Compound mutation method for inosine-producing strain

A technology for compound mutagenesis and production of bacteria, applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of toxicity or carcinogenicity, harm to humans and the environment, etc., and achieves obvious mutagenesis effect. Ease of operation and improved conversion rate

Inactive Publication Date: 2015-04-22
肇东星湖生物科技有限公司
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Problems solved by technology

Traditional mutation breeding methods are basically toxic or

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1: the mutagenesis selection method of Bacillus subtilis protoplast

[0018] (1) Activation culture of the strain: transfer the glycerin species of Bacillus subtilis stored at -72°C to the slant medium for activation culture, and culture at a constant temperature of 34-37°C for 20-24h; slant medium formula (g / L): peptone 3. Beef extract 5, yeast extract 5, agar 20, pH7.0-7.2, sterilized at 121°C for 15 minutes.

[0019] (2) Liquid cultivation of strains in shake flasks: pick a ring of bacterial lawn from the activated cultured slant and inoculate it into a 500mL Erlenmeyer flask containing 50mL of seed medium, culture at 34-37°C, 180rpm for 16h. Then transfer 1 mL of bacterial liquid into 50 mL of fresh seed medium, and culture at 34-37° C., 180 rpm for 5 hours to enter the logarithmic growth phase. Seed medium formula (g / L): glucose 20, potassium dihydrogen phosphate 1, yeast powder 1, urea 2, pH7.0-7.2, sterilized at 121°C for 15 minutes.

[0020](3) Bact...

Embodiment 2

[0025] Embodiment 2: the mensuration of inosine productive rate

[0026] The method for determining the yield of inosine is a direct detection method for 24-well plate screening, and a paper chromatography method for re-screening in shake flask fermentation.

[0027] The direct detection method is to centrifuge the 24-well plate at 3500rpm for 10min, take the supernatant and dilute it by 2 times, and then use a multifunctional microplate reader to detect the ultraviolet absorption value at a wavelength of 260nm.

[0028] The method of paper chromatography is to centrifuge the fermentation broth at 10,000 rpm for 15 minutes, take the supernatant and dilute it by 2 times, then take 10 μL of the sample needle and spot it on the filter paper. The chromatographic solution is n-butanol: acetic acid: water = 4:1:1 After developing for 2 hours, the inosine spots were cut out and soaked in a hydrochloric acid eluent in a water bath at a constant temperature of 70°C for 30 minutes, and ...

Embodiment 3

[0029] Embodiment 3: genetic stability test

[0030] Activation culture of low-temperature preserved glycerin species is the first generation; the inoculation ring from the first generation slant seed is transferred to the new slant culture, which is the second generation; the second generation slant seed is inoculated The ring-picking-ring bacteria were transferred to a new slant culture, which was the third generation; and so on, passed to the fiftieth generation. Wherein seven generations of bacterial strains are carried out to shake flask fermentation test, detect fermented liquid inosine productivity by the method of embodiment 2, and detect and correct with high performance liquid chromatography method, its inosine productivity of seven generations of bacterial strains is all in 70-74g / L about.

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Abstract

The invention discloses a compound mutation method for an inosine-producing strain. Bacillus subtilis protoplast is subjected to atmospheric and room temperature plasma mutation. The compound mutation method comprises the following steps: (1) activation culture of the strain; (2) shake flask liquid culture of the strain; (3) enzymolysis and wall digestion of thallus; (4) atmospheric temperature plasma mutation; (5) regeneration culture of protoplast; (6) 24-well plate screening; and (7) shake flask fermentation and rescreening. After the strain is subjected to passage for fifty times, the genetic characters are stable. Compared with the original strain, the mutant strain has the advantages that the inosine-producing amount is increased from 60g/L to 72g/L, the conversion rate is increased by 20% and the significant effect is achieved.

Description

technical field [0001] The invention belongs to the technical field of mutagenesis and breeding of microbial strains, and in particular relates to a method for compound breeding of high-yield inosine-producing strains by protoplasts and normal-pressure room-temperature plasma mutagenesis. According to this method, high-yielding inosine yields can be obtained by breeding strains. Background technique [0002] Inosine, also known as inosine, has the molecular formula C 10 h 12 N 4 o 5 , molecular weight 268.23. Inosine has very important uses in the fields of medicine and food: inosine is a component of ATP, coenzyme A, ribonucleic acid and deoxyribonucleic acid in the body, and participates in the material metabolism and energy metabolism of the body. Inosine has good permeability to the cell membrane and can directly enter the cell. Inosine is converted into inosinic acid and adenosine triphosphate in the body, participates in energy metabolism and protein synthesis of ...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12R1/125
Inventor 蔡友华陆最青莫文滔严杰能
Owner 肇东星湖生物科技有限公司
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