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Method for fast and efficient screening of high-yield 5-aminolevulinic acid mutant strain

A technology of aminolevulinic acid and mutant strains, which is applied in the field of bioengineering, can solve the problems such as difficulty in maintaining the blindness and excellent production traits of mutation breeding, increasing the difficulty of mutation breeding screening, and large screening workload, etc. The effect of screening workload, shortening screening cycle, and high screening accuracy

Inactive Publication Date: 2013-08-07
DALIAN SANYI ANIMAL MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The key to improving the output of 5-aminolevulinic acid is the screening of high-yield 5-aminolevulinic acid mutant strains. The existing screening method is to screen after cultivating strains in shake flasks, but due to the blindness and mutagenesis of mutation breeding The excellent production traits produced are not easy to maintain, which increases the difficulty of mutation breeding screening, resulting in a large screening workload and low screening efficiency

Method used

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  • Method for fast and efficient screening of high-yield 5-aminolevulinic acid mutant strain

Examples

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Embodiment 1

[0011] a. The Rhodobacter sphaeroides that has undergone physical or (and) chemical mutagenesis treatment (the treatment method is the same as the prior art) to produce 5-aminolevulinic acid ( Rhodobacter sphaeroides ) (CICC No: 10278) Wash with physiological saline, resuspend in liquid medium, incubate at 32°C and dilute to make 10 4 A / ml bacterial suspension, apply the plate to a static culture, cultivate in the dark at 31°C for 4 days, and then form colonies on the plate;

[0012] b. Pick a single colony with a large and bright colony and inoculate it in a liquid medium in 96 wells of a 96 deep-well plate for 40 hours, and then add glycine, succinic acid and levulinic acid to the liquid medium, per liter Add 30mmol of glycine, 30mmol of succinic acid, and 45mmol of levulinic acid to the medium, and continue culturing for 24h; the liquid medium contains 9.0g glucose, 3.8g sodium glutamate, and 0.5 potassium hydrogen phosphate per liter of water. g, 0.5g potassium dihydrogen pho...

Embodiment 2

[0015] a. will undergo physical or (and) chemical mutagenesis (existing technology) to produce 5-aminolevulinic acid Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) (CICC No: 10278) Wash with physiological saline, resuspend in liquid medium, incubate at 32°C and dilute to make 10 3 A / ml bacterial suspension, apply the plate to static culture, cultivate in the dark at 32°C for 4 days, colonies will be formed on the plate;

[0016] b. Pick a single colony with a large and bright colony and inoculate it in a 96-well liquid medium in a 96-deep-well plate for 48 hours, and then add glycine, succinic acid and levulinic acid to the liquid medium in an amount of Glycine 15mmol / L, succinic acid 20mmol / L, levulinic acid 30mmol / L, continue to cultivate for 24h; the liquid medium contains: glucose 9.0g, sodium glutamate 3.8g, and dipotassium hydrogen phosphate 0.5 per liter of water g, 0.5g potassium dihydrogen phosphate, 0.8g ammonium sulfate, 0.2g magnesium sulfate, 0.053g calcium chlo...

Embodiment 3

[0019] a. will undergo physical or (and) chemical mutagenesis (existing technology) to produce 5-aminolevulinic acid Rhodobacter sphaeroides ( Rhodobacter sphaeroides ) (CICC No: 10278) Wash with physiological saline, resuspend in liquid medium, culture at 31°C and dilute to make 10 4 A / ml bacterial suspension, apply the plate to static culture, cultivate in the dark at 32°C for 4 days, colonies will be formed on the plate;

[0020] b. Pick a single colony with a large and bright colony and inoculate it in a 96-well liquid medium in a 96-deep-well plate for 36 hours, and then add glycine, succinic acid and levulinic acid to the liquid medium in the amount of glycine 45mmol / L, succinic acid 40mmol / L, levulinic acid 60mmol / L, continue to cultivate for 24h; the liquid medium contains: glucose 9.0g, sodium glutamate 3.8g, and dipotassium hydrogen phosphate 0.5g per liter of water , Potassium dihydrogen phosphate 0.5g, ammonium sulfate 0.8g, magnesium sulfate 0.2g, calcium chloride 0....

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Abstract

The invention discloses a method for fast and efficient screening of a high-yield 5-aminolevulinic acid mutant strain. The method guarantees screening accuracy, reduces screening workload and improves screening efficiency. The method comprises the following steps of mutagenizing a rhodobacter sphaeroides strain by a physical or chemical mutagenesis method, washing the mutagenized strain by a stroke-physiological saline solution, carrying out liquid medium re-suspension, carrying out culture at a temperature of 32 DEG C, carrying out dilution to obtain a bacterial suspension, coating the bacterial suspension on a flat plate, picking a single bacterial colony from the flat plate, transferring it into corresponding holes of a sterile 96-deep well plate, carrying out shaking culture at a temperature of 32 DEG C in the dark for 48h, adding a precursor and succinic acid into the culture products, carrying out re-culture for 24h, and detecting spectrophotometric values at wavelength of 553nm of the bacterial liquid in all the wells by an ALA microscale development method (deep-well plate method), wherein the bacterial liquid having the maximum spectrophotometric value contains the high-yield 5-aminolevulinic acid mutant strain.

Description

Technical field [0001] The invention belongs to the field of bioengineering, and in particular is a method for quickly and efficiently screening high-yield 5-aminolevulinic acid mutant strains that can ensure screening accuracy, reduce screening workload, and improve screening efficiency. Background technique [0002] 5-aminolevulinic acid (ALA), also known as δ-aminolevulinic acid and δ-aminopentanonic acid, has a wide range of uses and has been used in agriculture and medicine. For example, it is used as a light-activated green and pollution-free herbicide, insecticide, antimicrobial agent and plant growth promoter in the agricultural field; in medicine, it has been applied to the photodynamic treatment and diagnosis of cancer. In addition, it can also As an external medicine and hair growth agent for acne treatment. Nishikawa et al. used NTG (nitrosoguanidine, CH 4 N 4 O) Repeated mutation of Rhodopseudomonas sphaeroides ( Rhodebacter sphaeroides ) And through the control of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/01C12N1/20C12R1/01
Inventor 庄国宏宋涛张琪
Owner DALIAN SANYI ANIMAL MEDICINE CO LTD
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