CN108823098A - A kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain - Google Patents
A kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain Download PDFInfo
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Abstract
The invention discloses a kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid (R-HPPA) synthesis bacterial strain, the method carries out as follows：By strain to be tested, the shaking flask culture in the fermentation medium containing substrate R-2- phenoxy propionic acid, takes fermentation liquid to be centrifuged, supernatant is as sample to be tested.Sample to be tested is mixed with color developing agent, stands reaction, extracts reaction solution and surveys OD420nm, R-HPPA content in sample to be tested is obtained according to R-HPPA standard curve, screening obtains R-HPPA and synthesizes bacterial strain.The method of the present invention reaction time is 30min, the range of R-HPPA measurement is 0.1-1g/L, detectable limit (LOD) and quantitation limit (LOQ) are 0.011g/L and 0.034g/L, the rate of recovery is in the range of 95-99%, efficiently, sensitive, reliable, the screening efficiency of microorganism fungus kind is greatly improved, the time is saved, reduces manpower and financial resources.
Description
(1) technical field
The present invention relates to one kind to be based on 96 microwell plate high-flux fast screening R-2- (4- hydroxyphenoxy) propionic acid (R-
HPPA) the method for synthesis bacterium belongs to the technical field of high flux screening wild mushroom and mutagenic species.
(2) background technique
Fragrant phenoxy base propionic acid (APP) class herbicide is a kind of selective depression plant acetyl coenzyme developed in the past 40 years
The herbicide of A carboxylase, it is efficient, less toxic, highly selective, to crop safety and be easy to the characteristics of degrading and greatly facilitated choosing
The development of selecting property herbicide.R-HPPA is the weight for synthesizing the APP class herbicides such as clodinafop-propargyl, haloxyfop-P-methyl and haloxyfop-r-methyl
Want intermediate.Its compound continues to introduce new, and the demand of the industrialized production chiral intermediate R-HPPA crucial to it is huge.
The main of this intermediate of industrialized production uses chemical method at present, and production process reaction step is more, the period
Long, reaction condition is just carved, and by-product is more, and environmental pollution is big, and yield is low.Biological catalysis stereoselectivity is single-minded, reaction condition
Mildly, environmental-friendly, the advantages that product purity is high, product can be easily separated extraction, therefore realize that bioanalysis prepares APP class herbicide
Crucial chiral intermediate has huge economic benefit and social value.
Bioanalysis prepares R-HPPA, and external report is less at present, reports wherein mainly being studied by BASF Corp. of Germany.1992
Year is reported by researchs such as BASF Corp. of Germany Bryan C, filters out a variety of micro- lifes for capableing of hydroxylating R-2- phenoxy propionic acid such as：
Aspergillus niger (Aspergillus niger), aspergillus flavus (Aspergillus flavus), Aspergillus carbonerius (Aspergillus
Carbonarius), aspergillus parasiticus (Aspergillus parasiticus Speare), streptomycete (Streptomyces), ghost
Agaric (Coprinus comatus), muscardine (Beauveria), Sclerotium rolfsii (Sclerotium rolfsii) and powder are quasi-
Penicillium notatum (Penicillium).BASF AG Bryan C in 1996 etc. by ultraviolet mutagenesis with nitrosoguanidine (NTG) is compound lures
Become and obtain beauveria bassiana LU700, carries out fermented and cultured using beauveria bassiana LU700, full cell hydroxyl substrate R-PPA is
R-HPPA can reach 1000 tons/year of yield, yield by this method at present>99%.2008, German Matthias
K etc. is by agrocybe peroxidase in addition H2O2Under the conditions of R-PPA hydroxyl turned into R-HPPA, as shown in Figure 1.It reacts item
Part：In the reaction system of 1mL, 0.5 μM of agrocybe peroxidase (AaP), 50mM phosphate buffer solution (pH 3-10), 0.5-
2mM substrate racemic modification 2- phenoxy propionic acid.With 1mM H2O2Start to react, with 0.1mL, 50% (w/v) trichloroacetic acid terminates anti-
It answers.Wherein reaction carries out regioselective isomeric purities close to 98%, and the R- isomers e.e. value for generating required HPPA is
60%.
The detection of past research, conventional R-HPPA generally uses efficient liquid-phase chromatography method.Although this method tool
There is the advantages that accurate, sensitive, but sample treatment is complicated, heavy workload is not suitable for all biology laboratories.Conventional R-
The screening of HPPA synthesis bacterial strain is related to inoculation and shaking flask culture repeatedly, and entire screening process is time-consuming, and efficiency is lower, cost
It is high.Therefore, it is necessary to establish a kind of high-throughput screening method of efficient, accurate and reliable R-HPPA synthesis bacterium.
(3) summary of the invention
It is an object of the present invention to provide a kind of application values with higher, simple and easy high-flux fast screening R-HPPA
The method of synthesis bacterium.
The technical solution adopted by the present invention is that：
The present invention provides the high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid (R-HPPA) synthesis bacterial strain, described
Method carries out as follows：Being that substrate is fermented with R-2- phenoxy propionic acid (R-PPA) by strain to be tested cultivates the hair obtained
Zymotic fluid centrifugation, takes supernatant as sample to be tested；Sample to be tested is mixed with color developing agent and (is carried out preferably in microwell plate),
Under the conditions of 40-60 DEG C stand reaction 5-70min, extract reaction solution survey 420nm place light absorption value, according to R-HPPA standard curve acquisition to
R-HPPA content in sample, screening obtain R-HPPA and synthesize bacterial strain；The color developing agent is by sodium nitrite (NaNO2) be dissolved in
Ionized water is configured to 1-9g/L solution, adjusts pH to 1.4-6.0 with 0.5mol/L sulfuric acid solution；The R-HPPA standard curve is
R-HPPA aqueous solution substitutes sample to be tested, surveys light absorption value at 420nm under the same conditions, with R-HPPA concentration of aqueous solution for horizontal seat
It marks, light absorption value is made at 420nm of ordinate.
Further, the preferably described color developing agent sodium nitrite solution concentration is 6.0g/L, pH 1.4.
Further, the preferably described reaction condition is that 60 DEG C of standings react 30min.
Further, the sample to be tested and color developing agent volume ratio are 1:1.
Further, the concentration of the substrate in the fermentation medium is 10.0g/L.
Further, the strain to be tested fermentation process condition is：28 DEG C, 150r/min fermented and cultured 7d obtain fermentation liquid.
Further, the following method preparation of R-2- (4- hydroxyphenoxy) the propionic acid standard curve：R-2- (4- hydroxy benzenes oxygen
Base) the following method preparation of propionic acid standard curve：Prepared respectively with deionized water 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L,
R-2- (4- hydroxyphenoxy) propionic acid mark of 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L and 1.0g/L concentration gradient
Quasi- solution takes the standard solution of various concentration to be added in the micropore (preferably 100 μ L) of 96 hole microwell plates, each micropore aobvious respectively
Toner reacts 30min under the conditions of 60 DEG C, measures light absorption value at 420nm, dense with R-2- (4- hydroxyphenoxy) propionic acid aqueous solution
Degree is abscissa, and light absorption value is that ordinate draws standard curve；In the micropore, color developing agent and standard solution volume ratio are 1:1,
The color developing agent is that sodium nitrite is dissolved in deionized water to be configured to concentration 6.0g/L, is 1.4 with sulphur acid for adjusting pH.
Further, the screening technique carries out as follows：The soil for taking sampled point is made suspended of physiological saline
Liquid, being then allowed to stand makes its sedimentation；It takes supernatant to be coated in PDA culture medium plate with pipette, is sealed plate with sealed membrane,
In 28 DEG C of cultures to growing bacterium colony；Single colonie in plate is chosen with oese be inoculated in fill the first enriched medium of 50mL
In 250mL triangular flask, 28 DEG C, first time enrichment culture 2-3d is carried out under 150r/min；With the inoculum concentration of volumetric concentration 2% by
The bacterium solution of enrichment culture is inoculated in the second enriched medium, and 28 DEG C, carry out second of enrichment culture 1- under 150r/min
2d；The bacterium solution of second of enrichment culture is diluted step by step and (takes 10 respectively-3With 10-5Two 100 μ L of gradient dilution liquid) after be coated on
On PDA plate containing 10.0g/L R-2- phenoxy propionic acid, and in 28 DEG C of constant temperature stationary culture 3-4d；By the single bacterium on plate
It falls and is seeded to seed culture medium, 28 DEG C of culture 72h one by one as strain to be tested；Then it is forwarded to the phenoxy group of R-2- containing 10.0g/L
The fermentation medium of propionic acid, 28 DEG C, 150r/min culture 7d, obtains fermentation liquid；By fermentation liquid centrifugation (1000rpm, centrifugation
15min), take supernatant as sample to be tested；By sample to be tested and color developing agent with volume ratio 1:1 mixing, it is anti-under the conditions of 60 DEG C
30min is answered, light absorption value at detection 420nm is extracted reaction solution, standard curve obtains R-HPPA content in sample to be tested, and screening obtains R-
HPPA synthesizes bacterial strain.
Further, the fermentation medium group becomes：Glucose 5.0g/L, yeast extract 5.0g/L, ammonium sulfate 5.0g/
L, bitter salt 0.5g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate trihydrate 3.6g/
L, FeSO47H2O 2mg/L, 100 μ g/L of zinc sulfate (II) tetrahydrate, 300 μ g/L of boric acid, (II) six water of cobalt chloride
Close 200 μ g/L of object, 10 μ g/L of copper chloride (II) dihydrate, 20 μ g/L of nickel chloride (II) hexahydrate, sodium molybdate dihydrate
30 μ g/L, adjusting pH with the sodium hydroxide of 2M is 6.8, and solvent is distilled water；First enriched medium, the second enrichment culture
Base and seed culture medium composition are identical as fermentation medium composition.
Present invention discover that phenolic compound easily by nitrite-oxidizing generation nitroso compound, generates in acid medium
Substance all has certain chromophore, makes solution in yellow or brown color, and reaction mechanism (Fig. 1) devises one kind and is based on
The nitrite-oxidizing high-throughput screening method that chromogenic reaction occurs of hydroxyl on R-HPPA phenyl ring.Reaction time is 30min, R-
The range of HPPA measurement is 0.1-1g/L, average recovery rate 95%-99%.
High-throughput screening method screens R-HPPA and synthesizes bacterial strain, is based primarily upon bacterial strain to the tolerance and R-HPPA of R-PPA
Two factors of yield.Potato dextrose agar (PDA) solid sieve culture medium for obtaining single bacterium colony, bacterium
Strain is resistant to R-PPA concentration>10.0g/L, has highly selective and without byproduct, and cannot degrade R-PPA and R-HPPA, be suitable for
The amplification of strain improvement and system.Existing traditional screening R-HPPA synthesis bacterial strain screening method mainly passes through high performance liquid chromatography
Method is related to inoculation and shaking flask culture repeatedly.Single colonie on plate is inoculated into one by one and is free of by the high-throughput screening method
In 96 deep-well plates of R-PPA, 28 DEG C of culture 72h.Then next piece is transferred to as seed liquor to ferment containing R-POPA (10.0g/L)
In 96 deep-well plates of culture medium, 28 DEG C of culture 7d.By 96 hole deep-well plates in 1000rpm, it is centrifuged 15min, each hole takes on 100 μ L
Color developing agent is added in 96 hole microwell plates in final proof.The light absorption value of sample in microwell plate is detected by microplate reader.According to standard curve
The content of R-HPPA in each sample is calculated, the conversion ratio of bacterial strain conversion R-PPA is calculated.
Compared with prior art, the method for the present invention beneficial effect is mainly reflected in：
The present invention relates to a kind of method based on microwell plate high-flux fast screening R-HPPA synthesis strain, the method for the present invention
Reaction time is 30min, and the range of R-HPPA measurement is 0.1-1.0g/L, average recovery rate 95.0%-99.0%, has height
Effect property, sensitivity, reliability.High-throughput screening method of the present invention is compared with the method for traditional liquid chromatogram measuring, R-HPPA
Do not differed significantly in measurement result, but 96 orifice plate high-throughput screening methods of the invention can 1h detect 960 samples, and
Traditional high performance liquid chromatography then needs 160h, greatly improves the efficiency of screening, has saved time and cost.In addition, this
Invention can the intuitive and accurate bacterial strain that high yield R-HPPA is screened from wild mushroom and mutagenic strain, have it is easy, quickly,
The advantages that accurate, the ability of the bacterial strain with Large-scale Screening high yield R-HPPA provide to screen the bacterial strain of high yield R-HPPA
Effective strategy.
(4) Detailed description of the invention
Fig. 1 is R-HPPA structure.
Fig. 2 is that microbial hydroxylation R-PPA generates R-HPPA schematic diagram, A：R-PPA standard solution and NaNO2Aqueous solution；
B：R-HPPA marks product solution and NaNO2Aqueous solution；C：R-HPPA marks product solution；D：NaNO2Aqueous solution；E：Fermentation medium with
NaNO2Aqueous solution.
Fig. 3 is the influence that sodium nitrite concentration develops the color to R-HPPA.
Fig. 4 is the influence that reaction temperature develops the color to R-HPPA.
Fig. 5 is the influence that pH value develops the color to R-HPPA.
Fig. 6 is the colour development difference figure of various concentration R-HPPA.
Fig. 7 is the correlation curve of (R)-HPPA concentration and OD value.
Fig. 8 is that high-throughput screening method screens R-HPPA production bacterial strain flow chart.
Fig. 9 is the chromogenic reaction result figure of 2 microwell plate of embodiment, shows that the catalysis of corresponding 96 deep-well plates growth bacterial strain is living
Property it is strong and weak.
Figure 10 is the chromogenic reaction result figure of the different color developing agents of embodiment 3.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This：
Embodiment 1
One, experimental material
(1) reagent and instrument
R-HPPA standard items are purchased from Aladdin company, and substrate R-PPA is provided by Shandong Run Feng limited liability company, remaining examination
Agent is that domestic analysis is pure.
The preparation of standard solution：The R-HPPA standard solution of 5.0g/L and the R- of 5.0g/L are accurately prepared with deionized water
POPA standard solution.
Instrument：SpectraMax M2 multi-function microplate reader (Molecular Device Company), 96 microwell plates, 96
Deep-well plates.
Two, the analysis program of the microplate reader detection of R-HPPA
(1) selection of absorption spectrum and characteristic wavelength
Take respectively the R-HPPA standard solution of 0.5g/L, the R-PPA standard solution of 0.5g/L, 100 μ L of fermentation medium in
96 microwell plates, then the NaNO that isometric concentration is 3.0g/L is added to each hole2Aqueous solution, in order to exclude color developing agent and R-
Influence of the HPPA to developing solution absorption value, takes the NaNO of 3.0g/L2Aqueous solution and each 200 μ L of the R-HPPA standard solution of 0.5g/L
In 96 microwell plates, then by 96 microwell plates in 40 DEG C of standing 30min, carried out in 200~900nm wave-length coverage with microplate reader complete
Length scanning excludes interference of the different material to absorption value, finally determines Detection wavelength (such as Fig. 2).
As shown in Figure 2, A, B, C, D and E map maximum absorption wavelength is in 230nm, wherein the OD of A, C, D and E420nm=0, B
OD420nm=0.57, and due to R-HPPA standard solution and NaNO2Solution reacts, solution displaing yellow, in order to eliminate R-
HPPA、R-PPA、NaNO2The influence of solution and fermentation medium to substance that show color absorption value, therefore select 420nm as measurement
Wavelength.
(2) color developing agent NaNO2The optimization of concentration
By prepared NaNO2Aqueous solution (10.0g/L), be diluted to 1.0 respectively, 2.0,3.0,4.0,5.0,6.0,
7.0,8.0,9.0g/L take various concentration NaNO2Aqueous solution and R-HPPA standard solution (0.5g/L) each 100 μ L are in 96 micropores
Plate, 40 DEG C of standing 30min survey OD with microplate reader420nm(three groups parallel) draws NaNO according to data2Concentration of aqueous solution and
OD420nmCurve graph (such as Fig. 3), determines optimum N aNO2Concentration of aqueous solution.
From the figure 3, it may be seen that NaNO2Concentration of aqueous solution is within the scope of 1.0g/L-6.0g/L, OD420nmIt gradually rises；Work as NaNO2
When concentration of aqueous solution is greater than 6.0g/L, OD420nmTend to be invariable, thus it is speculated that possible NaNO2The amount of solution can be complete by R-HPPA
It is oxidized.Therefore NaNO2Aqueous solution optimum dose is 100 μ L, 6.0g/L.
(3) optimization of chromogenic reaction temperature
Take the NaNO of 6.0g/L2Aqueous solution and each 100 μ L of the R-HPPA standard solution of 0.5g/L are in 96 microwell plates, it is contemplated that
The microplate reader operability of test, respectively at 40 DEG C, 50 DEG C and 60 DEG C, every 20min surveys OD with microplate reader420nmIt (does three groups to put down
Row), according to data, time and OD are drawn out respectively420nmCurve graph (such as Fig. 4), determines optimal reaction temperature.
As shown in Figure 4, OD at 40 DEG C420nmIncrease slowly, illustrates NaNO2Aqueous solution reacts slow with R-HPPA；At 50 DEG C
OD420nmIncrease comparatively fast compared to 40 DEG C, illustrates NaNO at a temperature of this2Aqueous solution reacts very fast at being reacted with R-HPPA than 40 DEG C；
0-30min, OD at 60 DEG C420nmGrowth is most fast, after 30min, gradually tends towards stability, illustrates NaNO at this time2Aqueous solution and R-
HPPA reaction tends to be complete.Therefore select 60 DEG C as chromogenic reaction temperature.
(4) optimization of chromogenic reaction pH value and time study on the stability
The NaNO for the 6.0g/L that secure ph is 1.4,2.4,3.4,4.4,5.4 and 6.42Aqueous solution takes different pH's
NaNO2Aqueous solution and each 100 μ L of R-HPPA (0.5g/L) standard solution are in 96 microwell plates, 60 DEG C of standings, every 10min microplate reader
Survey OD420nm(do three groups parallel) draws pH value and OD according to data420nmCurve graph (such as Fig. 5), determines optimal pH and colour developing
Reaction reaches the stable time.
As shown in Figure 5, when pH is 1.4, OD420nmRapid development, OD after 30min420nmIt increasess slowly；When pH is 2.4
When, OD420nmGrowth trend is more slow, OD after 30min420nmIt increasess slowly；PH is 3.4,4.4,5.4 and 6.4, OD420nmIncrease
Trend is slow, and OD when 30min420nmStill gradually increase.In conclusion the NaNO that pH is 1.42Aqueous solution reacts fast with R-HPPA
Speed, OD420nmIt tends towards stability after 30min, so NaNO2The optimal ph of aqueous solution is 1.4, reaction time 30min.
(5) drafting of standard curve
By prepared R-HPPA (10.0g/L) standard solution be diluted to concentration be 0.1,0.2,0.3,0.4,0.5,
0.6,0.7,0.8,0.9,1.0,2.0,3.0g/L, take the R-HPPA standard solution of various concentration and the NaNO of pH1.42Aqueous solution
(6.0g/L) each 100 μ L detects OD after 96 microwell plates, 60 DEG C of standing 30min, with microplate reader420nm(do three groups parallel), according to
OD420nmWith the average value of three groups of data of R-HPPA concentration of standard solution, using concentration as X-axis, OD is Y-axis, draws Y-X standard curve
(A) (such as Fig. 6 and 7).
Fig. 6 discovery is observed, R-HPPA concentration is 0.1g/L-1.0g/L, and color becomes brown color from yellow, works as R-HPPA
Concentration is 0.7g/L-1.0g/L, and color does not change significantly.
It is found from Fig. 7, R-HPPA concentration range is 0.1g/L-1.0g/L, R-HPPA concentration and OD420nmIt is in a linear relationship,
Its linear equation is Y=2.41818*X+0.09636, R2=0.9946, R2Numerical value shows absorption value OD420nmWith R-HPPA concentration
With good linear relationship.
(6) detectable limit (LOD) and quantitation limit (LOQ)
LOD and LOQ is to carry out analysis program verification according to following equation by international coordination meeting (ICH), quasi- for examining
The sensitivity parameter of construction method：
LOD=3.3 σ/S (formula 2-1)
LOQ=10 σ/S (formula 2-2)
σ is the intercept standard deviation of standard curve；S is slope of standard curve.
Three groups of data OD that (5) are detected420nmWith R-HPPA concentration of standard solution, standard curve A is drawn out respectively1、A2
And A3, then calculate A1、A2And A3The intercept standard deviation sigma of three groups of standard curves, according to formula 2-1 and 2-2 calculate LOD and
LOQ is 0.011g/L and 0.034g/L, shows this method with preferable sensitivity.
(7) investigation of preci-sion and accuracy
In order to verify the content that the method can be used to R-HPPA in test sample, it is added into the sample of measurement known dense
The R-HPPA standard sample of degree, measures the rate of recovery of this method.Bacterial strain is inoculated into respectively equipped with 50mL fermentation medium
In 250mL triangular flask, and 150r/min cultivates 7d between 28 DEG C of constant-temperature tables.Centrifuging and taking supernatant simultaneously dilutes a certain concentration, takes
The NaNO of pH1.4,6.0g/L is added in 96 hole microwell plates in the diluted upper final proof of 100 μ L in each micropore2100 μ L of aqueous solution,
60 DEG C of reaction 30min calculate R- in sample according to standard curve with light absorption value of the microplate reader test sample at 420nm
HPPA concentration C 1.It takes the supernatant of certain volume that the standard sample of the R-HPPA of C2 concentration is added, then takes 100 μ L ultrapure waters
The upper final proof of the R-HPPA of the appropriate diluted concentration containing C2 be added in 96 hole microwell plates, in each micropore color developing agent (pH1.4,
The NaNO of 6.0g/L2Aqueous solution) 100 μ L, 60 DEG C of reaction 30min, with light absorption value of the microplate reader test sample at 420nm.Meter
R-HPPA concentration C 3 in sample after calculating addition mark product.
1 NaNO of table2The preci-sion and accuracy (n=8) of development process
It is found by table 1, for the rate of recovery obtained in the range of 95-99%, ICH requires good accuracy should be
Within the scope of the 95-102% of actual value, so the result shows that the method proposed is accurate.
Resulting relative standard deviation (RSD) is respectively less than 5%, shows that proposed method is accurate.
Embodiment 2：
(1) culture medium
Fermentative medium formula：Glucose 5g/L, yeast extract 5g/L, ammonium sulfate 5g/L, bitter salt 0.5g/
L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate 1.5g/L, dipotassium hydrogen phosphate trihydrate 3.6g/L, FeSO47H2O
2mg/L, 100 μ g/L of zinc sulfate (II) tetrahydrate, 300 μ g/L of boric acid, 200 μ g/L of cobalt chloride (II) hexahydrate, copper chloride
(II) 10 μ g/L of dihydrate, 20 μ g/L of nickel chloride (II) hexahydrate, 30 μ g/L of sodium molybdate dihydrate, with the hydroxide of 2M
It is 6.8 that sodium, which adjusts pH, and solvent is distilled water.
Potato glucose agar medium (PDA culture medium)：Peeled potatoes 200g stripping and slicing is added 1L deionized water and boils
The filtering of 20min double gauze, glucose 20g, agar 15-20g are added into filtrate, and deionized water is added and supplies 1L, with 2M's
It is 6.8 that sodium hydroxide, which adjusts pH,.
(2) screening of the process referring to shown in Fig. 8
1) it cultivates：Suspension, standing sedimentation is made with physiological saline in the soil sample 10.0g for taking each sampled point respectively.Take 1mL
Supernatant is in the triangular flask of the 250mL equipped with the first enriched medium of 25.0mL, in 28 DEG C, 150r/min shaking table culture 1-
2d.The bacterium solution of first time enrichment culture is transferred with 2.0% inoculum concentration of volumetric concentration in newly shaking equipped with the second enriched medium
Second of enrichment culture is carried out in bottle, in 28 DEG C, 150r/min shaking table culture 1-2d.By the bacterium solution difference of second of enrichment culture
Dilution 10-3With 10-5Two gradients take the dilution of 100 μ L to be coated on the PDA plate of the substrate containing 10.0g/L, and at 28 DEG C
Stationary culture 3-4d in constant incubator.Each single colonie sterile toothpick picking on the plate for cultivating 3-4d is contained in inoculation
28 DEG C in 96 deep-well plates of the fermentation medium of 10.0g/L R-2- phenoxy propionic acid, 150r/min cultivate 7d, obtain fermentation liquid.
2) selective mechanisms：96 deep-well plates are centrifuged 15min in 1000r/min, every hole takes 100 μ L suitably dilute with deionized water
The upper final proof released and (be diluted to detection range) in 96 hole microwell plates, in each micropore be added color developing agent (pH1.4,6.0g/L's
NaNO2Aqueous solution) 100 μ L, 60 DEG C of reaction 30min, microwell plate is (letter represents row, number representative column) as shown in Figure 9 after reaction,
It was found that the relatively deeply aobvious brown color of hole B1, B2, B3, B4, B5, C1, C and C9 color.With microplate reader test sample at 420nm
Light absorption value calculates the content of R-HPPA in each sample according to R-HPPA standard curve prepared by embodiment 1, calculates bacterial strain and turns
Change the conversion ratio of R-PPA, while with the content of R-HPPA in HPLC method test sample B1, B2, B3, B4, B5, C1, C and C9.
HPLC testing conditions：Erie's spy's C18 chromatographic column：250mm×4.6mm；Mobile phase：V (phosphate aqueous solution pH=2)：V
(acetonitrile)=6:4；Flow velocity：1mL/min；Detector DAD；Detection wavelength：220nm；Sample volume：5μL；Column temperature：30℃.
3) result：The results are shown in Table 2.Analytical table 2, high flux screening detects R-HPPA concentration obtained by bacterial strain and HPLC is examined
R-HPPA concentration comparable is surveyed, illustrates that this high-throughput screening method is more accurate.R-HPPA concentration obtained by B3 is most in these bacterial strains
Height, high flux screening method result are 4.50g/L, and HPLC result is 4.56g/L, conversion ratio 45.59%.
2 R-HPPA testing result (n=3) of table
More than the 30 plants of bacterial strains with catalysis activity are had been obtained for by this high-throughput screening method.96 orifice plate high throughputs sieve
Choosing method 1h is able to detect 960 samples, and traditional high performance liquid chromatography then needs 160h, greatly improves the effect of screening
Rate.
Embodiment 3：Different color developing agents detect R-HPPA
Prepare the R- of 0,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9 and 1.0g/L respectively with deionized water
PPA and R-HPPA standard solution and other 8 kinds of color developing agents, respectively 0.2mol/L copper-bath, 0.375g/L potassium permanganate
Solution, 0.75mol/L bromic acid potassium solution, ferrous tartrate standard solution (ferrous tartrate solution allocation method：Weigh 1.00g
Ferrous sulfate (FeSO4·7H2) and 5.00g sodium potassium tartrate tetrahydrate (C O4H4O6NaK·4H2O), deionized water dissolving is added after mixing, it is fixed
Hold 1000mL), 32.4g/L liquor ferri trichloridi, 7mol/L nitric acid solution and 30% hydrogenperoxide steam generator.R-PPA is taken respectively
The 100 above-mentioned color developing agents of μ L are added in the micropore of 96 microwell plates, each micropore with 100 μ L of R-HPPA standard solution to be reacted.
The result is shown in Figure 10, after copper-bath is added in R-PPA and R-HPPA titer, solution is all light blue color, with copper-bath face
Color no significant difference illustrates that copper sulphate is not suitable for quantitative determining R-HPPA.
R-HPPA and potassium permanganate reaction generate yellow-brownish solution, and reaction temperature and time are respectively 30 DEG C and 10min, pH
It is 11.0.But R-HPPA and potassium permanganate are reacted as the increase of R-HPPA concentration can generate precipitating, and change over time OD
Value differs greatly, deficient in stability, therefore potassium permanganate is not suitable for quantitative determining R-HPPA.
R-HPPA and potassium bromate illustrate that potassium bromate is not suitable for quantitative determining R-HPPA without color reaction.
After ferric trichloride standard solution is added in R-PPA and R-HPPA titer, solution is all in yellowish-brown, with ferric trichloride mark
Quasi- solution colour no significant difference.Illustrate that ferric trichloride is not suitable for quantitative determining R-HPPA.
R-HPPA and nitric acid reaction generate pale yellow solution, but with the extension of reaction time or the raising of temperature, color
It can take off, deficient in stability, therefore nitric acid is not suitable for quantitative determining R-HPPA.
R-PPA and R-HPPA and hydroperoxidation illustrate that hydrogen peroxide is not suitable for quantitative determining without color change
R-HPPA。
By embodiment 3, illustrate that the present invention is based on 96 microwell plate NaNO2The method tool of the high-throughput detection R-HPPA of colour developing
It is creative.
Claims (9)
1. a kind of high-throughput screening method of R-2- (4- hydroxyphenoxy) propionic acid synthesis bacterial strain, it is characterised in that the method is pressed
Following steps carry out：Strain to be tested is cultivated in the fermentation medium containing substrate R-2- phenoxy propionic acid, the fermentation of acquisition
Liquid centrifugation, takes supernatant as sample to be tested；Sample to be tested is mixed with color developing agent, reacts 5- under conditions of 40-60 DEG C
70min is extracted reaction solution and is surveyed light absorption value at 420nm, obtains sample to be tested according to R-2- (4- hydroxyphenoxy) propionic acid standard curve
Middle R-2- (4- hydroxyphenoxy) propionic acid content, screening obtain R-2- (4- hydroxyphenoxy) propionic acid and synthesize bacterial strain；The colour developing
Agent is that sodium nitrite is dissolved in deionized water to be configured to concentration 1-9g/L, is 1.4-6.0 with sulphur acid for adjusting pH；R-2- (the 4-
Hydroxyphenoxy) propionic acid standard curve be with R-2- (4- hydroxyphenoxy) propionic acid aqueous solution substitute sample to be tested, in identical item
Light absorption value at 420nm, using R-2- (4- hydroxyphenoxy) propionic acid concentration of aqueous solution as abscissa, light absorption value at 420nm are surveyed under part
It is made of ordinate.
2. the method as described in claim 1, it is characterised in that the color developing agent sodium nitrite concentration is 6.0g/L, pH 1.4.
3. the method as described in claim 1, it is characterised in that reaction condition is that 60 DEG C of standings react 30min.
4. the method as described in claim 1, it is characterised in that the sample to be tested is 1 with color developing agent volume ratio:1.
5. the method as described in claim 1, it is characterised in that the concentration of the substrate in the fermentation medium is 10.0g/L.
6. the method as described in claim 1, it is characterised in that the strain to be tested fermentation process condition is：28 DEG C, 150r/
Min fermented and cultured 7d obtains fermentation liquid.
7. the method as described in claim 1, it is characterised in that R-2- (4- hydroxyphenoxy) the propionic acid standard curve is as follows
Method preparation：Prepared respectively with deionized water 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L,
R-2- (4- hydroxyphenoxy) propionic acid standard solution of 0.8g/L, 0.9g/L and 1.0g/L concentration gradient, takes various concentration respectively
Standard solution color developing agent is added in the micropore of 96 hole microwell plates, each micropore, react 30min under the conditions of 60 DEG C, measure
Light absorption value at 420nm, using R-2- (4- hydroxyphenoxy) propionic acid concentration of aqueous solution as abscissa, light absorption value is that ordinate draws mark
Directrix curve；In the micropore, color developing agent and standard solution volume ratio are 1:1, the color developing agent be sodium nitrite is dissolved in from
Sub- water is configured to concentration 6.0g/L, is 1.4 with sulphur acid for adjusting pH.
8. the method as described in claim 1, it is characterised in that the screening technique carries out as follows：Take the mud of sampled point
Suspension, standing sedimentation is made with physiological saline in soil；Supernatant is taken to be coated in PDA culture medium plate, with sealed membrane by plate
It seals, in 28 DEG C of cultures to growing bacterium colony；Single colonie in plate is chosen with oese to be inoculated in the first enriched medium, 28 DEG C,
First time enrichment culture 2-3d is carried out under 150r/min；With the inoculum concentration of volumetric concentration 2% by the bacterium solution of first time enrichment culture
It is inoculated in the second enriched medium, 28 DEG C, second of enrichment culture 1-2d is carried out under 150r/min；By second of enrichment culture
Bacterium solution dilution after be coated on the PDA plate of the phenoxy propionic acid of R-2- containing 10.0g/L, and in 28 DEG C of constant temperature stationary culture 3-
4d；It is seeded to seed culture medium, 28 DEG C of culture 72h one by one using the single colonie on plate as strain to be tested；Then it is forwarded to and contains
The fermentation medium of 10.0g/L R-2- phenoxy propionic acid, 28 DEG C, 150r/min culture 7d, obtains fermentation liquid；By fermentation liquid from
The heart takes supernatant as sample to be tested；By sample to be tested and color developing agent with volume ratio 1:1 mixing, reacts under conditions of 60 DEG C
30min extracts reaction solution light absorption value at detection 420nm, is obtained according to R-2- (4- hydroxyphenoxy) propionic acid standard curve to test sample
R-2- (4- hydroxyphenoxy) propionic acid content in product, screening obtain the bacterial strain of high yield R-2- (4- hydroxyphenoxy) propionic acid.
9. method according to claim 8, it is characterised in that the fermentation medium group becomes：Glucose 5.0g/L, yeast
Extract 5.0g/L, ammonium sulfate 5.0g/L, bitter salt 0.5g/L, manganese sulfate monohydrate 0.05g/L, potassium dihydrogen phosphate
1.5g/L, dipotassium hydrogen phosphate trihydrate 3.6g/L, FeSO47H2O 2mg/L, 100 μ g/L of zinc sulfate tetrahydrate, boron
300 μ g/L of acid, 200 μ g/L of cobalt chloride hexahydrate, 10 μ g/L of copper chloride dihydrate, 20 μ g/L of nickel chloride hexahydrate, molybdenum
Sour 30 μ g/L of sodium dihydrate, adjusting pH with the sodium hydroxide of 2M is 6.8, and solvent is deionized water；The seed culture medium,
One enriched medium and the second enriched medium composition are formed with fermentation medium.
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