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Aquilaria sinensis terpene synthase

A technology for sesquiterpenes and compounds, which is applied in the field of terpenoid synthases of serrata, can solve the problems of staying, not reaching industrialized production, and many impurities, and achieves a corresponding high yield, high-efficiency catalytic activity, and broad industrial practical prospects. Effect

Active Publication Date: 2021-08-10
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, sesquiterpenoids are mainly extracted from plants or chemically synthesized, but there are many impurities, and the biosynthesis process of sesquiterpenoids has not yet met the requirements of industrial production, and is still in the stage of basic research in the laboratory

Method used

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  • Aquilaria sinensis terpene synthase
  • Aquilaria sinensis terpene synthase
  • Aquilaria sinensis terpene synthase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1, recombinant expression of sesquiterpene synthase

[0053] Step 1. Synthesize the modified sesquiterpene synthase gene shown in SEQ ID No.1 or SEQ ID No.3 by whole gene synthesis, connect it to the pET-28a vector, and identify it by sequencing and double enzyme digestion Extract plasmids for correct clones for later use.

[0054] Step 2: The recombinant plasmid identified as positive was transformed into Escherichia coli Transetta (DE3) competent cells, and the PET-21a empty plasmid was also transformed into Escherichia coli Transetta (DE3) competent cells as a control.

[0055] Step 3: After the bacteria are picked and activated, transfer a large number of shaken bacteria so that the initial OD value is lower than 0.1, and measure the OD value with an ultraviolet spectrophotometer. Shake the bacteria at 37°C and 200rpm for 2h and 40min, add 0.5mM IPTG when the OD value rises to about 0.5, then induce at 16°C and 150rpm for 16h, then centrifuge at 12000rpm...

Embodiment 2

[0057] Embodiment 2, the purification of sesquiterpene synthase

[0058] According to the established induction conditions, the protein is expressed in large quantities.

[0059] Step 1: Centrifuge at 5000rpm for 20min to collect the cells for the next step of affinity purification.

[0060] Step 2: Resuspend the cells with 40 mL binding buffer (containing protease inhibitors), and sonicate. Centrifuge at 8000r / min at 4°C for 30min, filter the supernatant with a 0.45μm filter, and store the precipitate on ice for later use.

[0061] Step 3: Take 1mL Ni-NTA His Bind resin, equilibrate it with binding buffer, add it to the filtered supernatant, mix the filtered supernatant with 1mL Ni 2+ The resin homogenate was mixed and combined on ice for 4h.

[0062] Step 4, put the above mixture on the column and let the solution flow out.

[0063] Step 5. Wash the resin with washing buffer, 10 mL each time, repeat 3 times.

[0064] Step 6: Elute the target protein with elution buffer,...

Embodiment 3

[0068] Embodiment 3, the in vitro enzymatic reaction of sesquiterpene synthase

[0069] The reaction system contained 25mM Tris-HCl (pH7.0), 10% glycerol, 100mM Mg2SO4, 5mMDTT, 46uMFPP or GPP (purchased from sigma company), 50uL of purified protein, and ddH2O to make up to 200μL. After mixing the above solution, put it in a metal bath at 30°C for 1h.

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Abstract

The invention relates to an aquilaria sinensis terpene synthase, and provides a modified sesquiterpene synthase gene, which enables common host microorganisms to be efficiently expressed through a gene engineering technology: a synthase of a sesquiterpene compound with high catalytic synthesis efficiency of a target product and good solubility is provided; therefore, progress and development of a biological enzyme catalytic synthesis technology of the sesquiterpenoids are promoted.

Description

technical field [0001] The invention relates to a white wood terpene synthase, and also relates to its catalytic product and application, belonging to the field of biotechnology. Background technique [0002] Agarwood is a resinous wood induced by the injury of Agarwood or Pseudo-Agarwood in the family Daphneaceae. It is a rare and endangered southern medicine and a high-value global resource. Domestic agarwood is mainly produced in Guangdong, Hainan, Yunnan, Guangxi and other places in my country. Aquilaria sinensis (Lour.) Gilg is the only authentic plant source of agarwood in my country. [0003] According to the Chinese Pharmacopoeia (2010 edition), agarwood has the effects of promoting qi and relieving pain, warming the middle and relieving vomiting, inducing qi and relieving asthma, and has been one of the rare medicinal materials in my country since ancient times. However, the yield of agarwood is extremely low under natural conditions. For a long time, wild agarwood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12P5/02C12P5/00
CPCC12N9/1051C12P5/026C12P5/002C12Y204/01216
Inventor 徐艳红魏建和唐小琳余翠翠
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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