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Beta-elemene synthetase, encoding gene thereof, carrier, engineering bacterium and application of beta-elemene synthetase

A technology of genetically engineered bacteria and coding genes, applied in the field of β-elemene synthase, can solve the problems of low yield, high pollution of highly toxic substances, and long time consumption

Inactive Publication Date: 2013-11-27
李海峰
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These separation methods require a lot of materials, take a long time, have low purity, and low yield, and cannot fully meet the needs of the market.
Compared with the characteristics of elemene obtained by plant extraction method, which is a mixture of β-, γ-, and δ-elemenes with different components, chemical synthesis can be single-synthesized for specific substances. There are reports abroad using double asymmetric folding The stereoselective total synthesis of elemene has been achieved by the method of alkylation of enol with allyl-constrained lactone, but the synthesis steps are cumbersome, and the synthesis process uses highly toxic substances such as sodium cyanide, causing great pollution. limitations of this method for the commercial production of β-elemene

Method used

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  • Beta-elemene synthetase, encoding gene thereof, carrier, engineering bacterium and application of beta-elemene synthetase
  • Beta-elemene synthetase, encoding gene thereof, carrier, engineering bacterium and application of beta-elemene synthetase
  • Beta-elemene synthetase, encoding gene thereof, carrier, engineering bacterium and application of beta-elemene synthetase

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Construction of a soil genome library. Strains from the metagenomic library were randomly selected for sequencing. Determine the nucleotide sequence of the open reading frame (ORF) of the β-elemene synthase gene through literature review and sequence analysis, design primers to amplify the complete coding reading frame, and introduce restrictions on the upstream and downstream primers Endonuclease sites (depending on the carrier selected). The β-elemene synthase gene of the nucleotide sequence shown in SEQ ID NO.2 was obtained by PCR technique. PCR amplification program 95°C, pre-denaturation for 5min, 32 cycles (denaturation at 98°C for 10sec; annealing at 58°C for 30sec; extension at 68°C for 2min), hold at 16°C. The gene was connected with pET28a expression vector, and then transformed into E. Coil BL21 to obtain a recombinant strain.

Embodiment 2

[0078] Transfer the obtained recombinant strain to LB liquid medium containing 10 μg / ml kanamycin, induce overnight at 30°C with 0.4mM IPTG, and use the strain without inducer as a control; take 1.5ml culture solution at 12000rpm Centrifuge for 1 min, add 300 μl of 0.5 mM pH7.5 Tris-HCl buffer solution, and ultrasonically disrupt the cells. Take 100 μl of broken samples for whole cells, supernatant, and pellets and add 20 μl of 5× protein electrophoresis loading buffer. For control samples, take 100 μl of broken whole cells and add 20 μl of 5× Protein electrophoresis Loading buffer, boiled in a boiling water bath for 5 minutes; after the sample is cooled to room temperature, use 15% SDS-PAGE gel for protein electrophoresis to detect protein expression, the results are shown in figure 1 .

[0079] Depend on figure 1 It can be seen that β-elemene synthase is an intracellular enzyme, which mainly exists in the inclusion bodies of bacteria.

[0080] Preparation of LB liquid medi...

Embodiment 3

[0082] Transfer the recombinant strain to 100ml LB liquid medium containing 10μg / ml kanamycin, wait for OD 600 When it reaches 0.8, add 400 μl of 10mM IPTG and seal the bottle mouth completely with a parafilm, place it at 30°C for 16-20 hours, take the culture solution and use solid phase microextraction (SPME) to extract it for 30 minutes and then perform gas chromatography-mass spectrometry detection. The results are shown in figure 2 . While the blank E.Coil BL21 strain was used as a negative control, no β-elemene production was detected.

[0083] Depend on figure 2 It can be seen that the initial expression peak area of ​​β-elemene produced by the recombinant strain is 4,067,531. According to the standard product β-elemene 1 μl, the peak area detected after adsorption for 30 minutes under the same conditions of solid-phase microextraction is 278,992,338. After conversion, β-elemene - Elemene yield was 128 μg / L.

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Abstract

The invention provides a beta-elemene synthetase, an encoding gene thereof, a carrier with the encoding gene, an engineering bacterium with the encoding gene and an application of the beta-elemene synthetase. The beta-elemene synthetase has an amino acid sequence as shown in SEQ ID No.1, and the encoding gene of the beta-elemene synthetase has a sequence as shown in SEQ ID No.2. The invention provides the beta-elemene synthetase and the encoding gene thereof and provides the basis for producing the beta-elemene synthetase by using a biological enzyme method; the quality of a beta-elemene synthetase product can be improved, the production cost of the beta-elemene synthetase can be reduced, the production efficiency can be increased, and the technical precondition is provided for the industrial biosynthesis of the beta-elemene synthetase.

Description

(1) Technical field [0001] The invention relates to a β-elemene synthetase, a gene encoding the enzyme, a recombinant vector containing the encoding gene, a genetically engineered bacterium and applications thereof. (2) Background technology [0002] β-elemene (β-elemene) belongs to the oxygen-free sesquiolefins. It is an oily monomer separated from the volatile oil of zedoary. Its chemical name is 1-methyl-1-vinyl-2. 4-diisopropylcyclohexane, the structural formula is shown in Formula 1. β-elemene has been proven to have anti-tumor activity against adenocarcinoma, human neurokeratinoma, liver cancer, lung cancer, colon cancer, gastric cancer, ovarian cancer, etc. In addition, some studies have also shown that β-elemene also has analgesic and Radiation sensitization. Elemene milk with β-elemene as the main component began to be widely used in the clinical treatment of malignant serous cavity effusion, lung cancer, digestive tract tumors, brain tumors and other superficial ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/63C12N1/15C12N1/19C12N1/21C12P5/00
Inventor 李海峰黄黎锋蓝袁洋高允允
Owner 李海峰
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