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A kind of doramectin producing bacteria and application thereof
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A doramectin and bacteria-producing technology, which is applied in the direction of bacteria, microorganisms, microorganisms, etc., can solve the problems of low specificity of reaction substrates, doramectin production efficiency and influence of doramectin purification, etc. Improve and simplify the effect of purification process
Active Publication Date: 2021-08-24
武汉回盛生物科技股份有限公司
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Since the synthesis of doramectin involves multi-step enzymatic reactions and the specificity of the synthetase to the reaction substrate is not strong, the doramectin-producing bacteria will simultaneously produce a variety of doramectins during the process of synthesizing doramectin Structural analogs, which affect the production efficiency of doramectin and the purification of doramectin
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Embodiment 1
[0035] Example 1 Screening of doramectin-producing bacteria 643a31
[0036] Using the doramectin-producing strain suk17-643a as the initial starting strain, proceed according to the following method steps:
[0037] A. Culture medium preparation
[0038] MS medium: Boil 10g of soybean cake powder with distilled water for 2.5 hours, filter with gauze to obtain 250ml of filtrate, put the filtrate into a 500ml Erlenmeyer flask containing 5g of agar and 5g of mannitol, adjust the pH to 7.2-7.3 with NaOH solution. After sterilization, each 10ml was dispensed into test tubes to prepare slant MS medium. After sterilization, each 30ml was dispensed onto a plate, and when the temperature dropped to about 60 degrees Celsius, lithium chloride with a final concentration of 0.05% was added to prepare a plate MS medium containing lithium chloride.
[0049] Seed culture medium: boil 10g of soybean cake powder with distilled water for 2.5 hours, filter with gauze to obtain 250ml of filtrate, divide each 25ml of filtrate into a 250ml Erlenmeyer flask containing 0.5g of cornstarch, and adjust the pH to 7.2-7.3 with NaOH solution.
[0050] Fermentation medium: Take 120g of corn starch, 30g of soybean meal powder, 6g of yeast powder, 0.01g of cobalt chloride and 0.01g of amylase, add tap water to make up to 1L. Divide into 500ml conical flasks, 50ml per bottle, and sterilize.
[0051] B. Preparation of Shake Flask Seeds
[0052] Take a slant of doramectin-producing bacteria 643a31 rich in spores, add 10ml of sterile saline in an aseptic method, scrape off the spores with a sterile inoculation loop, and make a spore suspension. According to the inoculum amount of 1%, inoculate the seed shake flask...
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Abstract
The invention belongs to the field of biotechnology, and specifically discloses a doramectin-producing bacteria. The doramectin-producing bacteria is preserved in the China Center for Type Culture Collection, and the preservation date is September 14, 2017, and the preservation number is CCTCC NO. : M 2017506, which is classified as Streptomyces avermectin 643a31 ( Streptomyces avermitilis 643a31). The invention also discloses the application of the strain, that is, the method for preparing doramectin by using the strain. The ratio of doramectin to doramectin structural analogs produced by the strain of the invention is significantly increased, and the doramectin purification process can be simplified. The strain of the present invention is a mutant strain with an increased proportion of doramectin obtained by the inventor by performing 4 rounds of ultraviolet composite lithium chloride mutagenesis screening on the doramectin-producing strain suk17-643a independently deposited by the inventor. Finally, strain 643a31 was obtained by screening, the proportion of doramectin can reach more than 85%, and the fermentation titer of shake flask can reach more than 700μg / ml.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to a doramectin-producing bacterium and its application. Background technique [0002] Doramectin is a macrolide animal-specific broad-spectrum antiparasitic drug in vitro and in vivo. Doramectin is produced by fermentation of microorganisms, and microorganisms with doramectin synthesis genes use cyclohexane formic acid as the starting material to synthesize doramectin. Since the synthesis of doramectin involves multi-step enzymatic reactions and the specificity of the synthetase to the reaction substrate is not strong, the doramectin-producing bacteria will simultaneously produce a variety of doramectins during the synthesis of doramectin Structural analogues affect the production efficiency of doramectin and the purification of doramectin. The types and proportions of structural analogues produced by different doramectin-producing bacteria are not the same. According to literature rep...
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