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Actinoplanes of high-yielding sirolimus

A technology of actinomycetes and rapamycin, applied in the direction of bacteria, microorganism-based methods, microorganisms, etc., can solve the problem of low biological fermentation unit of rapamycin, inability to fully meet industrial production, unfavorable extraction and purification, etc. problems, to achieve the effects of reduced production costs, high production capacity, and high genetic stability

Active Publication Date: 2014-04-23
天津北洋百川生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the deficiency in the biological fermentation production of rapamycin mainly lies in that the biological fermentation unit of rapamycin is not high, which is not conducive to extraction and purification, and cannot fully meet the requirements of industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Rapamycin-producing strain hn-016 screened from soil

[0030] 1. Morphology of the bacteria: The bacteria are Gram-positive bacteria without aerial hyphae, and reproduce in the form of spores. The spores are round and most of them have extremely tufted hairs and can swim.

[0031] 2. Colony morphology: On the plate, the base filament surface of the colony is granular and orange-yellow. With the extension of culture time, the colony will change from orange-yellow to yellow-brown.

[0032] 3. Culture characteristics: This bacterium is a highly aerobic bacterium, the optimum growth temperature is 28°C, and the optimum growth pH is 7.0-7.2; when the shaker speed is 210-240r / min and cultured for 7-8 days, Rapamycin The yield of rapamycin was the highest, and dissolved oxygen had a significant effect on the production of rapamycin during the fermentation process.

Embodiment 2

[0034] Using Actinomycetes hn-016 as the starting strain, the strain BCStr-096 was screened by plasma mutagenesis at room temperature at atmospheric pressure

[0035] 1. Obtaining of mutagenized strains

[0036] (1) Select the well-growing rapamycin-producing actinomycetes hn-016 slant strain, wash the spores with normal saline, filter through gauze, and make 10 6 Individual / mL spore suspension;

[0037] (2) Use a pipette gun to pipette 10 μL of the spore suspension in (1) onto a circular iron plate with a diameter of 1 cm, and place it in a laboratory with helium as the working gas, a power supply of 110 W, and a working gas flow rate of 10 L / min. In the normal pressure and room temperature plasma mutagenesis system, the treatment distance is 2mm, and the 0s, 20s, 40s, 60s, 80s, 100s, 120s, 140s, 160s, 180s are respectively treated, and the treated spore suspension is gradiently diluted and spread on a plate to make lethality curve;

[0038] (3) According to the lethality ...

Embodiment 3

[0051] Cultivation of Actinomycetes BCStr-096

[0052] 1. Preparation of culture medium

[0053] (1) Preservation and separation medium: 3% oatmeal, 2% glucose, 1% yeast powder, 1% peptone, 0.25% calcium carbonate, supplemented with distilled water, pH 7.0-7.2, sterilized by high-pressure steam at 121°C for 20 minutes;

[0054] (2) Seed medium: 2.8% soluble starch, 0.8% peptone, 2% glucose, 1% soybean cake powder, 0.3% calcium carbonate, supplemented with distilled water, pH 7.0-7.2, sterilized by high pressure steam at 121°C for 20 minutes;

[0055] (3) Fermentation medium: corn starch 2%, glucose 2.5%, soybean cake powder 3%, peptone 0.8%, dipotassium hydrogen phosphate 0.5%, potassium dihydrogen phosphate 0.5%, magnesium sulfate 0.2%, sodium chloride 0.5% , 1% glycerin, 0.3% calcium carbonate, 0.2% distilled water, make up the deficiency with distilled water, pH 7.0-7.2, sterilize by autoclaving at 121℃ for 20min.

[0056] 2. Activation of strains

[0057] Transfer the...

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PUM

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Abstract

The invention discloses actinoplanes CGMCC NO.8430 of high-yielding sirolimus, belonging to the technical field of fermentation engineering. The bacterium is generated by mutating actinoplanes hn-016 of high-yielding sirolimus screened in a soil sample of Hainan Island as an original strain by plasmas at constant pressure and room temperature. Compared with conventional mutation methods, the mutant strain BCStr-096 disclosed by the invention is higher in hereditary stability and higher in production capacity of sirolimus, and the output is improved by 175% compared with the output 200mg / L of the original strain hn-016. The output is improved by about 37% in the highest fermentation level of existing sirolimus, so that the production cost is greatly lowered.

Description

technical field [0001] The invention belongs to the technical field of fermentation engineering, and in particular relates to a mutagenic strain BCStr-096 of a high-yield rapamycin motile actinomycetes. Background technique [0002] Rapamycin (RPM), also known as sirolimus, is a nitrogen-containing triene macrolide antibiotic with a unique mechanism of action. It has antifungal and antiproliferative effects. According to reports, Luan FL et al. Animal experiments have found that rapamycin also has anti-tumor effects. Rapamycin is currently the most promising new powerful immunosuppressant in the world. Its molecular structure is similar to FK506. Cyclosporin is dozens of times stronger and is the immunosuppressant with the lowest nephrotoxicity. It can also be used to treat autoimmune diseases such as rheumatoid arthritis and lupus erythematosus. It is currently developed as a gene therapy and anti-tumor drug with broad applications prospect. [0003] Originally, RPM was ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/18C12R1/045
Inventor 乔长晟石漫漫李雪朱明
Owner 天津北洋百川生物技术有限公司
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