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Mutagenic screening method of strain mortierella alpina for generating arachidonic acid

A technology of arachidonic acid and Mortierella alpina, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem that AA production cannot be greatly improved, mechanism research is not perfect, arachidonic acid Low acid production and other problems, to achieve high economic and social benefits, good prospects for industrialization, and high improvement

Inactive Publication Date: 2010-05-19
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, in CN1362522A, Mortierella alpina was used as the starting strain, and a mutagenic strain with better activity was obtained through N+ ion implantation mutagenesis. After fermentation, the biomass could reach 30g / L, the oil 10.8g / L, peanut The yield of arachidonic acid is 4.65g / L. On the one hand, the disadvantage of this patent is that the mechanism research on the interaction between ion implantation and mold is not perfect;
Publication No. CN1587378A discloses a method for isolating and screening high-yielding arachidonic acid bacteria. The effect is obvious, but this is only a natural breeding process, and the AA production cannot be greatly improved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] ①Activation of seeds:

[0037] Mortierella alpina strain ATCC 16266 was inoculated on a PDA+PE+BE slope at 28°C for 5 days.

[0038] ②Cultivation of seeds:

[0039] After ① the slope is full, wash the mycelia with 10 mL of sterile water, inoculate in the seed medium with an inoculation amount of 20% (v / v), and incubate at 25°C for 60 hours. At this time, the seeds have grown to the logarithmic growth phase.

[0040] ③Pretreatment of seed solution:

[0041] Pretreatment method: ② Put 5mL of the obtained seed solution into a sterilized 250mL fluted Erlenmeyer flask covered with glass beads, add 45mL of 0.9g / 100mL sterile saline, shake at 25°C and 250r / m Treat for 30 minutes, filter with sterilized 4 layers of gauze, take 5mL of the obtained bacterial suspension and add it into a sterile Erlenmeyer flask filled with 5mL of LiCl physiological saline solution with a concentration of 0.6-2.0g / 100mL, and place the Erlenmeyer flask into Treat in a shaking incubator at 25°C w...

Embodiment 2

[0048] Example 2: Investigation of genetic stability.

[0049] The five high-yielding strains obtained in Example 1 were investigated for genetic stability, passed down for 10 times, and a shake flask fermentation experiment was carried out for each passage, and their biomass, oil production and AA production were measured. Experiments have shown that after multiple subcultures and fermentation verification, the biomass, oil production and AA production are equivalent to the fermentation level of one activation. Among them, HM-5 has the highest AA production and the strongest growth vigor, at an initial sugar concentration of 60g / Under the condition of L, the biomass was stable at about 35g / L, the oil output was maintained at about 18g / L, and the AA output was maintained at about 9g / L.

Embodiment 3

[0050] Example 3: Comparison of fermentation performance of strains before and after mutagenesis.

[0051] Strain HM-5 was fermented in a laboratory 50mL shake flask at a temperature of 25°C, a rotational speed of 120rpm, an initial sugar concentration of 60g / L, and fermentation for 7 days. See Table 1 for comparison of the fermentation results before and after mutagenesis.

[0052] Table 1 Comparison of fermentation results before and after strain mutagenesis

[0053]

[0054] From the above table, after the mutagenesis, the growth activity of the strain increased, the fermentation time was significantly shortened, and the AA yield increased from 0.03g / L·h to 0.058g / L·h.

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Abstract

The invention discloses a mutagenic screening method of strain mortierella alpina for generating arachidonic acid. After analyzing a microbe glycolytic pathway, a tricarboxylic acid transferring system, a fat acid synthesizing pathway and other related pathways in the process of producing arachidonic acid (AA) by fermenting the mortierella alpina, iodoacetic acid and acetylsalicylic acid are used as screening agents, and by a plurality of steps of activation of seeds, culture of the seeds, pretreatment of a seed solution, mutation of the treated seed solution, primary screening, secondary screening, third screening and the like, a strain with high growth rate of the arachidonic acid, short fermenting period and large increase range of the yield of the arachidonic acid can be obtained. The method of the invention is an ideal rational strain selective breeding technology and enjoys large economic benefits and social benefits and good industrialization prospects.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to the field of producing arachidonic acid by fermentation of Mortierella alpina, and specifically relates to a mutagenesis screening method for obtaining a strain of Mortierella alpina with high yield. Background technique [0002] Arachidonic acid (AA) is an important unsaturated fatty acid belonging to the omega-6 series. It is an important precursor for the synthesis of prostaglandins in the human body. It is also the most abundant and widely distributed polyunsaturated fatty acid in the human body. It has unique biological activities in reducing blood lipids, inhibiting platelet aggregation, anti-inflammation, anti-cancer, anti-lipid oxidation, and promoting brain tissue development. Therefore, it has a wide range of applications and has aroused great interest. Arachidonic acid widely exists in animals and microorganisms in a small amount. At present, commercial arachido...

Claims

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Application Information

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IPC IPC(8): C12N15/01C12N13/00C12P7/64C12R1/645
Inventor 黄和常淑梅彭超纪晓俊任路静刘欣尤江英范益春丛蕾蕾路金淼胡龄
Owner NANJING UNIV OF TECH
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