A bifunctional enzyme with wax ester synthesis activity and triglyceride synthesis activity, its encoding gene and application

A bifunctional enzyme and triglyceride technology, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of application limitation, huge demand in industry, etc., and high cost

Active Publication Date: 2019-10-15
OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Extracting wax esters from whales is also a traditional way to obtain liquid wax esters. However, natural resources are very limited and the demand from industries is huge, which limits its application. Liquid wax esters are also called liquid gold.
Currently, wax esters are expensive to synthesize using chemical methods

Method used

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  • A bifunctional enzyme with wax ester synthesis activity and triglyceride synthesis activity, its encoding gene and application
  • A bifunctional enzyme with wax ester synthesis activity and triglyceride synthesis activity, its encoding gene and application
  • A bifunctional enzyme with wax ester synthesis activity and triglyceride synthesis activity, its encoding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Cloning of TrWSD4 gene and TrWSD5 gene

[0074] 1.1 Cloning of TrWSD4 gene

[0075] 1.1.1 The RNA of Thraustochytrium roseum (ATCC28210) was extracted, and the cDNA template was obtained by RT-PCR.

[0076] 1.1.2 Use TrWSD4-for and TrWSD4-rev as primers (see Table 1 for the sequence), and use the obtained cDNA as a template to perform PCR with high-fidelity enzyme (Phusion High-Fidelity DNA Polymerase; Thermo), 50 μl PCR reaction system: 5 ×HF buffer 10μl, dNTP (10mM) 1μl, TrWSD4pET-23b-for (10mM) 1μl, TrWSD4pET-23b-rev (10mM) 1μl, Phusion DNA Polymerase 1μl, cDNA 2μl, ddH 2 O make up to 50 μl.

[0077] The reaction program was set according to Phusion High-Fidelity DNA Polymerase, as follows: 98°C for 30s, 98°C for 10s, 55°C for 30s, 72°C for 30s, 30 cycles, 72°C for 10min.

[0078] Table 1

[0079]

[0080]

[0081] 1.1.3 Take 3 μl of the PCR amplification product, and carry out agarose gel electrophoresis to detect whether the band is correct for the target f...

Embodiment 2

[0097] Expression and purification of TrWSD4 and TrWSD5 enzymes

[0098] 2.1 Expression and purification of TrWSD4 enzyme

[0099] 2.1.1 The recombinant expression vector pET-23(b+)::TrWSD4 was transformed into Escherichia coli Rosetta DE3, and the transformation method was carried out according to the instructions of Transetta DE3 competent cells. Escherichia coli (TrWSD4 positive expression strain) containing pET-23(b+)::TrWSD4 vector was obtained, inoculated in LB liquid medium (containing Amp), cultivated overnight at 37°C, and transferred into Expand culture in 100ml of LB+Amp until the bacteria concentration grows to OD 600 When the value is 0.6, add 1mMIPTG to induce TrWSD4 enzyme expression (the C-terminus expression of TrWSD4 protein has the His-Tag tag, and the effect of the His-Tag tag is on the Ni in the Ni column 2+ combination facilitates subsequent purification). Then placed at 28° C., 120 rpm to induce culture for 6 h.

[0100] 2.1.2 Under the conditions of...

Embodiment 3

[0108] 3.1 Determination of Kinetic Parameters and Substrate Preference Analysis of Nine Fatty Acyl-CoA Enzymes with Different Carbon Chain Lengths by TrWSD4 Enzyme

[0109]Measuring principle of enzyme activity: Ws / dgat belongs to transferase, which conducts nucleophilic attack on the hydroxyl group on the ester bond in order to transfer fatty acyl. Released free CoA reacts with DTNB (5,5'-dithiobis(2-nitrobenzoic acid)) to generate NTB 2- , the ion is yellow and has an absorbance value at 412nm. According to the change of its OD value with the reaction time, the corresponding enzymatic data are measured.

[0110] The enzyme activity assay reaction system of TrWSD4 enzyme (or TrWSD5 enzyme) includes: phosphate buffer, DTNB, palmitic alcohol (C16alcohol), fatty acyl-CoA (C8:0-CoA, C10:0-CoA, C12:0-CoA, One of C14:0-CoA, C16:0-CoA, C18:0-CoA, C18:3-CoA, C20:5-CoA and C20:4-CoA), enzyme solution (containing TrWSD4 enzyme or TrWSD5 enzyme ).

[0111] Add 15 μl 1mg / ml DTNB (dis...

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Abstract

The invention discloses a bifunctional enzyme having a wax ester synthesis activity and a triglyceride synthesis activity, and a coding gene and an application thereof, and relates to the biotechnical field. The amino acid sequence of the bifunctional enzyme having a wax ester synthesis activity and a triglyceride synthesis activity is represented by SEQ ID NO: 1 or SEQ ID NO: 3. The bifunctional enzyme (TrWSD4) represented by the SEQ ID NO: 1 and the bifunctional enzyme (TrWSD5) represented by the SEQ ID NO: 3 are a novel enzyme combining the activities of wax ester synthetase and triglyceride synthetase, the wax ester synthetase and the triglyceride synthetase can synthesize wax ester and / or triglyceride, and the bifunctional enzyme provides a new idea for the basic research of wax ester synthases, and can lay a foundation and provide a new way and idea for the production of the wax esters and / or triglycerides.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bifunctional enzyme with wax ester synthesis activity and triglyceride synthesis activity, its coding gene and application. Background technique [0002] Triacylglyceride (TAG) and wax ester (Wax Ester, WE) are a kind of neutral oil, which are formed by the esterification of fatty alcohol, diacylglyceride and long-chain fatty acid respectively. The carbon chain length of the components of the wax ester is generally medium or long chain, such as C16, C18, C20 or longer carbon chains. [0003] Wax esters come from a wide range of sources in nature and there are many kinds. And it is widely used in various fields with its good performance, for example, it has very important application value in food nutrition, industry, cosmetic industry and pharmaceutical industry. [0004] Wax esters widely exist in plants, animals, microorganisms, etc., and have important functions in organisms a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N15/81C12N1/21C12N1/19C12P7/64C12R1/19C12R1/01C12R1/865
Inventor 龚阳敏万霞张南南黄凤洪陈文超
Owner OIL CROPS RES INST CHINESE ACAD OF AGRI SCI
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