A kind of bacillus subtilis tolerant to limulusin and preparation method thereof

A technology of Bacillus subtilis and lysate, which is applied in the field of Bacillus subtilis and its preparation, can solve the problems of not being able to overcome the degradation effect of lysine I to inhibit and kill lysate I, and achieve the effect of strong tolerance

Active Publication Date: 2020-08-04
SHENZHEN POLYTECHNIC
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Problems solved by technology

[0005] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a kind of Bacillus subtilis tolerant to limulusin and a preparation method thereof, aiming at solving the problem that expression hosts in the prior art cannot overcome the inhibition of limulusin I and the inhibition of limulusin I. The problem of the degradation of factor I

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  • A kind of bacillus subtilis tolerant to limulusin and preparation method thereof
  • A kind of bacillus subtilis tolerant to limulusin and preparation method thereof
  • A kind of bacillus subtilis tolerant to limulusin and preparation method thereof

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[0041](1) Detect the MIC value of WB800 original bacteria (i.e. Bacillus subtilis) in BLM medium, and the minimum inhibitory concentration (MIC) of Limulin to WB800 original bacteria is 10 μg / mL, as shown in Table 1 below. In this embodiment, half of the MIC value (ie 1 / 2 MIC, 5 μg / mL) was used as the initial induction concentration.

[0042] (2) First, pick Bacillus subtilis species and culture them in 30 mL of BLM liquid medium at 37° C. in a shaking incubator at 200 rpm until the OD600 value of the Bacillus subtilis solution reaches 1.25. Then, add TAL to a new 5 mL BLM medium to make a final concentration of 5 μg / mL TLB. Then, 50 μL of cultured fresh Bacillus subtilis was inserted into the medium of 5 μg / mL of lysate, and cultured in a shaking incubator at 37° C. at 200 rpm. Finally, by the method of streak culture, that is, repeated induction 25 times at a concentration of 5 μg / mL of lysate, the strain can reach a stable tolerance, and pure Bacillus subtilis tolerant to ...

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Abstract

A limulusin-tolerant Bacillus subtilis and a preparation method thereof of the present invention, comprising the steps of: culturing Bacillus subtilis species in a culture medium to obtain Bacillus subtilis; adding limulusin to a new culture medium to prepare Different mediums with different concentrations of lysate; first inoculate the obtained Bacillus subtilis in the medium with lower lysate concentration for streak culture to obtain Bacillus subtilis tolerant to a lower concentration of lysate; then in the next lysate Insert the obtained Bacillus subtilis tolerant to lower concentration of TAL into the culture medium with higher concentration for streak culture, and so on, gradually increase the concentration of TAL, and cultivate Bacillus subtilis that can tolerate higher concentration TAL . In the invention, the culture medium added with gradient concentration of limulus is continuously used for long-term directional breeding of bacillus subtilis, and the anti-limulusin resistant strain is obtained. The strains obtained by the long-term continuous induction of limulus have a strong tolerance to limulus.

Description

technical field [0001] The invention relates to the field of microbes, in particular to a limulusin-tolerant bacillus subtilis and a preparation method thereof. Background technique [0002] Limulusin is a cationic antibacterial peptide isolated from small granules of Limulus blood cells. It has biological activities such as antibacterial, antifungal, antiviral, inhibiting tumor cell proliferation and inducing cancer cell differentiation. It is an antibacterial peptide with great potential application value. peptide. Limulusin mainly has three production methods: separation and purification from Limulus blood cells, chemical synthesis and genetic engineering expression. Due to the scarcity of horseshoe crab resources and the high cost of chemical synthesis, the first two methods are difficult to carry out large-scale production. Therefore, the production of horseshoe crab by genetic engineering expression is the only way to realize its industrialization. [0003] The resea...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/36C12N1/20C12R1/125
CPCC12N1/36C12N1/205C12R2001/125
Inventor 代建国唐勇军金刚
Owner SHENZHEN POLYTECHNIC
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