CN108823098B

CN108823098B - High-throughput screening method of R-2- (4-hydroxyphenoxy) propionic acid synthetic strain

Info

Publication number: CN108823098B
Application number: CN201810702857.6A
Authority: CN
Inventors: 周海岩; 郑裕国; 姜瑞; 李一作; 薛亚平; 王远山
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-06-30
Filing date: 2018-06-30
Publication date: 2021-04-06
Anticipated expiration: 2038-06-30
Also published as: CN108823098A

Abstract

The invention discloses a high-throughput screening method of an R-2- (4-hydroxyphenoxy) propionic acid (R-HPPA) synthetic strain, which comprises the following steps: carrying out shake flask culture on the strain to be detected in a fermentation medium containing a substrate R-2-phenoxypropionic acid, centrifuging fermentation liquor, and taking supernatant as a sample to be detected. Mixing the sample to be tested with a color developing agent, standing for reaction, and taking reaction liquid to test OD_420nmAnd obtaining the content of the R-HPPA in the sample to be detected according to the R-HPPA standard curve, and screening to obtain the R-HPPA synthetic strain. The method has the advantages of reaction time of 30min, measurement range of R-HPPA of 0.1-1g/L, detection Limit (LOD) and quantification Limit (LOQ) of 0.011g/L and 0.034g/L, recovery rate of 95-99%, high efficiency, sensitivity and reliability, greatly improved screening efficiency of microbial strains, time saving, and reduced manpower and financial resources.

Description

High-throughput screening method of R-2- (4-hydroxyphenoxy) propionic acid synthetic strain

(I) technical field

The invention relates to a method for rapidly screening R-2- (4-hydroxyphenoxy) propionic acid (R-HPPA) synthetic bacteria at high flux based on a 96-microporous plate, belonging to the technical field of high-flux screening of wild bacteria and mutagenized bacteria.

(II) background of the invention

Aryloxy phenoxy propionic Acid (APP) herbicides are herbicides which are developed in recent 40 years and selectively inhibit plant acetyl coenzyme A carboxylase, and the development of selective herbicides is greatly promoted by the characteristics of high efficiency, low toxicity, high selectivity, safety to crops and easiness in degradation. The R-HPPA is an important intermediate for synthesizing APP herbicides such as clodinafop-propargyl, haloxyfop-R-methyl and high-efficiency galingale. The compound is continuously promoted to be new, and the industrial production has great demand on the key chiral intermediate R-HPPA.

At present, the chemical method is mainly adopted for the industrial production of the intermediate, and the production process has the disadvantages of more reaction steps, long period, harsh reaction conditions, more byproducts, great environmental pollution and low yield. The biological catalysis method has the advantages of specific stereoselectivity, mild reaction conditions, environmental friendliness, high product purity, easiness in separation and extraction of products and the like, so that the preparation of the key chiral intermediate of the APP herbicide by the biological method has great economic benefit and social value.

The biological preparation of R-HPPA is reported in less foreign countries, wherein the biological preparation is mainly reported by research of BASF company in Germany. In 1992, Bryan C, et al, which is a company of BASF, Germany, have reported that various microorganisms capable of hydroxylating R-2-phenoxypropionic acid were selected such as: aspergillus niger (Aspergillus niger), Aspergillus flavus (Aspergillus flavus), Aspergillus carbonarius (Aspergillus carbonarius), Aspergillus parasiticus (Aspergillus parasitism Speare), Streptomyces (Streptomyces), Coprinus comatus (Coprinus comatus), Beauveria bassiana (Beauveria), Sclerotium rolfsii (Sclerotium rolfsii) and Penicillium farinosum (Penicillium). In 1996, Beauveria bassiana LU700 is obtained by ultraviolet mutagenesis and Nitrosoguanidine (NTG) compound mutagenesis by BASF company Bryan C and the like, fermentation culture is carried out by using the Beauveria bassiana LU700, a whole-cell hydroxylation substrate R-PPA is R-HPPA, and the yield and the productivity of 1000 tons/year can be achieved by the method>99 percent. In 2008, Matthias K et al, Germany added H via Agrocybe cylindracea peroxidase₂O₂Hydroxylating R-PPA to R-HPPA under the conditions shown in FIG. 1. The reaction conditions are as follows: in a 1mL reaction system, Agrocybe aegerita peroxidase (AaP) 0.5. mu.M, 50mM phosphate buffer solution (pH 3-10), 0.5-2mM substrate racemate 2-phenoxypropionic acid. With 1mM H₂O₂The reaction was started and stopped with 0.1mL of 50% (w/v) trichloroacetic acid. The specific chosen isomeric purity in which the reaction is carried out is close to 98% and yields the desired R-isomer e.e. value of HPPA of 60%.

In past research, the conventional R-HPPA detection generally adopts a high performance liquid chromatography method. Although the method has the advantages of accuracy, sensitivity and the like, the sample processing is complex, the workload is large, and the method is not suitable for all biological laboratories. The conventional screening of the R-HPPA synthetic strain involves repeated inoculation and shake flask culture, and the whole screening process is time-consuming, low in efficiency and high in cost. Therefore, a high-efficiency, accurate and reliable high-throughput screening method for R-HPPA synthetases needs to be established.

Disclosure of the invention

The invention aims to provide a simple and feasible method for rapidly screening R-HPPA synthetic bacteria with high flux and high application value.

The technical scheme adopted by the invention is as follows:

the invention provides a high-throughput screening method of an R-2- (4-hydroxyphenoxy) propionic acid (R-HPPA) synthetic strain, which comprises the following steps: centrifuging fermentation liquor obtained by fermentation culture of a strain to be detected by taking R-2-phenoxypropionic acid (R-PPA) as a substrate, and taking supernatant as a sample to be detected; mixing a sample to be detected with a color developing agent (preferably in a microporous plate), standing for 5-70min at 40-60 ℃, taking a reaction solution to measure the absorbance at 420nm, obtaining the content of R-HPPA in the sample to be detected according to an R-HPPA standard curve, and screening to obtain an R-HPPA synthetic strain; the developer is sodium nitrite (NaNO)₂) Dissolving in deionized water to prepare 1-9g/L solution, and adjusting pH to 1.4-6.0 with 0.5mol/L sulfuric acid solution; the R-HPPA standard curve is prepared by replacing a sample to be detected with an R-HPPA aqueous solution, measuring the absorbance at 420nm under the same condition, and taking the concentration of the R-HPPA aqueous solution as the abscissa and the absorbance at 420nm as the ordinate.

Further, the concentration of the developer sodium nitrite solution is preferably 6.0g/L, and the pH value is preferably 1.4.

Further, the reaction condition is preferably a standing reaction at 60 ℃ for 30 min.

Further, the volume ratio of the sample to be detected to the color developing agent is 1: 1.

Further, the concentration of the substrate in the fermentation medium was 10.0 g/L.

Further, the fermentation method conditions of the strain to be detected are as follows: fermenting and culturing at 28 deg.C and 150r/min for 7d to obtain fermentation liquid.

Further, the standard curve of the R-2- (4-hydroxyphenoxy) propionic acid is prepared as follows: the R-2- (4-hydroxyphenoxy) propionic acid standard curve is prepared as follows: preparing 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L and 1.0g/L of R-2- (4-hydroxyphenoxy) propionic acid standard solutions with concentration gradients by using deionized water respectively, putting the standard solutions with different concentrations into micropores (preferably 100 mu L) of a 96-pore microporous plate respectively, adding a color developing agent into each micropore, reacting for 30min at the temperature of 60 ℃, determining the light absorption value at 420nm, and drawing a standard curve by taking the concentration of the R-2- (4-hydroxyphenoxy) propionic acid aqueous solution as a horizontal coordinate and the light absorption value as a vertical coordinate; in the micropores, the volume ratio of the color developing agent to the standard solution is 1:1, the color developing agent is prepared by dissolving sodium nitrite in deionized water to prepare the concentration of 6.0g/L, and the pH value is adjusted to 1.4 by using sulfuric acid.

Further, the screening method comprises the following steps: taking soil at a sampling point, preparing a turbid liquid by using physiological saline, and standing to settle; taking the supernatant by a pipette, coating the supernatant on a PDA culture medium flat plate, sealing the flat plate by a sealing film, and culturing at 28 ℃ until a bacterial colony grows out; inoculating a single colony in an inoculating loop picked plate into a 250mL triangular flask containing 50mL of first enrichment culture medium, and carrying out first enrichment culture at 28 ℃ and 150r/min for 2-3 d; inoculating the bacterial liquid of the first enrichment culture into a second enrichment culture medium by an inoculation amount with the volume concentration of 2%, and carrying out the second enrichment culture for 1-2d at the temperature of 28 ℃ and at the speed of 150 r/min; gradually diluting the bacterial liquid obtained by the second enrichment culture (10 are respectively taken^-3And 10^-5100 mu L of two gradient dilutions) and then spread on a PDA plate containing 10.0g/L R-2-phenoxypropionic acid, and the PDA plate is kept still and cultured for 3-4d at the constant temperature of 28 ℃; inoculating single colonies on the plate as strains to be detected into a seed culture medium one by one, and culturing for 72h at 28 ℃; then transferring to a fermentation culture medium containing 10.0g/L R-2-phenoxypropionic acid, and culturing at 28 ℃ and 150r/min for 7d to obtain fermentation liquor; centrifuging the fermentation liquor (1000rpm, 15min), and taking the supernatant as a sample to be detected; mixing a sample to be detected and a color developing agent in a volume ratio of 1:1, reacting for 30min at 60 ℃, taking a reaction solution to detect a light absorption value at 420nm, obtaining the content of R-HPPA in the sample to be detected by a standard curve, and screening to obtain the R-HPPA synthetic strain.

Further, the fermentation medium consists of: 5.0g/L of glucose, 5.0g/L of yeast extract, 5.0g/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, 2mg/L of ferrous sulfate heptahydrate, 100 mu g/L of zinc sulfate (II) tetrahydrate, 300 mu g/L of boric acid, 200 mu g/L of cobalt chloride (II) hexahydrate, 10 mu g/L of copper chloride (II) dihydrate, 20 mu g/L of nickel chloride (II) hexahydrate and 30 mu g/L of sodium molybdate dihydrate, wherein 2M sodium hydroxide is used for adjusting the pH to be 6.8, and the solvent is distilled water; the composition of the first enrichment medium, the second enrichment medium and the seed culture medium is the same as that of the fermentation medium.

The invention discovers that in an acidic medium, phenolic compounds are easy to be oxidized by nitrous acid to generate nitroso compounds, generated substances all have certain chromogenic groups, so that a solution is yellow or brownish yellow, and the reaction mechanism (figure 1) of the chromogenic reaction is designed based on the oxidation of hydroxyl nitrous acid on a benzene ring of R-HPPA. The reaction time is 30min, the range of R-HPPA measurement is 0.1-1g/L, and the average recovery rate is 95% -99%.

The high-throughput screening method is used for screening R-HPPA synthetic strains and is mainly based on two factors of the tolerance of the strains to R-PPA and the yield of the R-HPPA. The Potato Dextrose Agar (PDA) solid sieve culture medium is used for obtaining single colonies, the strain can tolerate the concentration of R-PPA to be more than 10.0g/L, has high selectivity and no by-product, can not degrade the R-PPA and the R-HPPA, and is suitable for strain improvement and system amplification. The traditional screening method for screening R-HPPA synthetic strains mainly adopts high performance liquid chromatography and relates to repeated inoculation and shake flask culture. According to the high-throughput screening method, single colonies on a plate are inoculated into a 96-deep-well plate without R-PPA one by one and cultured for 72h at 28 ℃. Then, the seed solution was transferred to the next 96-well plate containing R-POPA (10.0g/L) fermentation medium and cultured at 28 ℃ for 7 days. Centrifuging a 96-well deep-well plate at 1000rpm for 15min, sampling 100 mu L of supernatant in a 96-well microplate, and adding a color-developing agent. And detecting the light absorption value of the sample in the microporous plate by using a microplate reader. And calculating the content of the R-HPPA in each sample according to the standard curve, and calculating the conversion rate of the strain to convert the R-PPA.

Compared with the prior art, the method has the beneficial effects that:

the invention relates to a method for rapidly screening R-HPPA synthetic strains based on a microporous plate in high flux, the reaction time of the method is 30min, the detection range of the R-HPPA is 0.1-1.0g/L, the average recovery rate is 95.0-99.0%, and the method has high efficiency, sensitivity and reliability. Compared with the traditional liquid chromatography determination method, the high-throughput screening method has no obvious difference in the R-HPPA determination result, but the 96-pore plate high-throughput screening method can detect 960 samples in 1h, while the traditional high performance liquid chromatography needs 160h, so that the screening efficiency is greatly improved, and the time and the cost are saved. In addition, the method can intuitively and accurately screen the strains with high yield of the R-HPPA from wild bacteria and mutagenized strains, has the advantages of simplicity, convenience, rapidness, accuracy and the like, has the capacity of screening the strains with high yield of the R-HPPA on a large scale, and provides an effective strategy for screening the strains with high yield of the R-HPPA.

(IV) description of the drawings

FIG. 1 is an R-HPPA structure.

FIG. 2 is a schematic diagram of the microbial hydroxylation of R-PPA to R-HPPA, A: R-PPA standard solution and NaNO₂An aqueous solution; b: R-HPPA standard solution and NaNO₂An aqueous solution; c: a R-HPPA standard solution; d: NaNO₂An aqueous solution; e: fermentation Medium and NaNO₂An aqueous solution.

FIG. 3 is a graph showing the effect of sodium nitrite concentration on R-HPPA color development.

FIG. 4 shows the effect of reaction temperature on R-HPPA color development.

FIG. 5 is a graph showing the effect of pH on R-HPPA color development.

FIG. 6 is a graph showing the difference in color development between different concentrations of R-HPPA.

FIG. 7 is a graph of the dependence of (R) -HPPA concentration on OD value.

FIG. 8 is a flow chart of the high throughput screening method for screening R-HPPA producing strains.

FIG. 9 is a color reaction result of the microplate of example 2, showing that the catalytic activity of the corresponding 96 deep well plate growth strain is strong or weak.

FIG. 10 is a graph showing the results of color development reactions of different color developers in example 3.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1

First, experimental material

(1) Reagent and apparatus

The R-HPPA standard is purchased from Aladdin, the substrate R-PPA is provided by Shandong Runfeng GmbH, and other reagents are domestic analytical purifications.

Preparation of a standard solution: 5.0g/L of R-HPPA standard solution and 5.0g/L of R-POPA standard solution are accurately prepared by deionized water.

The instrument comprises the following steps: SpectraMax M2 multifunctional microplate reader (Molecular Device Company), 96 microwell plate, 96 deep well plate.

II, analysis program for enzyme label instrument detection of R-HPPA

(1) Absorption spectrum and selection of characteristic wavelengths

Respectively putting 0.5g/L of R-HPPA standard solution, 0.5g/L of R-PPA standard solution and 100 mu L of fermentation medium in a 96-microporous plate, and adding equal volume of NaNO with the concentration of 3.0g/L into each well₂In order to eliminate the influence of the developer and R-HPPA on the absorption value of the color developing solution, 3.0g/L of NaNO was used as the aqueous solution₂Respectively putting 200 mul of the water solution and 0.5g/L of R-HPPA standard solution in a 96 micro-porous plate, standing the 96 micro-porous plate for 30min at 40 ℃, carrying out full-wavelength scanning by using an enzyme-labeling instrument within the wavelength range of 200-900 nm, eliminating the interference of different substances on the absorption value, and finally determining the detection wavelength (as shown in figure 2).

As can be seen from FIG. 2, the absorption maxima at A, B, C, D and E are 230nm, where the OD of A, C, D and E_420nmOD of B0_420nm0.57, due to the standard solution of R-HPPA and NaNO₂The solution reacts, and the solution is yellow to eliminate R-HPPA, R-PPA and NaNO₂The influence of the solution and the fermentation medium on the absorbance of the chromogenic substance was chosen to be 420nm as the measurement wavelength.

(2) Color developing agent NaNO₂Optimisation of concentration

The prepared NaNO is added₂Diluting the water solution (10.0g/L) to 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0g/L respectively, and collecting N with different concentrationsaNO₂Placing 100 μ L of the water solution and 0.5g/L of R-HPPA standard solution in 96 microporous plate, standing at 40 deg.C for 30min, and measuring OD with microplate reader_420nm(three parallel groups), based on the data, NaNO was plotted₂Aqueous solution concentration and OD_420nmGraph (see FIG. 3), determining optimal NaNO₂Concentration of aqueous solution.

As can be seen from FIG. 3, NaNO₂The concentration of the aqueous solution is in the range of 1.0g/L to 6.0g/L, OD_420nmGradually increasing; when NaNO is present₂OD at an aqueous solution concentration of more than 6.0g/L_420nmTend to be constant, presumably NaNO₂The amount of solution is such as to completely oxidize the R-HPPA. Thus NaNO₂The optimal dosage of the aqueous solution is 100 mu L and 6.0 g/L.

(3) Optimization of the temperature of the color reaction

Taking 6.0g/L NaNO₂The aqueous solution and 0.5g/L R-HPPA standard solution are respectively 100 μ L in 96 micro-porous plate, and OD is measured by microplate reader at 40 deg.C, 50 deg.C and 60 deg.C every 20min in consideration of test microplate reader operability_420nm(three parallel sets are made), and time and OD are respectively drawn according to the data_420nmGraph (see fig. 4), determine the optimum reaction temperature.

As can be seen from FIG. 4, OD at 40 ℃ is_420nmSlow increase, indicating NaNO₂The aqueous solution slowly reacts with the R-HPPA; OD at 50 ℃_420nmThe increase is faster compared to 40 ℃, which indicates that NaNO is present at this temperature₂The reaction of the aqueous solution and the R-HPPA is faster than that at the temperature of 40 ℃; OD at 60 deg.C for 0-30min_420nmThe increase is fastest, and gradually becomes stable after 30min, which indicates that the NaNO is in the process₂The aqueous solution reacts towards completion with R-HPPA. Therefore, 60 ℃ was selected as the color reaction temperature.

(4) Optimization of pH value of color reaction and time stability investigation

6.0g/L NaNO with pH values of 1.4, 2.4, 3.4, 4.4, 5.4 and 6.4 is prepared₂Aqueous solution of NaNO at different pH₂Placing 100 μ L of the water solution and 0.5g/L of R-HPPA standard solution in 96 microporous plate, standing at 60 deg.C, and measuring OD every 10min with microplate reader_420nm(three parallel sets were made) and based on the data, the pH and OD were plotted_420nmGraph (see FIG. 5), determining optimum pH and developingThe color reaction reaches a stable time.

As can be seen from FIG. 5, OD was measured at pH1.4_420nmIncrease rapidly, OD after 30min_420nmThe growth is slow; OD at pH 2.4_420nmThe growth trend is slow, OD after 30min_420nmThe growth is slow; pH of 3.4, 4.4, 5.4 and 6.4, OD_420nmThe growth trend is slow, and the OD at 30min_420nmAnd still gradually increases. As described above, NaNO at pH1.4₂The aqueous solution reacts rapidly with R-HPPA, OD_420nmAfter 30min, the NaNO tends to be stable₂The optimum pH value of the aqueous solution is 1.4, and the reaction time is 30 min.

(5) Drawing of standard curve

Diluting the prepared R-HPPA (10.0g/L) standard solution to the concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0 and 3.0g/L, and mixing the R-HPPA standard solution with different concentrations with NaNO of pH1.4₂ Placing 100 μ L of each aqueous solution (6.0g/L) in 96 microporous plate, standing at 60 deg.C for 30min, and detecting OD with microplate reader_420nm(three parallel groups were made) according to OD_420nmThe mean values of the three data sets with respect to the concentration of the R-HPPA standard solution were plotted as a Y-X standard curve (A) with respect to the concentration on the X-axis and the OD on the Y-axis (see FIGS. 6 and 7).

As can be seen from the observation of FIG. 6, the color of the blend changed from yellow to brown when the concentration of R-HPPA was 0.1g/L to 1.0g/L, and the color did not change significantly when the concentration of R-HPPA was 0.7g/L to 1.0 g/L.

From FIG. 7, it was found that the concentration of R-HPPA ranged from 0.1g/L to 1.0g/L, the concentration of R-HPPA related to OD_420nmIn a linear relationship, the linear equation is Y-2.41818X +0.09636, R²＝0.9946，R²The values indicate the absorption OD_420nmHas good linear relation with the concentration of the R-HPPA.

(6) Limit of detection (LOD) and limit of quantitation (LOQ)

LOD and LOQ were validated by the international harmonization conference (ICH) for analysis according to the following formula to examine sensitivity parameters of the proposed method:

LOD 3.3 σ/S (formula 2-1)

LOQ 10 σ/S (formula 2-2)

σ is the intercept standard deviation of the standard curve; s is the standard curve slope.

The three groups of data OD detected in (5)_420nmRespectively drawing a standard curve A with the concentration of the R-HPPA standard solution₁、A₂And A₃Then calculate A₁、A₂And A₃The standard deviation sigma of the intercept of the three groups of standard curves is calculated according to the formulas 2-1 and 2-2, and the LOD and the LOQ are 0.011g/L and 0.034g/L, which shows that the method has better sensitivity.

(7) Examination of precision and accuracy

In order to verify that the method can be used for detecting the content of the R-HPPA in a sample, a standard sample of the R-HPPA with a known concentration is added to the measured sample, and the recovery rate of the method is measured. The strains are respectively inoculated into 250mL triangular flasks filled with 50mL fermentation medium, and are cultured for 7d at a constant temperature of 28 ℃ and 150r/min in shaking tables. Centrifuging to obtain supernatant, diluting to a certain concentration, collecting 100 μ L diluted supernatant, loading into 96-well microporous plate, and adding NaNO with pH of 1.4 and 6.0g/L into each microporous plate₂The water solution is 100 mu L, the reaction is carried out for 30min at 60 ℃, the light absorption value of the sample at 420nm is detected by a microplate reader, and the concentration C1 of R-HPPA in the sample is calculated according to a standard curve. Adding a standard sample of R-HPPA with C2 concentration into a certain volume of supernatant, then taking 100 μ L of supernatant containing R-HPPA with C2 concentration and diluted with ultrapure water, and placing the supernatant into a 96-well microplate, and adding a color-developing agent (NaNO with pH1.4 and 6.0g/L) into each well₂Aqueous solution) 100 mu L, reacting for 30min at 60 ℃, and detecting the light absorption value of the sample at 420nm by using a microplate reader. The concentration of R-HPPA C3 in the sample after the addition of the standard was calculated.

TABLE 1 NaNO₂Precision and accuracy of color development (n ═ 8)

From table 1 it is found that the recoveries obtained are in the range of 95-99% and ICH requires a good accuracy should be in the range of 95-102% of the actual value, so the results show that the proposed method is accurate.

The obtained Relative Standard Deviations (RSD) are all less than 5%, which indicates that the proposed method is precise.

Example 2:

(1) culture medium

The fermentation medium formula comprises: 5g/L glucose, 5g/L yeast extract, 5g/L ammonium sulfate, 0.5g/L magnesium sulfate heptahydrate, 0.05g/L manganese sulfate monohydrate, 1.5g/L potassium dihydrogen phosphate, 3.6g/L dipotassium hydrogen phosphate trihydrate, 2mg/L ferrous sulfate heptahydrate, 100 mu g/L zinc sulfate (II) tetrahydrate, 300 mu g/L boric acid, 200 mu g/L cobalt chloride (II) hexahydrate, 10 mu g/L copper chloride (II) dihydrate, 20 mu g/L nickel chloride (II) hexahydrate and 30 mu g/L sodium molybdate dihydrate, wherein the pH is adjusted to be 6.8 by 2M sodium hydroxide, and the solvent is distilled water.

Potato dextrose agar medium (PDA medium): cutting peeled potato into 200g pieces, adding 1L deionized water, boiling for 20min, filtering with double-layer gauze, adding glucose 20g and agar 15-20g into the filtrate, adding deionized water to make up for 1L, and adjusting pH to 6.8 with 2M sodium hydroxide.

(2) Screening with reference to the flow chart shown in FIG. 8

1) Culturing: 10.0g of soil sample at each sampling point is respectively taken, and is made into turbid liquid by using physiological saline, and the turbid liquid is stood for sedimentation. Taking 1mL of supernatant, and shake culturing at 28 ℃ and 150r/min for 1-2d in a 250mL triangular flask containing 25.0mL of the first enrichment medium. Transferring the bacterial liquid of the first enrichment culture to a new shake flask filled with a second enrichment culture medium by an inoculum size with the volume concentration of 2.0 percent for second enrichment culture, and performing shake culture at 28 ℃ and 150r/min for 1-2 d. Will be enriched for the second timeSeparately diluting the cultured bacterial solutions 10^-3And 10^-5Two gradients, 100. mu.L of the dilution was spread on PDA plates containing 10.0g/L substrate and incubated at rest in a constant temperature incubator at 28 ℃ for 3-4 days. And picking each single colony on the plate cultured for 3-4d with a sterile toothpick to a 96 deep-well plate inoculated with a fermentation medium containing 10.0g/L R-2-phenoxypropionic acid, and culturing at 28 ℃ and 150r/min for 7d to obtain a fermentation liquid.

2) Screening and detecting: centrifuging 96 deep-well plate at 1000r/min for 15min, collecting 100 μ L supernatant diluted with deionized water to the detection range in 96-well plate, and adding color-developing agent (pH1.4, 6.0g/L NaNO) into each well₂Aqueous solution) was reacted at 60 ℃ for 30min, and the wells were as shown in FIG. 9 (letters representing rows and numbers representing columns) after the reaction, and it was found that the wells B1, B2, B3, B4, B5, C1, C and C9 were relatively dark in color and appeared brown-yellow. The absorbance of the samples at 420nm was measured using a microplate reader, the content of R-HPPA in each sample was calculated according to the R-HPPA standard curve prepared in example 1, the conversion rate of the strain to convert R-PPA was calculated, and the content of R-HPPA in samples B1, B2, B3, B4, B5, C1, C and C9 was measured using an HPLC method.

HPLC detection conditions: illite C18 column: 250mm × 4.6 mm; mobile phase: v (aqueous phosphoric acid pH 2): v (acetonitrile) ═ 6: 4; flow rate: 1 mL/min; a detector DAD; detection wavelength: 220 nm; sample introduction amount: 5 mu L of the solution; column temperature: at 30 ℃.

3) As a result: the results are shown in Table 2. The analysis table 2 shows that the concentration of R-HPPA obtained by high-throughput screening and detecting the strains is similar to that of R-HPPA detected by HPLC, which indicates that the high-throughput screening method is more accurate. The concentration of R-HPPA obtained by B3 in the strains is the highest, the result of a high-throughput screening method is 4.50g/L, the result of HPLC is 4.56g/L, and the conversion rate is 45.59%.

TABLE 2R-HPPA assay results (n ═ 3)

More than 30 strains with catalytic activity have been obtained by this high throughput screening method. The 96-well plate high-throughput screening method can detect 960 samples in 1h, and the traditional high performance liquid chromatography requires 160h, so that the screening efficiency is greatly improved.

Example 3: detection of R-HPPA by different color-developing agents

Respectively preparing 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0g/L of R-PPA and R-HPPA standard solutions and other 8 kinds of color developing agents by using deionized water, wherein the standard solutions are respectively 0.2mol/L copper sulfate solution, 0.375g/L potassium permanganate solution, 0.75mol/L potassium bromate solution and ferrous tartrate standard solution (preparation method of ferrous tartrate solution: weighing 1.00g ferrous sulfate (FeSO)₄·7H₂O) and 5.00g of sodium potassium tartrate (C)₄H₄O₆NaK·4H₂O), mixing, adding deionized water for dissolving, and fixing the volume to 1000mL), 32.4g/L ferric trichloride solution, 7mol/L nitric acid solution and 30% hydrogen peroxide solution. And (3) respectively taking 100 mu L of R-PPA and R-HPPA standard solutions to micropores of a 96 micropore plate, and adding 100 mu L of the color developing agent into each micropore for reaction. The results are shown in FIG. 10, and the R-PPA and R-HPPA standard solutions are both light blue after being added into the copper sulfate solution, and have no obvious difference from the color of the copper sulfate solution, which indicates that the copper sulfate is not suitable for quantitative determination of R-HPPA.

The R-HPPA reacts with potassium permanganate to generate a yellow brown solution, the reaction temperature and the reaction time are respectively 30 ℃ and 10min, and the pH value is 11.0. However, the reaction of R-HPPA and potassium permanganate can generate precipitates along with the increase of the concentration of the R-HPPA, and the OD value is greatly different along with the change of time and the potassium permanganate is lack of stability, so that the potassium permanganate is not suitable for quantitatively determining the R-HPPA.

The R-HPPA and potassium bromate have no color reaction, which indicates that the potassium bromate is not suitable for quantitatively determining the R-HPPA.

After the standard solutions of the R-PPA and the R-HPPA are added into the standard solution of the ferric trichloride, the solutions are yellow brown, and the color of the solutions is not obviously different from that of the standard solution of the ferric trichloride. Indicating that ferric trichloride is not suitable for the quantitative determination of R-HPPA.

The reaction of R-HPPA with nitric acid produces a pale yellow solution, but the color fades with the increase of the reaction time or the temperature, and the stability is lacked, so that the nitric acid is not suitable for the quantitative determination of R-HPPA.

The R-PPA and R-HPPA reacted with hydrogen peroxide with no color change, indicating that hydrogen peroxide is not suitable for quantitative determination of R-HPPA.

Example 3 illustrates the invention based on 96 microwell plates NaNO₂The method for chromogenic high-throughput detection of R-HPPA is inventive.

Claims

1. A high-throughput screening method of R-2- (4-hydroxyphenoxy) propionic acid synthetic strains is characterized by comprising the following steps: culturing a strain to be detected in a fermentation medium containing a substrate R-2-phenoxypropionic acid, centrifuging the obtained fermentation liquor, and taking the supernatant as a sample to be detected; mixing a sample to be detected with a color developing agent, reacting for 5-70min at 40-60 ℃, taking reaction liquid to measure the light absorption value at 420nm, obtaining the content of R-2- (4-hydroxyphenoxy) propionic acid in the sample to be detected according to the standard curve of the R-2- (4-hydroxyphenoxy) propionic acid, and screening to obtain a synthetic strain of the R-2- (4-hydroxyphenoxy) propionic acid; the color developing agent is prepared by dissolving sodium nitrite in deionized water to prepare 1-9g/L, and adjusting the pH value to 1.4-6.0 by using sulfuric acid; the standard curve of the R-2- (4-hydroxyphenoxy) propionic acid is prepared by replacing a sample to be detected with an aqueous solution of the R-2- (4-hydroxyphenoxy) propionic acid, and measuring the light absorption value at 420nm under the same condition, wherein the concentration of the aqueous solution of the R-2- (4-hydroxyphenoxy) propionic acid is used as the abscissa and the light absorption value at 420nm is used as the ordinate.

2. The method according to claim 1, characterized in that the developer sodium nitrite concentration is 6.0g/L, pH to 1.4.

3. The method of claim 1, wherein the reaction conditions are 60 ℃ and 30min of standing reaction.

4. The method of claim 1, wherein the volume ratio of the sample to be tested to the chromogenic agent is 1: 1.

5. The method of claim 1, wherein the substrate is present in the fermentation medium at a concentration of 10.0 g/L.

6. The method according to claim 1, wherein the test strain is fermented under the following conditions: fermenting and culturing at 28 ℃ and 150r/min for 7d to obtain fermentation liquor.

7. The method of claim 1, wherein said R-2- (4-hydroxyphenoxy) propionic acid standard curve is prepared by: preparing 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L and 1.0g/L of R-2- (4-hydroxyphenoxy) propionic acid standard solutions with concentration gradients by using deionized water respectively, putting the standard solutions with different concentrations into micropores of a 96-hole micropore plate respectively, adding a color developing agent into each micropore, reacting for 30min at the temperature of 60 ℃, measuring the light absorption value at 420nm, and drawing a standard curve by taking the concentration of the R-2- (4-hydroxyphenoxy) propionic acid aqueous solution as a horizontal coordinate and the light absorption value as a vertical coordinate; in the micropores, the volume ratio of the color developing agent to the standard solution is 1:1, the color developing agent is prepared by dissolving sodium nitrite in deionized water to prepare the concentration of 6.0g/L, and the pH value is adjusted to 1.4 by using sulfuric acid.

8. The method of claim 1, wherein the screening method is performed by: taking soil at a sampling point, preparing a turbid liquid by using physiological saline, and standing and settling; coating the supernatant on a PDA culture medium plate, sealing the plate with a sealing film, and culturing at 28 deg.C until bacterial colony grows out; inoculating a single colony in an inoculating loop picked plate into a first enrichment culture medium, and carrying out first enrichment culture at 28 ℃ and 150r/min for 2-3 d; inoculating the bacterial liquid of the first enrichment culture into a second enrichment culture medium by an inoculation amount with the volume concentration of 2%, and carrying out the second enrichment culture for 1-2d at the temperature of 28 ℃ and at the speed of 150 r/min; diluting the bacterial liquid of the second enrichment culture, then coating the diluted bacterial liquid on a PDA plate containing 10.0g/L R-2-phenoxypropionic acid, and standing and culturing the bacterial liquid at the constant temperature of 28 ℃ for 3-4 days; inoculating single colonies on the plate as strains to be detected into a seed culture medium one by one, and culturing for 72h at 28 ℃; then transferring to a fermentation culture medium containing 10.0g/L R-2-phenoxypropionic acid, and culturing at 28 ℃ and 150r/min for 7d to obtain fermentation liquor; centrifuging the fermentation liquor, and taking the supernatant as a sample to be detected; mixing a sample to be detected and a color developing agent in a volume ratio of 1:1, reacting for 30min at 60 ℃, taking reaction liquid to detect a light absorption value at 420nm, obtaining the content of R-2- (4-hydroxyphenoxy) propionic acid in the sample to be detected according to an R-2- (4-hydroxyphenoxy) propionic acid standard curve, and screening to obtain a strain with high yield of R-2- (4-hydroxyphenoxy) propionic acid.

9. The method of claim 8, wherein the fermentation medium consists of: 5.0g/L of glucose, 5.0g/L of yeast extract, 5.0g/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, 2mg/L of ferrous sulfate heptahydrate, 100 mu g/L of zinc sulfate tetrahydrate, 300 mu g/L of boric acid, 200 mu g/L of cobalt chloride hexahydrate, 10 mu g/L of copper chloride dihydrate, 20 mu g/L of nickel chloride hexahydrate and 30 mu g/L of sodium molybdate dihydrate, wherein 2M sodium hydroxide is used for adjusting the pH to be 6.8, and the solvent is deionized water; the seed culture medium, the first enrichment culture medium and the second enrichment culture medium are all composed of the same fermentation culture medium.