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High-yielding penicillin strain as well as screening method and application thereof

A technology for high-yield strains and screening methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, fungi, etc., can solve the problems of increased phenylacetic acid addition, large irradiation dose, and low improvement rate, and achieves the mutagenesis method Suitable, low radiation dose, strong resistance

Active Publication Date: 2017-10-27
NORTH CHINA PHARMA COMPANY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Wang Cheng and others disclosed a mutagenesis method using high-energy electrons to irradiate penicillin spores (Wang Cheng, Zhang Junjian, Yin Guichao. High-energy electron mutagenesis and breeding of Penicillium chrysogenum. Coal and Chemical Industry 2014, 37(2): 68- 69.), this method uses high-energy electrons to directly mutate penicillin spores. Due to the thickness of the fungal spore wall, it has the disadvantages of large irradiation dose, high lethality, and low mutation rate; use high-concentration phenylacetic acid plates to screen for those resistant to high-concentration phenylacetic acid Mutant strain, the fermentation unit of the screened mutant strain No. 13-6-55 increased by 3.6% compared with the control, the increase rate was low, and the addition of phenylacetic acid increased

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, (screening of penicillin high-yielding bacterial strain)

[0047] Preparation of mycelia medium: Weigh 2.5g sucrose, 1.5g peptone, 2.0g yeast powder, 0.3g ammonium sulfate, 0.4g potassium dihydrogen phosphate, add distilled water to 100mL, and then use 30% hydrogen to oxidize Adjust the pH to 6.0±0.2 with sodium solution, fill each 250mL Erlenmeyer flask with 30mL mycelial medium, seal it with 8 layers of gauze, sterilize at 121°C for 20min, and obtain the mycelial medium after cooling;

[0048] (A) Protoplast preparation:

[0049] A-1) Inoculate the spore liquid of the starting strain B1123 (the self-produced strain of North China Pharmaceutical Hebei Huamin Pharmaceutical Co., Ltd.) into a 30mL mycelium culture medium shake flask, and inoculate it at 25±0.5°C and a relative humidity of 40% to 60°C. % under the condition of culturing for 2 days to obtain mycelial culture fluid;

[0050] A-2) Take 10mL of culture solution and centrifuge at 3000rpm for 10...

Embodiment 2

[0062] Embodiment 2, (the genetic stability investigation of penicillin high-yield bacterial strain)

[0063] The high-yielding Penicillium chrysogenum B131536 preserved in Example 1 was inoculated on the slant culture medium prepared in Example 1, and cultured in the dark at 25±0.5° C. and relative humidity of 40% to 60% for 7 days, and the slant strains were matured. It was re-inoculated in the slant medium for cultivation, and repeated four times; the five cultured strains were respectively transferred to the seed medium prepared in Example 1, at 25 ± 0.5 ° C, relative humidity 40% ~ 60 %, 220rpm under the condition of cultivating 40 hours, after the strains are mature, they are respectively transferred to the fermentation medium prepared in Example 1, and cultivated for 7 days at 25±0.5°C, relative humidity 40%~60%, 220rpm, Five groups of fermentation broth containing penicillin were prepared, and the potency of penicillin in the fermentation broth was determined by HPLC. ...

Embodiment 3

[0064] Embodiment 3, (production application of penicillin high-yielding bacterial strain)

[0065] Millet spore medium preparation: weigh 6g sucrose, 10g corn steep liquor, 2g sodium chloride, 0.008g magnesium sulfate, 0.005g ferrous sulfate, dilute to 1000mL with distilled water, then use 30% sodium hydroxide solution Adjust the pH to 6.8±0.2, pour it into a pot with 2400 grams of dried millet, stir it well, steam it in a disinfection cabinet at 100±2°C for 20 minutes, put the steamed millet in the fan mouth to cool, rub it, and put it in Put the millet into eggplant bottles (20g / bottle), put the eggplant bottles in a disinfection cabinet, sterilize at 121°C for 30 minutes, and cool to obtain millet spore culture medium;

[0066] Seed tank medium preparation: Weigh 375kg sucrose, 900kg corn steep liquor, 300kg ammonium sulfate, 200kg calcium carbonate, and dilute to 13m with deep well water 3 , then use 30% sodium hydroxide solution to adjust the pH to 6.0±0.2, sterilize th...

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PUM

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Abstract

The invention discloses a high-yielding penicillin strain, a screening method of the strain, and application of the strain in penicillin production, and belongs to the technical field of antibiotics production and application. The strain is characterized in that penicillin fermenting unit is increased by 13.2%, so that the yield of penicillin is effectively increased, and the production cost is decreased. The screening method comprises the following steps: (A) preparing a protoplast; (B) performing high-energy electronic mutagenesis; (C) rationally screening; and (D) screening with a shake bottle. The penicillin strain screening method is high in purposiveness, and high in mutation probability.

Description

technical field [0001] The invention relates to an industrial strain used for antibiotic production, a screening method thereof and application in the field of fermentation, in particular to a penicillin high-yielding bacterial strain, a screening method and application thereof. Background technique [0002] Penicillin (Penicillin) belongs to β-lactam antibiotics, is an important antibiotic with high efficiency, low toxicity and wide clinical application; penicillin can also be used as raw material to synthesize a series of semi-synthetic penicillins and semi-synthetic cephalosporin antibiotics. Penicillin is synthesized by secondary metabolism in the fermentation process of Penicillium Chrysogenum. At present, the breeding methods of excellent strains of Penicillium chrysogenum usually adopt hybrid breeding, chemical mutagen mutation breeding, physical mutagen mutation breeding and genetic engineering breeding, etc. Mold strains have low production capacity. Wang Cheng an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N1/02C12N15/01C12N13/00C12P37/00C12R1/82
CPCC12N1/02C12N13/00C12N15/01C12P37/00C12N1/145C12R2001/82
Inventor 段志钢王平尹贵超王成张军剪王淑琳王云
Owner NORTH CHINA PHARMA COMPANY
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