Epimedium source galactosyl transferase and application of epimedium source galactosyl transferase to preparation of hyperoside

A technology of glycosyltransferase and galactoside, applied in the direction of glycosyltransferase, transferase, application, etc., can solve the problem of not synthesizing hyperoside

Active Publication Date: 2020-07-17
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the glycosyltransferase from Epimedium that catalyzes the O-galactosylation of the 3-position of flavonoids has not been reported, and there is no article on the use of related enzymes to enzymatically catalyze the synthesis of hyperin

Method used

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  • Epimedium source galactosyl transferase and application of epimedium source galactosyl transferase to preparation of hyperoside
  • Epimedium source galactosyl transferase and application of epimedium source galactosyl transferase to preparation of hyperoside
  • Epimedium source galactosyl transferase and application of epimedium source galactosyl transferase to preparation of hyperoside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Cloning of Epimedium Korean galactosyltransferase gene

[0048] Pick fresh Epimedium Korean plants, wash them twice with deionized water to remove the attached soil and microorganisms. Different tissues are separated according to roots, stems, leaves, and flowers, and are quickly frozen in liquid nitrogen and stored in liquid nitrogen tanks. According to the TRIzol method, total RNA was extracted from different tissues, and cDNA was synthesized using a rapid cDNA reverse transcription kit. Dilute the cDNA to an appropriate concentration (50-100ng / μL), use the diluted cDNA as a template, use the upstream primer GalT-F: ATGGGAACCAACCAACAA, the downstream primer GalT-R: TCAGCAGCTAGTGATTATC, and PCR amplify the target sequence to obtain the sequence as SEQ ID NO .4 Gene sequence shown. The obtained fragments were ligated with the T vector, transformed into Escherichia coli JM109, and positive clones were picked and sent for sequencing.

Embodiment 2

[0049] Embodiment 2 Construction of recombinant galactosyltransferase expression vector and recombinant Saccharomyces cerevisiae

[0050] Pick the positive clone with correct sequencing and use it as a template, use the upstream primer pY13-GalT-F: CCC CCGGGC TGC AGG AAT TCA TGG GAA CCA ACC AAC AA, the downstream pY13-GalT-R: TAC ATG ACT CGAGGT CGA CTC AGC AGC TAG TGA TTA TC, to amplify galactosyltransferase, carry out SpeI and Sal I double enzyme digestion on the vector pY13, purify the amplification product and the enzyme-cut vector respectively, and construct the vector pY13-GalT by using a one-step cloning kit , transform Escherichia coli JM109. Pick positive clones and extract plasmids after correct sequencing. According to the experimental guidelines of yeast genetics methods, the plasmid pY13-GalT was transformed into Saccharomyces cerevisiae C800, spread on SD-His for positive clone screening, and Saccharomyces cerevisiae Y-GalT was constructed. The enzyme activity o...

Embodiment 3

[0051] Example 3 Catalytic Synthesis of Hyperin by Recombinant Saccharomyces cerevisiae

[0052] The recombinant galactosidyl transferase expression Saccharomyces cerevisiae Y-GalT constructed in Example 2 was streaked on the SD-His medium, a single colony was picked and transferred into the YPD medium, and cultured at 30° C. and 220 rpm for 18 hours. Transfer to fresh 25mL YPD medium to control the initial OD 600 The value was 2.0, and the culture was carried out at 30° C. and 220 rpm. After 24h, culture OD 600 value is about 50.0, add quercetin to the culture, so that the final concentration of quercetin in the culture system is 100 mg / L, and continue to cultivate. After fermenting and cultivating 120h, collect culture, measure hyperin content by HPCL and be 125.6mg / L ( figure 2 ).

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Abstract

The invention discloses epimedium source galactosyl transferase and an application of the epimedium source galactosyl transferase to the preparation of hyperoside, and belongs to the technical field of gene engineering and biological medicines. The invention provides galactosyl transferase and an application of recombinant saccharomyces cerevisiae expressing the galactosyl transferase to the catalytic synthesis of flavonoid compounds. The constructed recombinant saccharomyces cerevisiae can be enabled to catalyze the transfer of UDP-galactose to various flavone substrates such as quercetin, kaempferol, myricetin, dihydroquercetin, dihydrokaempferol, dihydromyricetin, fisetin, morin and icaritin.

Description

technical field [0001] The invention relates to galactosyltransferase derived from epimedium and its application in preparing hyperin, belonging to the technical fields of genetic engineering and biomedicine. Background technique [0002] Hyperoside (quercetin-3-O-galactoside, Hyperoside) is a flavonol glycoside compound, which was first isolated from Cornus officinalis and is an important plant natural product. It widely exists in various plants such as Labiatae, Hypericaceae, Rosaceae, Platycodonaceae, Fabaceae, etc., but the relative content of a single plant is low. Hyperin has excellent biological functions and exerts a variety of pharmacological activities, including anti-inflammation, anti-depression, reducing cerebral ischemic damage, inhibiting tumors, and protecting myocardium. Hyperin is an important component of a variety of traditional Chinese medicine preparations, such as Tongmai Ciwujia Capsules, Xin'an Capsules, Xinxuening Dropping Pills, Yukexin Capsules a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/81C12N1/19C12P17/06C12P19/60C12R1/865
CPCC12N9/1051C12P17/06C12P19/60C12Y204/01037
Inventor 周景文陈坚吕云斌曾伟主堵国成
Owner JIANGNAN UNIV
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