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A high-throughput method for rapid screening of (r)-2-(4-hydroxyphenoxy)propionic acid-producing strains

A hydroxyphenoxy, high-throughput technology, used in biochemical equipment and methods, measuring devices, and microbial determination/inspection, etc., can solve the problems of large workload, high cost, low efficiency, etc., saving time, The effect of improving efficiency

Active Publication Date: 2022-03-18
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)-HPOPA hydroxylase-producing strains involves repeated inoculation and shake flask culture. The entire screening process is time-consuming, inefficient and costly.

Method used

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  • A high-throughput method for rapid screening of (r)-2-(4-hydroxyphenoxy)propionic acid-producing strains
  • A high-throughput method for rapid screening of (r)-2-(4-hydroxyphenoxy)propionic acid-producing strains
  • A high-throughput method for rapid screening of (r)-2-(4-hydroxyphenoxy)propionic acid-producing strains

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] 1. Experimental materials

[0032] (1) Reagents and instruments

[0033] The (R)-HPOPA standard was purchased from Aladdin Company, the substrate (R)-POPA was provided by Shandong Runfeng, and the rest of the reagents were of domestic analytical grade.

[0034] Preparation of standard solution: Accurately prepare 5g / L (R)-HPOPA standard solution and 5g / L (R)-POPA standard solution with deionized water.

[0035] Preparation of 6g / L 4-AAP reagent: 6g / L 4-AAP, 18.0mM Na 2 CO 3 , 2.0M NaOH to adjust the pH to 10.0, and the solvent is deionized water. Store in a brown bottle, protected from light.

[0036] Preparation of 0.06g / mL chromogenic agent: K 3 [Fe(CN) 6 ] was added to 6g / L 4-AAP reagent to prepare a final concentration of 0.06g / mL.

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

[0038] 2. Analytical procedure for (R)-HPOPA microplate reader detection

[0039] (1) Se...

Embodiment 2

[0058] (1) culture medium

[0059] 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.

[0060] 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.

[0061] (2) Refer to Figure 6 Shown process filter

[006...

Embodiment 3

[0068] Embodiment 3: Quantitative determination of (R)-HPOPA with ferric chloride

[0069] Use deionized water to prepare (R)-HPOPA standard solutions with concentration gradients of 0.5g / L, 1.0g / L, 1.5g / L, 2.0g / L, 2.5g / L, 3.5g / L, and 4.0g / L respectively And 32.4g / L ferric chloride solution. Make three groups in parallel, add 100 μL of (R)-HPOPA standard solution concentration from low to high to the microwells of the 96-well microplate, and add 100 μL ferric chloride solution to each microwell. (R)-HPOPA reacted with ferric chloride to generate a green solution, which was scanned at a full wavelength, and the measurement wavelength was selected as 580nm. The reaction temperature and time were 30° C. and 20 min, respectively, and the pH was 7.0. Determination of OD 580nm Value and draw (R)-HPOPA standard curve. The linearity of the standard curve is not enough, and its reason is that (R)-HPOPA and ferric chloride reaction will produce precipitation with the increase of con...

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Abstract

The invention discloses a method for high-throughput rapid screening of high-yield R-2-(4-hydroxyphenoxy)propionic acid strains, the method is carried out as follows: the strain to be tested is treated with R-2-phenoxy Propionic acid is used as the substrate to centrifuge the fermentation broth obtained by fermentation, and the supernatant is taken as the sample to be tested; the sample to be tested is mixed with the chromogenic agent, and reacted at 25-50°C and pH7.0-10.0 for 15- 45min, get reaction solution and measure the absorbance value at 550nm, obtain (R)-HPOPA content in the sample to be tested according to (R)-HPOPA standard curve, screen and obtain the bacterial strain of high yield (R)-HPOPA; The inventive method reaction time is 35 minutes , (R)-HPOPA has a detection range of 0.5-4g / L, and an average recovery rate of 98.5%-100.9%. It has high efficiency, sensitivity, and reliability, which greatly improves the efficiency of screening and saves time.

Description

(1) Technical field [0001] The invention relates to a high-throughput rapid screening method for biosynthetic (R)-2-(4-hydroxyphenoxy)propionic acid ((R)-HPOPA) high-yield bacteria based on micro-orifice plate, which belongs to high-throughput screening wild The technical field of bacteria and mutagenic strains. (2) Background technology [0002] 2-(4-Aryloxyphenoxy)propionic acid (APP) herbicides are a class of herbicides that selectively inhibit plant acetyl-CoA carboxylase developed in the past 40 years, with high efficiency, low toxicity and high selectivity. , safety to crops and ease of degradation have greatly promoted the development of selective herbicides. (R)-HPOPA is an important intermediate in the synthesis of aryloxypropionic acid herbicides such as clodinafop-propargyl, haloxyfop-p-p, and high-efficiency gatriol. Its compounds are constantly being introduced, and industrial production has a huge demand for its key chiral intermediate (R)-HPOPA. [0003] At...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/04C12Q1/26
CPCC12Q1/04C12Q1/26G01N2333/90245C12Q2326/96
Inventor 薛亚平郑裕国王美欣胡海峰王远山周海岩
Owner ZHEJIANG UNIV OF TECH
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