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Ocean micro-alga delta5 aliphatic acid desaturase and application thereof

A technology of desaturase and marine microalgae, which is applied in the field of Δ5 fatty acid desaturase, can solve the problems of substrate specificity, low enzyme activity, and unretrieved problems, and achieve high enzyme activity and substrate specificity sex high effect

Inactive Publication Date: 2008-07-02
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the substrate specificity and enzyme activity of the Δ5 desaturase gene obtained from this algae were not high (Domergue et al.2002)
In addition, no relevant patents related to marine microalgae Δ5 desaturase gene were found in domestic and foreign patent databases

Method used

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  • Ocean micro-alga delta5 aliphatic acid desaturase and application thereof
  • Ocean micro-alga delta5 aliphatic acid desaturase and application thereof
  • Ocean micro-alga delta5 aliphatic acid desaturase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Obtaining of Δ5 desaturase gene

[0021] Specific steps include:

[0022] (1) Cultivation of Phaeodactylum tricornutum

[0023] 1) The seawater used for cultivation is the natural seawater off the coast of Qingdao. The seawater is filtered through absorbent cotton and boiled at 100°C for 3 minutes. The 250ml Erlenmeyer flask and pipette were autoclaved (121°C, 15min) before use.

[0024] 2) Prepare f / 2 mother liquor (see accompanying drawing 3 of the description) and sterilize it.

[0025] 3) Add f / 2 mother liquor to the sterilized seawater at a ratio of 1:1000 (v / v), insert algae species at a ratio of 1:10 (v / v), and place them in a light incubator for cultivation without aeration. Shake the flask several times. See Table 1 for specific culture conditions.

[0026] Table 1 The cultivation conditions of Phaeodactylum tricornutum

[0027]

[0028] f / 2 mother liquor preparation

[0029] 1. Basic elements:

[0030] NaNO 3 78.4g+1L DDW (do...

Embodiment 2

[0114] Example 2 Construction of expression vector pYD5-2 and transformation of Saccharomyces cerevisiae

[0115] 1 Brief introduction of Saccharomyces cerevisiae expression vector pYES2 and yeast strain INVSc1

[0116] The Saccharomyces cerevisiae expression vector pYES2 (Invitrogen) used in the experiment was 5856bp long ( figure 1 ), with the GAL1 promoter, which is inducible, and the transcription is inhibited in the presence of glucose in the medium, while the addition of galactose to the glucose can induce transcription. In addition, cells can also be grown in medium with raffinose as a carbon source. Raffinose neither induces nor inhibits transcription. Adding galactose to the medium in its presence can also induce transcription. And the induction speed is faster than cells initially cultured with glucose as carbon source.

[0117] The genotype of yeast INVSc1 is MATa his3Δ1 leu2 trp1-289ura3-52 / MATαhis3Δ1 leu2 trp1-289 ura3-52, and the phenotype is His - , Leu - ,T...

Embodiment 3

[0151] Example 3 Induced expression and fatty acid analysis of yeast strains

[0152] 1 induced expression

[0153] 1) Streak the positive clone containing the recombinant plasmid and the empty plasmid control bacteria on the SC-U (2% glucose) plate again, and culture at 30°C until colonies grow;

[0154] 2) Insert a single clone into 3 mL of SC-U (2% raffinose) liquid medium, shake overnight at 30°C;

[0155] 3) Take an appropriate amount of bacterial solution and centrifuge, add 3.6ml SC-U (2% galactose) to the initial OD 600 About 0.2, add 400 μL of 10% NP-40 (final concentration 1%), and add fatty acid substrate (20:4n3) to the final concentration of 50 μM at the same time, and induce at 20°C for 72 hours.

[0156] 2 Fatty acid extraction and analysis

[0157] 2.1 Fatty acid extraction

[0158] 1) Collect the induced bacterial solution by centrifugation at 5000rpm for 3min;

[0159]2) Grinding with liquid nitrogen, transfer the ground dry powder into an EP tube, add 3...

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Abstract

The invention provides a Delta 5 fatty acid desaturase from ocean microalgae, and coding gene and an application thereof. The inventive Delta 5 fatty acid desaturase has amino acid sequence shown in SEQ ID NO.2 or amino acid subjected to substitution, deletion or addition of one or more amino acid residues of the amino acid sequence and having the same functions. The inventive Delta 5 fatty acid desaturase can effectively catalyze the conversion of ETA(20:4 Delta 8,11,14,17) to EPA(20:5 Delta 5,8,11,14,17), and has the advantages of high specificity of substrate and high enzyme activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a Δ5 fatty acid desaturase of marine microalgae Phaeodactylum tricornutum, its encoding gene and application. Background technique [0002] Fatty acids are monocarboxylic acids with carbon and hydrogen chains that play important roles in many biological processes. Fatty acids rarely exist in free form, but are lipidated into various major lipid components, such as phospholipids and triacylglycerols. Fatty acids are mainly divided into two categories: saturated fatty acids and unsaturated fatty acids, which are further divided into monounsaturated fatty acids and polyunsaturated fatty acids, and their carbon and hydrogen chains contain one or more cis double bonds (C=C). [0003] The formation of unsaturated bonds requires fatty acid desaturase (fatty acid desaturase) catalysis. Fatty acid desaturase exists in almost all plants, animals and microorganisms, and can conver...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/63C12N15/82C12N1/12C12P7/64
Inventor 潘克厚石娟朱葆华于文功宫倩红杨官品
Owner OCEAN UNIV OF CHINA
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