Mutagenic screening method of DHA-producing strain crypthecodinium. cohnii

A dinoflagellate, mutagenic screening technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problem of low DHA oil production, achieve excellent industrial production prospects, and reduce screening links and work. The effect of improving the amount and biosynthesis

Inactive Publication Date: 2012-03-28
XIAMEN KINGDOMWAY GROUP
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Problems solved by technology

Among the currently published patents on the production of DHA using marine microalgae, the yield of DHA o

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Embodiment Construction

[0025] A kind of mutagenesis screening method of DHA producing bacterium dinoflagellates of the present invention, its steps are as follows:

[0026] 1. Seed cultivation and seed solution pretreatment

[0027] Dinoflagellates ( Crypthecodinium. cohnii ATCC30556) was inoculated in the seed medium, the inoculum volume percentage was 10%, the culture temperature was 26°C, the shaker speed was 200r / min, and cultured for 36h to the logarithmic phase.

[0028]Take 2.5 mL of the above-mentioned cells cultured to the logarithmic phase, centrifuge at 4000 r / min for 10 min, wash 3 times with 0.2 mol / L phosphate buffer, and dilute 50 times, break up with sterile glass beads, 6 layers without Single-cell suspension was obtained by filtering with bacterial gauze.

[0029] 2. Preparation of protoplasts and ultraviolet mutagenesis treatment

[0030] The protoplastization reaction solution was 5 mL, including 0.2 mL of single cell suspension, 0.2 mL of helicase enzyme solution and 4.6...

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Abstract

The invention discloses a mutagenic screening method of a DHA-producing strain crypthecodinium. cohnii. According to the invention, a protoplast of a DHA-producing strain crypthecodinium. cohnii ATCC30556 is prepared; single or composite mutagenesis of ultraviolet + LiCl and diethyl sulfate is carried out upon the protoplast; a basic thought of metabolism regulation and fermentation cultivation is adopted, and oriented screening is carried out by using malonic acid and/or a high-glucose resistance plate; and preliminary screening is carried out by using a solid cultivation medium of red tetrazoline (TTC), such that screening efficiency can be effectively improved, screening links can be reduced, and work load can be reduced. The strain obtained from the preliminary screening step is processed through shake-flask secondary screening and hereditary stability verification. As a result, the growth activity of the strain after mutagenesis is improved. Compared to an initial strain, the DHA biosynthesis amount of an YVD-4 mutagenic strain is improved by 4.7 times, and reaches 45.3g/L. Therefore, the method has an excellent prospect of industrialized production.

Description

technical field [0001] The invention relates to a method for mutagenesis screening of DHA-producing bacteria dinoflagellates. Background technique [0002] DHA (docosahexaenoic acid) is a polyene fatty acid with a special structure (6 cis-configured double bonds in a non-conjugated system), which belongs to ω according to the position of the last double bond at its methyl end -3 series of typical long-chain highly unsaturated fatty acids. DHA in the human body is mainly distributed in the gray matter of the brain, the outer segment of retinal cells, the nervous system, breast milk, myocardium, eosinophilic leukocytes, sperm, etc., and its physiological functions include: inhibiting platelet aggregation; reducing neutral lipids in the blood; reducing extremely low High-density lipoprotein and low-density lipoprotein cholesterol and increase high-density lipoprotein cholesterol; reduce blood viscosity; nourish the brain and prevent Alzheimer's disease; improve vision and ...

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Application Information

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IPC IPC(8): C12N1/12C12N15/01C12R1/89
Inventor 陈金卿陈俊煌吴美琼林超
Owner XIAMEN KINGDOMWAY GROUP
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