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High-throughput screening method for R-2-(4-hydroxy phenoxy)propionic acid synthetic strain

A technology of hydroxyphenoxy and phenoxypropionic acid, applied in the measurement of color/spectral characteristics, separation of microorganisms, etc., can solve problems such as large workload, high cost, and inapplicability to biological laboratories, saving time and cost , the effect of improving efficiency

Active Publication Date: 2018-11-16
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has the advantages of accuracy and sensitivity, the sample processing is complicated and the workload is heavy, so it is not suitable for all biological laboratories
The screening of conventional R-HPPA synthetic strains involves repeated inoculation and shake flask culture, and the whole screening process is time-consuming, inefficient and costly

Method used

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  • High-throughput screening method for R-2-(4-hydroxy phenoxy)propionic acid synthetic strain
  • High-throughput screening method for R-2-(4-hydroxy phenoxy)propionic acid synthetic strain
  • High-throughput screening method for R-2-(4-hydroxy phenoxy)propionic acid synthetic strain

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Experimental materials

[0034] (1) Reagents and instruments

[0035] The R-HPPA standard was purchased from Aladdin Company, the substrate R-PPA was provided by Shandong Runfeng Co., Ltd., and the rest of the reagents were of domestic analytical grade.

[0036] Preparation of standard solution: accurately prepare 5.0g / L R-HPPA standard solution and 5.0g / L R-POPA standard solution with deionized water.

[0037] Instrument: SpectraMax M2 multifunctional microplate reader (Molecular Device Company), 96 microwell plates, 96 deep well plates.

[0038] 2. Analytical procedure of R-HPPA microplate reader detection

[0039] (1) Selection of absorption spectrum and characteristic wavelength

[0040] Take 0.5g / L R-HPPA standard solution, 0.5g / L R-PPA standard solution, and 100μL of fermentation medium in a 96-well plate, and then add an equal volume of NaNO with a concentration of 3.0g / L to each well. 2 Aqueous solution, in order to eliminate the influence of the chromoge...

Embodiment 2

[0071] (1) culture medium

[0072] Fermentation medium formula: glucose 5g / L, yeast extract 5g / L, ammonium sulfate 5g / L, 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, ferrous sulfate heptahydrate 2mg / L, zinc(II) sulfate tetrahydrate 100μg / L, boric acid 300μg / L, cobalt(II) chloride hexahydrate 200μg / L , copper (II) chloride dihydrate 10 μg / L, nickel (II) chloride hexahydrate 20 μg / L, sodium molybdate dihydrate 30 μg / L, adjust the pH to 6.8 with 2M sodium hydroxide, and the solvent is distilled water.

[0073] Potato glucose agar medium (PDA medium): 200g of peeled potatoes, cut into pieces, add 1L of deionized water, boil for 20min, filter with double-layer gauze, add 20g of glucose and 15-20g of agar to the filtrate, add deionized water to make up 1L, and use 2M of NaOH to adjust the pH to 6.8.

[0074] (2) Refer to Figure 8 Shown process filter

[007...

Embodiment 3

[0082] Embodiment 3: Different chromogenic agents detect R-HPPA

[0083] Prepare 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 g / L R-PPA and R-HPPA standard solutions and other 8 kinds of color developers with deionized water, respectively 0.2mol / L copper sulfate solution, 0.375g / L potassium permanganate solution, 0.75mol / L potassium bromate solution, ferrous tartrate standard solution (ferrous tartrate solution configuration method: weigh 1.00g ferrous sulfate (FeSO 4 ·7H 2 O) and 5.00g potassium sodium tartrate (C 4 h 4 o 6 NaK·4H 2 (2), add deionized water to dissolve after mixing, be settled to 1000mL), 32.4g / L ferric chloride solution, 7mol / L nitric acid solution and 30% hydrogen peroxide solution. Take 100 μL of R-PPA and R-HPPA standard solutions in the microwells of a 96-microwell plate, and add 100 μL of the above-mentioned chromogen to each microwell for reaction. see results Figure 10 , After the R-PPA and R-HPPA standard solutions were added to the...

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Abstract

The invention discloses a high-throughput screening method for a R-2-(4-hydroxy phenoxy) propionic acid (R-HPPA) synthetic strain. The method is carried out according to the following steps of: carrying out shake-flask culture on a to-be-detected strain in a fermentation culture medium containing a substrate R-2-phenoxypropionic acid, taking fermentation liquor to carry out centrifugation, and using supernate as a to-be-detected sample; mixing the to-be-detected sample with a color developing agent, performing a standing reaction, taking reaction liquid to measure OD420 nm, obtaining R-HPPA content in the to-be-detected sample according to a R-HPPA standard curve, and screening to obtain the R-HPPA synthetic strain. According to the method disclosed by the invention, reaction time is 30 min, a R-HPPA measurement range is 0.1 to 1 g / L, a LOD (Limit Of Detection) and a LOQ (Limit Of Quantitation) are 0.011 g / L and 0.034 g / L, a recovery rate is in a range of 95 to 99%, the method is efficient, sensitive and reliable, screening efficiency of the microorganism strain is greatly improved, time is saved, and human and financial resources are reduced.

Description

(1) Technical field [0001] The invention relates to a high-throughput rapid screening method for R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) synthetic bacteria based on a 96-microwell plate, which belongs to high-throughput screening of wild bacteria and mutant bacteria kinds of technical fields. (2) Background technology [0002] Aryloxyphenoxypropionic acid (APP) herbicides are a class of herbicides that selectively inhibit plant acetyl-CoA carboxylase developed in the past 40 years. The characteristics of degradation have greatly promoted the development of selective herbicides. R-HPPA is an important intermediate for the synthesis of APP herbicides such as clodinafop-propargyl, haloxyfop-p-p and high-efficiency gazolone. Its compounds are constantly being introduced, and industrial production has a huge demand for its key chiral intermediate R-HPPA. [0003] At present, the industrial production of this intermediate mainly adopts chemical methods, and the production...

Claims

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Application Information

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IPC IPC(8): C12N1/02G01N21/31
CPCC12N1/02G01N21/31
Inventor 周海岩郑裕国姜瑞李一作薛亚平王远山
Owner ZHEJIANG UNIV OF TECH
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