Application of poplar glycosyl transferase PtGT2 to catalytic synthesis of phenylpropanoid glucosides

A technology of glycosyltransferase and phenylpropanoid glycoside, applied in directions such as transferase, fermentation, etc., can solve problems such as reports that have not yet been found to be applied, and achieve the effects of low efficiency and huge economic benefits

Inactive Publication Date: 2012-08-01
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it has been reported that there are more than 800 different glycosyltransferases in poplar, most of which can catalyze the formation of glycosides of small molecular compounds (Geisler-Lee

Method used

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  • Application of poplar glycosyl transferase PtGT2 to catalytic synthesis of phenylpropanoid glucosides
  • Application of poplar glycosyl transferase PtGT2 to catalytic synthesis of phenylpropanoid glucosides
  • Application of poplar glycosyl transferase PtGT2 to catalytic synthesis of phenylpropanoid glucosides

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 Cloning, prokaryotic expression and enzyme protein purification of poplar glycosyltransferase gene PtGT2

[0026] 1. Cloning of poplar glycosyltransferase gene PtGT2

[0027] The glycosyltransferase gene PtGT2 involved in the present invention is cloned from poplar through RT-PCR amplification technology. Firstly, RNA was extracted from young leaves of Populus tomentosa by TRIzol method, and then the coding region cDNA of the gene was amplified by RT-PCR method. A pair of primers used for amplification are: GT2-a: 5'-ATAGGATCCATGCAAAAC ACAAAACCTCA-3'; GT2-b: 5'-ATAGAGCTC CTATGCACCTTGAGCCTTTGC-3'. The RT-PCR amplification program is: 94°C (pre-denaturation), 5min; 94°C (denaturation), 10s; 55°C (annealing), 15s; 72°C (extension), 2min; 35cycle; 72°C (final extension), 10min. The amplified product is recovered and purified. The amplified target gene PtGT2 was connected to the EcoRV single-digested intermediate vector pBluescript SK to obtain the vector pBGT2...

Embodiment 2

[0036] Example 2 Poplar glycosyltransferase PtGT2 catalyzes the synthesis of phenylpropanoid glycosides

[0037] 1. Enzyme-catalyzed reaction of phenylpropanoid

[0038] The fusion protein of the purified poplar glycosyltransferase PtGT2 (GST-PtGT2, which can reflect the catalytic activity of PtGT2, which is a common practice) is used for in vitro enzyme-catalyzed reaction, and six kinds of phenylpropanoid compounds are selected as substrates, including Coniferyl alcohol, sinapyl alcohol, coniferyl aldehyde, sinapinaldehyde, sinapinic acid, ferulic acid. UDP-glucose was used as the sugar donor in the catalytic reaction. The enzymatic reaction system is as follows:

[0039]

[0040] Put the above mixed reaction system in a constant temperature water bath at 30°C and react for 3 hours. The reaction was then terminated by adding 20 μl of trichloroacetic acid at a concentration of 240 mg / ml. The reaction tubes were snap-frozen with liquid nitrogen and stored at -20°C until ...

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Abstract

The invention discloses application of poplar glycosyl transferase PtGT2 to catalytic synthesis of phenylpropanoid glucosides. The amino acid sequence of the poplar glycosyl transferase PtGT2 is shown in SEQ ID No.2; the phenylpropanoid is sinapinic alcohol, sinapinic aldehyde, coniferyl alcohol, coniferyl aldehyde, sinapinic acid or ferulic acid; and any one of the poplar glycosyl transferase PtGT2 and the phenylpropanoid is put in a reaction tube for enzymatic reaction, and the corresponding phenylpropanoid glucosides can be catalytically synthesized. According to the application disclosed by the invention, a feasible method is provided for synthesizing the phenylpropanoid glucosides efficiently and exclusively by utilizing an enzyme catalysis method; after the application is implemented, large economic benefit is brought for the synthetic industry of the phenylpropanoid glucosides.

Description

technical field [0001] The invention relates to the application of a glycosyltransferase, in particular to the application of a poplar glycosyltransferase PtGT2 in catalyzing the synthesis of phenylpropanoid glycosides; it belongs to the field of biotechnology. Background technique [0002] Among the natural ingredients of plants, there is a class of compounds in which a benzene ring and three straight-chain carbons are linked together, collectively known as phenylpropanoids (Phenylpropanoids). For example sinapyl alcohol, sinapaldehyde, coniferyl alcohol, coniferaldehyde, sinapic acid, ferulic acid, caffeic acid and so on. Many glycosides of phenylpropanoid are active ingredients of Chinese herbal medicine, and have important medicinal value in anti-oxidation, detoxification of liver, anti-asthma and anti-tumor. In the pharmaceutical industry or chemical industry, there are two main ways to obtain phenylpropanoid glycosides: extraction from plants and chemical synthesis. ...

Claims

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Application Information

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IPC IPC(8): C12P19/44C12N9/10
Inventor 侯丙凯韩萍
Owner SHANDONG UNIV
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