterial strains with conversion capability are subjected to preservation.
(2) access of primary dcreening operation bacterial strain is cultivated under 28 DEG C, 150rpm equipped in the 250mL shaking flask of 50mL seed culture medium
2-3d, then with the switching of 3% inoculum concentration of volumetric concentration in the fermentation medium equipped with 50mL R-2- containing 10g/L phenoxy propionic acid
Fermentation secondary screening is carried out in 250mL shaking flask, at 28 DEG C, after cultivating 7d under 150rpm, 10000rpm is centrifuged 10min, supernatant warp
Using HPLC detection, (Erie spy C18 reversed-phase column 250mm × 4.6mm, mobile phase V be (pH=2's after 0.22 μm of filtering with microporous membrane
Phosphate aqueous solution): V (acetonitrile)=3:2, flow velocity 1mL/min, detector DAD, Detection wavelength 210nm, 30 DEG C of column temperature) R-2-
The concentration of phenoxy propionic acid and R-2- (4- hydroxyphenoxy) propionic acid.R-2- phenoxy propionic acid and R-2-'s (4- hydroxyphenoxy)
Retention time is respectively 8.1min and 3.9min, sees Fig. 2.The bacterial strain with higher conversion capability is filtered out from 64 plants of bacterium, is obtained
Secondary screening bacterial strain ZJB16085 is obtained, slant preservation is carried out.
Wherein, the enriched medium composition of the present embodiment: glucose 5g/L, yeast extract 5g/L, ammonium sulfate 5g/L, seven
It is Magnesium sulfate heptahydrate 0.5g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate trihydrate 3.6g/L, micro-
Secondary element liquid 1mL/L, solvent are deionized water, adjust 6.8,115 DEG C of sterilizing 20min of pH value with the sodium hydroxide solution of 5M.
Liquid microelement composition: FeSO47H2O 2g/L, zinc sulfate (II) tetrahydrate 100mg/L, boric acid
300mg/L, cobalt chloride (II) hexahydrate 200mg/L, copper chloride (II) dihydrate 10mg/L, nickel chloride (II) hexahydrate
20mg/L, sodium molybdate dihydrate 30mg/L, using deionized water as solvent.
Screening and culturing medium: 20g/L agar is added in enrichment culture based formulas.
Slant medium (PDA culture medium): potato 200g/L, glucose 20g/L, agar 20g/L, solvent are deionization
Water, pH is naturally, 115 DEG C of sterilizing 20min.
Seed culture medium (PDA culture medium): potato 200g/L, glucose 20g/L, solvent are deionized water, pH naturally,
115 DEG C of sterilizing 20min.
Fermentation medium forms same enriched medium.
4-AA development process: taking 100 μ L samples to be tested to be added in 96 microwell plates, tries to 4-AA
The 0.06g/L potassium ferricyanide is added in agent, is configured to color developing agent.It is added 100 μ L color developing agents in each micropore, 30 DEG C of reaction 20min,
Then light absorption value of the microplate reader test sample at 550nm is used.
The preparation of the 4-AA reagent: 0.6g 4-AA, 2mL1mol/L NaOH,
10mL 20%Na2CO3, it is settled to 100mL.It is stored in brown bottle, is kept in dark place.
Embodiment 2: bacterial strain ZJB16085 identification
(1) colony morphological observation
With a small amount of spore of transfer needle picking slant preservation strain, PDA plate scribing line, 28 DEG C of culture 7d or so, single colonie
Form is shown in Fig. 3.Colony diameter is about 1.8cm, and bacterium colony is loose, and mycelia is shorter, quality villiform, intermediate projections, concentric circles, face
Color fades to cyan by blackish green, and most marginal portion is white.Bacterium colony dorsal edge part is white, and centre gradually becomes yellowish
Color.
(2) mycelium morphology is observed
A circular filter paper is spread in the culture dish bottom that diameter is 9.0cm, a U-shaped glass bar is put thereon, is put on glass bar
One piece of clean slide and two pieces of coverslips, cover ware lid, are inoculated with after sterilizing, in 28 DEG C of culture 7d or so, in optical microscopy
Hypha form is observed under 10 × 10 times, sees Fig. 4.Mycelia is longer, sparse, have it is branched, in mycelium top formed spore, spore
Subnumber is less.
(3) SEM Electronic Speculum is observed
SEM scanning electron microscope result is shown in Fig. 5.Bacterial strain mycelia is smooth, there is a branch, and 2.0-3.0 μm of hyphal diameter；Top capsule is expanded,
Conidiophore single-wheel is like wheat head shape, no separation.Conidium oval is subsphaeroidal, monospore, and 4.0-5.0 μm of diameter, according to
Morphology primarily determines that the bacterial strain is Eurotium (Aspergillus).
(4) Molecular Identification
Chromosomal DNA is extracted according to molecular kit operating guidance, using total DNA as template, with ITS1 (5 '-TC
CGTAGGTGAACCTGCGG-3 ') and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') be positive and reverse primer, PCR expands
Increase 18S rDNA sequence.PCR amplification system is (50mL): 10 × Taq Buffer (Mg2+)5 μ L, 10mM dNTPs 1 μ L, Taq
Archaeal dna polymerase each 0.5 μ L of 0.5 μ L, primer I TS1 and ITS4, template 1.5 μ L, ddH2O 41μL.PCR reaction condition are as follows: pre- to become
Property 94 DEG C of 2min, be denaturalized 94 DEG C of 30s, anneal 50 DEG C of 30s, extend 72 DEG C of 1min, 35 circulations, 72 DEG C of 10min, 4 DEG C of preservations.
(SEQ ID NO:1) is sequenced in PCR product commission Hangzhou Qing Ke Zi Xi Bioisystech Co., Ltd, then on the website NCBI
Retrieved with BLAST, select with the higher rDNA ITS sequence of the sequence similarity, utilize MEGA5 software Align by
Clustalw is automatically analyzed, and generates ortho position threaded tree (NJ phylogenetic tree) (Fig. 6) by 1000 repetitions.As the result is shown
The similitude of ZJB16085 and Asperillus versicolor strain ATCC9677 is 99%.Combining form identification
With the growth characteristics research to the bacterium, it is believed that be the mutation of aspergillus versicolor, be named as aspergillus versicolor (Aspergillus
Versicolor) ZJB16085, this bacterial strain have been deposited in China typical culture collection center, and deposit number is CCTCC NO:M
2018408, preservation date on June 28th, 2018, preservation address Wuhan, China Wuhan University, postcode 430072.
Nucleotide sequence shown in any couple of SEQ NO:1 carries out one or more nucleotide substitutions, missing or insertion process and obtains
The nucleotide sequence obtained belongs to protection model of the invention as long as it has 90% or more similitude with the nucleotide sequence
It encloses.
Embodiment 3: 2018408 microbe conversion R-2- phenoxy propionic acid of aspergillus versicolor CCTCC No:M produces R-2- (4- hydroxyl
Phenoxyl) propionic acid
Aspergillus versicolor CCTCC NO:M 2018408 is seeded to slant medium, in 28 DEG C of stationary culture 6-7d, is obtained
Slant strains.It is seeded in the 250mL shaking flask equipped with 50mL seed culture medium from picking thallus in inclined-plane, 28 DEG C, 150-
200rpm cultivates 2-3d, obtains seed liquor.Seed liquor is inoculated into the inoculum concentration of volumetric concentration 3% containing 50mL microbe conversion
In the 250mL shaking flask of culture medium, 28 DEG C, 150-200rpm culture 2-3d wet thallus content into fermentation liquid reach 20g/L, add
The R-2 phenoxy propionic acid of final concentration of 10g/L, continuation cultivate 6-7d under the conditions of 28 DEG C, 150-200rpm.Reaction solution warp
10000rpm is centrifuged 10min, and supernatant detects (Erie's spy's C18 reversed-phase column using HPLC after 0.22 μm of filtering with microporous membrane
250mm × 4.6mm, mobile phase V (phosphate aqueous solution of pH=2): V (acetonitrile)=3:2, flow velocity 1mL/min, detector DAD,
Detection wavelength 210nm, 30 DEG C of column temperature) R-2- phenoxy propionic acid and R-2- (4- hydroxyphenoxy) propionic acid concentration.The result shows that
At the end of reaction, R-2- (4- hydroxyphenoxy) propionate concentration is 4.5g/L；Mass transitions rate is 45%.
Wherein, the slant medium of the present embodiment, seed culture medium, fermentation medium composition are the same as embodiment 1.
Sequence table
<110>Zhejiang Polytechnical University
<120>application of aspergillus versicolor ZJB16085 and synthesis R-2- (4- hydroxyphenoxy) propionic acid
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 558
<212> DNA
<213>aspergillus versicolor (Aspergillus versicolor)
<400> 1
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)
- Aspergillus versicolor 1. (Aspergillus versicolor) ZJB16085, is preserved in China typical culture collection center, Deposit number CCTCC NO:M 2018408, preservation date on June 28th, 2018, preservation address Wuhan, China Wuhan University, postal Compile 430072.
- 2. aspergillus versicolor ZJB16085 described in a kind of claim 1 synthesizes R-2- (4- hydroxy benzenes oxygen in catalysis R-2 phenoxy propionic acid Base) application in propionic acid.
- 3. application as claimed in claim 2, it is characterised in that the application is with the fermented culture of aspergillus versicolor ZJB16085 The fermentation liquid of acquisition is catalyst and reaction medium, substrate R-2 phenoxy propionic acid is added, in 25-30 DEG C of temperature, shaking speed Conversion reaction is carried out under the conditions of 150-200rpm, obtains the conversion fluid for containing R-2- (4- hydroxyphenoxy) propionic acid, by conversion fluid point From purifying, R-2- (4- hydroxyphenoxy) propionic acid is obtained.
- 4. application as claimed in claim 3, it is characterised in that final concentration of 10-50g/L of the substrate in fermentation liquid；Institute Stating wet thallus content in fermentation liquid is 15~20g/L.
- 5. application as claimed in claim 3, it is characterised in that the hair that the fermented culture of aspergillus versicolor ZJB16085 obtains Zymotic fluid is prepared as follows: aspergillus versicolor ZJB16085 being seeded in fermentation medium, is turned in 25-30 DEG C of temperature, shaking table Fermented and cultured under the conditions of fast 150-200rpm obtains fermentation liquid；The fermentation medium composition: glucose 5-20g/L, yeast mention Take object 5-10g/L, ammonium sulfate 5-10g/L, bitter salt 0.5-1.0g/L, manganese sulfate monohydrate 0.05-0.12g/L, phosphoric acid Potassium dihydrogen 1.5-2.5g/L, dipotassium hydrogen phosphate trihydrate 3.6-6.0g/L, liquid microelement 1-5mL/L, solvent are deionized water, PH value 6.8；The liquid microelement composition: FeSO47H2O 2g/L, zinc sulfate tetrahydrate 100mg/L, boric acid 300mg/L, cobalt chloride hexahydrate 200mg/L, copper chloride dihydrate 10mg/L, nickel chloride hexahydrate 20mg/L, molybdic acid Sodium dihydrate 30mg/L, using deionized water as solvent.
- 6. application as claimed in claim 3, it is characterised in that the fermentation medium composition: glucose 5g/L, yeast extract Object 5g/L, ammonium sulfate 5g/L, bitter salt 0.5g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1.5g/L, three water Dipotassium hydrogen phosphate 3.6g/L, liquid microelement 1mL/L are closed, solvent is deionized water, adjusts pH value with the sodium hydroxide solution of 5M 6.8。
- 7. application as claimed in claim 3, it is characterised in that the aspergillus versicolor ZJB16085 first carries out inclined-plane before inoculation Activation and seed culture, then seed liquor is seeded to fermentation medium with the inoculum concentration of volumetric concentration 3-10%: by aspergillus versicolor ZJB16085 is seeded to slant medium, in 28 DEG C of stationary culture 6-7d, obtains slant strains；Picking thallus is inoculated with from inclined-plane Into the 250mL shaking flask equipped with 50mL seed culture medium, 28 DEG C, 150-200rpm culture 2-3d obtain seed liquor；The inclined-plane Culture medium: potato 200g/L, glucose 20g/L, agar 20g/L, solvent are deionized water, and pH is natural；The seed culture medium: Potato 200g/L, glucose 20g/L, solvent are deionized water, and pH is natural.
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| CN113881715A (en) * | 2021-09-15 | 2022-01-04 | 湖北工业大学 | Biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid |
| CN116355969A (en) * | 2023-05-23 | 2023-06-30 | 锦州四海生物化学有限公司 | Method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid |
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| CN113881715A (en) * | 2021-09-15 | 2022-01-04 | 湖北工业大学 | Biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid |
| CN113881715B (en) * | 2021-09-15 | 2023-06-06 | 湖北工业大学 | Biosynthesis method of R- (+) -2- (4-hydroxyphenoxy) propionic acid |
| CN116355969A (en) * | 2023-05-23 | 2023-06-30 | 锦州四海生物化学有限公司 | Method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid |
| CN116355969B (en) * | 2023-05-23 | 2023-12-15 | 锦州四海生物化学有限公司 | Method for biosynthesis of R- (+) -2- (4-hydroxyphenoxy) propionic acid |
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