Method for improving tolerance of erythromycin-producing strain against n-propanol through mutagenesis
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A technology for producing erythromycin and producing bacteria, which is applied in the field of bioengineering, can solve the problems of reducing antibiotics, production bacteria poisoning, unfavorable antibiotics, etc., and achieves the effects of increasing n-propanol concentration, improving tolerance and increasing yield
Inactive Publication Date: 2013-06-19
SHANDONG FANGMING PHARMACEUTICAL CO LTD
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Problems solved by technology
However, adding n-propanol in the early stage of fermentation will interfere with the growth of mycelium, thereby reducing the synthesis of antibiotics, and the residual
Method used
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Embodiment 1
[0025] The first mutagenesis and screening: culture the erythromycin-producing bacteria on the slant medium, and obtain fresh mature spores after 7 days of culture; use the fresh mature slant of the erythromycin-producing bacteria to prepare 10ml of spore suspension, and the spore quantity is 1×10 6 cells / mL, divided into two parts.
[0026] Take a portion (as a control) to spread plate medium, the formula of plate medium is: soluble starch 6g, corn steep liquor 7g, ammonium sulfate 3g, sodium chloride 3g, agar powder 20g, 100% n-propanol 15ml, add water to volume to 1000ml, adjust the pH value to 7.2, and analyze 2.5g of pure calcium carbonate. After culturing for 7 days, the erythromycin-producing bacteria did not grow.
[0027] Take another part (as a mutagenized part) of the spore suspension and move it to a petri dish, irradiate it with a 15W ultraviolet lamp and a fixed distance of 20cm for 3 minutes, perform gradient dilution after mutagenesis, and immediately spre...
Embodiment 2
[0030] Put the seed solution of the n-propanol-resistant erythromycin-producing bacteria screened out for the second time into the fermentation shaker medium containing 100% n-propanol 2.2%, and shake it for 7 days at 34°C and 260 rpm Around, the erythromycin content reaches 7500u / ml.
[0031] Common erythromycin-producing bacteria use this method, and the content of erythromycin can only reach 5500u / ml.
Embodiment 3
[0033] Put the seed liquid of the n-propanol-resistant erythromycin-producing bacteria screened out for the second time into a 100L fermenter containing 100% n-propanol 2.2% for cultivation, and cultivate it at 34°C for about 7 days, and the titer in the tank can reach 13000u / ml.
[0034] Common erythromycin-producing bacteria use this method, and the content of erythromycin can only reach 8000u / ml.
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Abstract
The invention discloses a method for improving the tolerance of erythromycin-producing strain against n-propanol through mutagenesis. The method comprises the steps that: (1) an erythromycin-producing strain is adopted as an initial strain, and is pre-cultured for 7 days on a slant culture medium; (2) the pre-cultured erythromycin-producing strain is subjected to ultraviolet mutagenesis, wherein the strain is irradiated for 3min under conditions of an ultraviolet lamp with a power of 15W and a fixed distance of 20cm; and the obtained product is instantly smeared on a culture medium plate; (3) live colonies after mutagenesis are subjected to ion beam irradiation mutagenesis, wherein a mutagenesis temperature is 25 DEG C and mutagenesis dose is 55Gy; after mutagenesis, culture medium plate pre-screening and shaking flask fermentation secondary-screening are carried out, and the obtained live colonies are the mutant erythromycin-producing strain with tolerance to n-propanol. With the method, the mutant erythromycin-producing strain with tolerance to high-concentration n-propanol is obtained. The n-propanol tolerance concentration is higher than 2%. Therefore, erythromycin-producing strain tolerance against n-propanol is improved, such that n-propanol concentration in a culture medium can be increased. Under a condition that producing strain conversion rate is unchanged, with the increasing of n-propanol concentration, erythromycin yield is increased. A draw-off titer of a 100L fermentation tank can be higher than 13000u/ml.
Description
technical field [0001] The invention relates to the field of bioengineering, in particular to a method for increasing the yield of erythromycin by means of microbial mutagenesis. Background technique [0002] Erythromycin is a macrolide antibiotic with broad-spectrum antibacterial activity. to G + Bacteria have a strong antibacterial effect, especially effective against penicillin-resistant Staphylococcus aureus. It has a strong inhibitory effect on rickettsia, mycoplasma and spirochete. It also has inhibitory effect on amoeba and trichomonas. [0003] Erythromycin is an alkaline antibiotic isolated from the culture broth of Streptomyces erythromycin, and later isolated from the fermentation broth, erythromycin A, B, C, D, E and other components, of which erythromycin Vitamin A is the main component, which has strong antibacterial activity and less toxic and side effects; other components have weak antibacterial activity and greater toxic and side effects than A. Common...
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