Breeding method of high-yielding strain of tetracycline

A tetracycline, high-yield technology, applied in the field of biology and medicine, can solve the problems of long breeding cycle, heavy workload, and the need for special equipment, and achieve the effects of high strain mutation rate, low environmental pollution, and high safety

Inactive Publication Date: 2013-01-02
NINGXIA QIYUAN PHARMA
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The above method has the defects of cumbersome process, long breeding cycle, heavy workload, special e...

Method used

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  • Breeding method of high-yielding strain of tetracycline

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Embodiment Construction

[0014] (1) Preparation of spore suspension

[0015] Add 15m sterile water to the well-grown Streptomyces aureus eggplant bottle slant to make a spore suspension, transfer it to a triangular flask containing glass beads, shake and cultivate for 20 minutes, filter and dilute.

[0016] (2) Mutagenesis treatment

[0017] Divide the diluted solution into four sterilized test tubes with 1ml each, then add 0.6ml EDTA solution at the same time, act for 3 minutes, 4 minutes, 5 minutes and 7 minutes respectively, then add 0.8ml lysozyme for 3 minutes . The treated spore fluid was serially diluted to 10 -5 , take 0.1, 0.3 and 0.5ml of each dilution of the spore liquid to smear the plate (agar medium), and cultivate it at an optimum temperature of 33-35°C for 90-100hr.

[0018] (3) Statistics positive variable rate and death rate respectively, select the plate with high positive variable rate as 5min action time, select single bacterium colony to be connected in slant medium (agar 2.5%...

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Abstract

The invention relates to a method for breeding high-yielding strain of tetracycline. The method is characterized by comprising the steps of: adding water into cultured Streptomyces aureus strains to prepare a spore suspension; shaking the flask for cultivation; filtrating; diluting; filling the suspension in four test tubes; adding a same amount of an EDTA solution and acting for respectively 3, 4, 5 and 7min; adding lysozyme and acting for 3 min; conducting gradient dilution to 10<-5>; and coating 0.1, 0.3 and 0.5ml spore liquid from each dilution on plates for cultivation; calculating a forward-mutation rate and a mortality rate; selecting an acting time with high forward-mutation rate; conducting further flask shaking screening; and selecting strains with high increase rate. The invention adopts a method, which first uses lysozyme to remove cell wall and then conducts EDTA treatment, thus breeding high-quality and high-yielding strain of tetracycline; and compared with an ultraviolet method and an ion implantation method, the method provided by the invention has advantages of simple operation, low cost, high safety, little environmental pollution, high mutation rate of strains, and high shake flask titer of the produced strain.

Description

technical field [0001] The invention belongs to the technical field of biology and medicine, in particular to a method for breeding tetracycline high-yielding bacteria. Background technique [0002] Tetracycline is an antibacterial substance isolated from the culture medium of the actinomycete Streptomyces aureofa-ciens, which is effective against Gram-positive bacteria, negative bacteria, Rickettsia, viral viruses, Spirochetes and even protozoa. It has a good inhibitory effect and is a broad-spectrum antibiotic, which is ineffective against Mycobacterium tuberculosis and Proteus. Its mechanism of action is to bind to the 30S subunit of the ribosome, thereby preventing aminoacyl-RNA from binding to the ribonucleosome. [0003] In the prior art, the natural selection process of tetracycline is as follows: determine the test plan → select the starting strain → prepare the plate separation medium → hatch the separation plate → prepare and sterilize the utensils → prepare the s...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12R1/485
Inventor 李学兵陈卓
Owner NINGXIA QIYUAN PHARMA
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