Screening method of high-producing strain of producing strain of echinocandins B

A technology of echinocandins and high-yield strains, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of large randomness, long natural breeding cycle, low efficiency, etc., and achieve simple steps, The effect of shortening the screening cycle and easy operation

Active Publication Date: 2018-03-09
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional natural breeding cycle is long, low efficiency, large randomness and a la...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Appropriately diluted Aspergillus nidulans spore suspension was subjected to ultraviolet mutagenesis treatment, and spread on a screening plate. In a constant temperature incubator, culture at 37°C for 4 days, select 900 well-growing strains, mark them, and pick half of the colonies and soak them in 150 microliters of ethanol for 2 hours, and save the remaining half of the colonies.

[0023] (2) From the leaching solution in step (1), take 10 microliters and add to a small filter paper piece, which is placed on the Candida albicans plate. After culturing at 28°C for 24 hours, observe the size of the bacteriostatic zone, select 30 strains with a larger bacteriostatic zone, and subculture half of the colony preserved in step (1) corresponding to the label according to the label.

[0024] (3) After the strains screened in step (2) are subcultured, the seed solution is prepared and inoculated in the fermentation shaker flask. After culturing at 26°C and 280rpm for 6 da...

Embodiment 2

[0028] (1) Appropriately diluted Aspergillus nidulans spore suspension was subjected to NTG mutagenesis treatment, and spread on a screening plate. In a constant temperature incubator, culture at 26°C for 8 days, select 750 well-growing colonies, mark them, and pick half of the colonies and soak them in 200 microliters of acetone for 4 hours, and save the remaining half of the colonies.

[0029] (2) From the leaching solution in step (1), take 10 microliters and add to a small filter paper piece, which is placed on the Candida albicans plate. After culturing at 28°C for 24 hours, observe the size of the inhibition zone, select 40 strains with a larger inhibition zone, and subculture half of the colony preserved in step (1) corresponding to the label according to the label.

[0030] (3) After the strains screened in step (2) are subcultured, the seed solution is prepared and inoculated in the fermentation shaker flask. After culturing at 26°C and 280rpm for 6 days, the fermentat...

Embodiment 3

[0034] (1) Appropriately diluted Aspergillus nidulans spore suspension was subjected to ion beam mutagenesis treatment, and spread on a screening plate. Incubate in a constant temperature incubator at 35°C for 2 days, select 750 well-growing colonies, mark them, and pick half of the colonies and soak them in 300 microliters of methanol for 0.5 hours, and save the remaining half of the colonies.

[0035] (2) From the leaching solution in step (1), take 10 microliters and add to a small filter paper piece, which is placed on the Candida albicans plate. After culturing at 28°C for 24 hours, observe the size of the inhibition zone, select 40 strains with a larger inhibition zone, and subculture half of the colony preserved in step (1) corresponding to the label according to the label.

[0036] (3) After the strains screened in step (2) are subcultured, the seed solution is prepared and inoculated in the fermentation shaker flask. After culturing at 26°C and 280rpm for 6 days, the...

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Abstract

The invention establishes a screening method of a high-producing strain of a producing strain of echinocandins B, and a strain obtained by screened is preserved. The screening method comprises the following steps: firstly, according to the characteristic of feedback inhibition of a product of the producing strain of the echinocandins B, adding an appropriate amount of echinocandins B into a screening plate for primary screening, so as to screen out strains with the characteristic of feedback inhibition of the product removed; next, soaking mycelia, inducing the strains into an organic solvent,extracting the echinocandins B in the mycelia, and rescreening the high-producing strain of the echinocandins B from the primarily screened strains according to the size of an inhibition zone by adopting the inhibition of the echinocandins B on candida albicans, so as to achieve the purpose of rescreening; finally, investigating the production capacity of the screened out high-producing strain with a fermentation shake flask. The screening method is simple, convenient and efficient, so that the screening period is greatly shortened, and the screening efficient of the strain is improved.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a screening method for high-yield strains of echinocandin B-producing bacteria. Background technique [0002] Humans discovered echinocandin drugs in the 1970s, which are secondary metabolites of Aspergillus nidulans. There are three main types, among which echinocandin B (Echinocandin B) is the most important type. Its molecular structure contains a cyclic hexapeptide core with a lipid side chain that can non-competitively inhibit the activity of fungal cell wall β-1,3-glucan synthase, resulting in cell wall glucan The emptying of glycans, osmotic instability and the dissolution of fungal cells exert its antifungal effect, showing the characteristics of broad antibacterial spectrum and strong activity. It is an important drug of choice for the treatment of fungal infections in immunosuppressed patients and patients with normal immunity. In Candida (Candidas) and Aspergillus (Asp...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N15/01C12N1/02C12P21/04C12R1/66
CPCC12N1/02C12N15/01C12P21/02C12N1/145C12R2001/66
Inventor 张贵民薛国希
Owner LUNAN PHARMA GROUP CORPORATION
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