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Marine micromonospora strain for fermenting to produce Rakicidin B and application thereof

A strain and marine technology, which is applied in the field of marine micromonospora strains that ferment and produce RakicidinB, can solve the problems of low fermentation yield and the like, and achieve the effects of increasing fermentation yield, reducing yield and satisfying industrialization needs.

Active Publication Date: 2018-06-08
FUJIAN INST OF MICROBIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] For this reason, the technical problem to be solved by the present invention is to provide a kind of marine micromonospora strain that produces Rakicidin B by fermentation, to solve the lower problem of the fermentation yield of Rakicidin B compound in the prior art

Method used

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  • Marine micromonospora strain for fermenting to produce Rakicidin B and application thereof
  • Marine micromonospora strain for fermenting to produce Rakicidin B and application thereof
  • Marine micromonospora strain for fermenting to produce Rakicidin B and application thereof

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Embodiment 1

[0030] The mutation breeding of embodiment 1 bacterial strain

[0031] The mutagenesis breeding method of marine Micromonospora FIM-R160708 described in the present embodiment comprises the steps:

[0032] (1) Preparation of spore suspension: add an appropriate amount of sterile saline to the fresh slant of the mature starting strain FIM02523 (preservation number CGMCC NO.12132), gently scrape it off with an inoculation spatula, and pour it into a sterile shaker with glass beads. Shake the bottle to disperse, filter the mycelia, and leave the spore suspension for later use;

[0033] (2) Ultraviolet mutagenesis: Take 2-4mL of the prepared bacterial solution and add it to a 9cm-diameter petri dish, put a sterile magnetic stirrer, and then place it on the magnetic stirrer at 30cm under 30W ultraviolet light; before formal irradiation, The ultraviolet light should be turned on for 10 minutes to preheat the ultraviolet lamp, and then the lid of the dish should be opened to formall...

Embodiment 2

[0038] Embodiment 2 starting strain FIM02523 and mutant strain FIM-R160708 shake flask fermentation comparison

[0039] Scrape the slant spores of freshly cultivated Micromonospora marina FIM02523 and the mutant strain FIM-R160708 and inoculate the suspension into the shake flask seed culture, cultivate at 32°C and 250rpm for 48 hours, and then inoculate at 5% inoculum Inoculate into the shake flask fermentation medium, culture at 30° C. and 250 rpm for 120 hours, release the bottle, and measure the fermentation product by HPLC.

[0040] Preparation of seed medium formula (mass fraction): 2.0% soluble starch, 1.0% glucose, 2.0% yeast powder, 1.0% peptone, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.005%, CuSO 4 ·5H 2 O 0.005%, CoCl 2 ·6H 2 O 0.0005%, CaCO 3 0.3%, prepared with tap water, adjust the pH value to 7.0-7.5; carry out seed liquid cultivation at 32°C.

[0041] Preparation of fermentation medium formula (mass fraction): 6.0% soluble starch, 1.0% sucrose, 3.0% soy...

Embodiment 3

[0046] Embodiment 3 mutant strain FIM-R160708 is fermented on 20L fermenter

[0047] Prepare seed medium and fermentation medium according to the recipe in Example 2.

[0048] The seed medium is shake flask seeds, 500ml shake flasks have a liquid volume of 100ml per bottle, cultivated at 32°C and 250rpm for 48 hours; then inoculate a 5% inoculum in a 20L fermenter (actual liquid volume 13L) for fermentation, 30 Cultivate at ℃, control tank pressure 0.03-0.05Mpa, start at 200rpm, gradually adjust to 350rpm after 48 hours according to the change of fermentation parameter DO, keep the DO value greater than 20% during the fermentation process, and the ventilation rate is 1:1vvm, until the end of fermentation About 96-120 hours.

[0049] During the fermentation process, the content of Rakicidins compounds in the fermentation broth was detected by HPLC. The final fermentation titer was: Rakicidin B1 content was 19.5mg / L, Rakicidin B content was 763.2mg / L, and the B1:B component rat...

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Abstract

The invention belongs to the technical field of microorganism fermenting, and particularly relates to a marine micromonospora strain for fermenting to produce Rakicidin B and application thereof. Theexisting marine micromonospora strain FIM02523 for producing Rakicidins compound is induced to breed, so as to obtain a mutation marine micromonospora strain FIM-R160708 for producing the Rakicidin Bat high output. The marine micromonospora strain has the advantages that the valence of the Rakicidin B in a fermenting solution can be effectively increased; in the fermenting experiment of a 20-1000L fermenting tank, the valence of the Rakicidin B fermented by the marine micromonospora strain FIM-R160708 reaches about 1000mg / L, the ratio of components B1 to B in the fermenting solution is smaller than or equal to 3:97, the fermenting output of the target product Rakicidin B is greatly increased, and the production rate of other Rakicidins byproducts is effectively reduced; the subsequent extracting and purifying of the Rakicidin B can be favorably performed, and the industrial requirements are met.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation, in particular to a marine Micromonospora strain producing Rakicidin B by fermentation and application thereof. Background technique [0002] Rakicidins are the fermentation products of Micromonospora marine. The currently isolated Rakicidins mainly contain Rakicidin A, Rakicidin A1, Rakicidin B1 and Rakicidin B. Their chemical structures and components are as follows: [0003] [0004] Rakicidin s compound has attracted attention because its 15-membered cyclic lipopeptide structure contains a rare 4-amino-2,4-pentadienoic acid octanamide and has anti-tumor cell activity. Yamazaki (2007) found that Rakicidin A has excellent hypoxic selective anti-tumor cell activity, and the anti-colon cancer HCT-8 cell activity under hypoxic conditions is 17.5 times that under normal oxygen conditions; Takeuchi (2011) also reported for the first time Rakicidin A can induce the apoptosis of myel...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P21/02C12R1/29
CPCC12P21/02C12N1/205C12R2001/29
Inventor 周剑林风江红连云阳江宏磊陈丽赵薇方志锴
Owner FUJIAN INST OF MICROBIOLOGY
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