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A new catalytic system for preparing rare ginsenosides and its application

A ginsenoside, a rare technology, applied in the fields of biotechnology and plant biology, can solve the problems of severe reaction conditions, limitation, and low specificity of chemical hydrolysis method.

Active Publication Date: 2019-11-05
SYNBIOTECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to severe reaction conditions, difficult control, poor specificity, and many by-products, chemical hydrolysis has been rarely used in recent years.
Both enzymatic hydrolysis and microbial fermentation hydrolysis are based on the action of glycosidase to hydrolyze ginsenoside sugar groups. Although compared with chemical methods, its specificity is improved, but the specificity for the hydrolysis of ginsenoside C3, C6, and C20 sugar chains is not very high. The degree of hydrolysis also needs to be controlled, and the hydrolyzed product may be a mixture of various saponins
[0005] In addition, the main problem faced by using GT to catalyze the glycosylation of small molecule compounds in vitro is that the sugar donor that GT can use is nucleoside diphosphate (UDP)-sugar, and such compounds are usually not commercialized. Some commercial compounds such as UDP-glucose are very expensive (about 4000RMB for 1g)
This greatly limits the application of using GT to synthesize glycosides

Method used

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  • A new catalytic system for preparing rare ginsenosides and its application
  • A new catalytic system for preparing rare ginsenosides and its application
  • A new catalytic system for preparing rare ginsenosides and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] Example 1. Cloning of glycosyltransferases UGTPg1, UGTPg5, UGTPg29, UGTPg50, BC10 and sucrose synthase SUS1

[0150] (1) Cloning of glycosyltransferases UGTPg1, UGTPg5, UGTPg29, UGTPg50 and BC10

[0151]Two primers respectively having the nucleotide sequences of SEQ ID NO:7 and SEQ ID NO:8 in the sequence listing were synthesized. Using the cDNA obtained by reverse transcription of RNA extracted from ginseng as a template, PCR was performed using primers SEQ ID NO:7 and SEQ ID NO:8. The DNA polymerase was selected from the high-fidelity KOD DNA polymerase of Treasure Bioengineering Co., Ltd. The PCR amplification program is: 94°C for 2min; 94°C for 15s, 58°C for 30s, 68°C for 2min, a total of 35 cycles; 68°C for 10min, then drop to 10°C. The PCR products were detected by agarose gel electrophoresis, and the results were as follows: figure 1 a. Under UV irradiation, the target DNA band is excised. Then Axygen GelExtraction Kit (AXYGEN Company) was used to recover DN...

Embodiment 2

[0166] Example 2. Glycosyltransferase and sucrose synthase SUS1 expression

[0167] (1) Expression of UGTPg1, UGTPg5, UGTPg29, UGTPg50 and BC10 in Escherichia coli

[0168] Two primers respectively having the nucleotide sequences of SEQ ID NO:19 and SEQ ID NO:20 in the sequence listing were synthesized. Two restriction sites, BamH I and Xho I, were respectively set at both ends of the synthesized primers SEQ ID NO:19 and SEQ ID NO:20, and PCR was performed using pMDT-UGTPg1 as a template. The PCR amplification procedure is the same as in Example 2. The PCR product was separated and recovered by agarose gel electrophoresis, and then digested with BamH I and Xho I, and then ligated into the pET28a vector (Novagen) that was also digested with BamH I and Xho I using T4 DNA ligase from NEB Company. The obtained recombinant plasmid was named pET28a-UGTPg1. The recombinant plasmid pET28a-UGTPg1 was transformed into Escherichia coli BL21(DE3) (Novagen Company) to construct the reco...

Embodiment 3

[0178] Example 3. Synthesis of rare ginsenosides from ginsenogenin catalyzed by sucrose synthase and glycosyltransferase

[0179] The reaction of glycosyltransferase catalyzing the synthesis of ginsenoside from ginsenogenin needs to add UDP-glucose as the glycosyl donor, and the current commercial UDP-glucose price is about 4000 RMB per gram (Sigma Company). In the reaction system, UDP-glucose needs to be added in excess (about 10 mM UDP-glucose needs to be added to the reaction system for converting 1 mM ginsenogenin) to ensure that the reaction proceeds. Moreover, UDP-glucose is in a state of continuous consumption during the reaction process and cannot be recycled. These comprehensive factors make it extremely expensive to directly use UDP-glucose as a glycosyl donor for glycosylation to prepare glycosides. In the present invention, by using plant-derived sucrose synthase, by adding UDP and sucrose to the reaction system, sucrose synthase catalyzes UDP and sucrose to synthe...

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Abstract

The invention discloses a co-catalytic reaction system for preparing rare ginsenosides. The co-catalytic reaction system comprises glycosyltransferase GT (a); sucrose synthase SUS (b); uridine diphosphate UDP (c); and saccharose (d). Experiments show that in the presence of the saccharose and little UDP, an enzyme combination composed of the glycosyltransferase and the saccharose is capable of replacing an in-vitro reaction system to synthesize UDP sugar, one of expensive materials for the rare ginsenosides, and efficiently and economically converting substrates such as protopanoxadiol or protopanaxatriol into the rare ginsenosides, wherein the UDP is about 1 / 4 of the UDP sugar in price and is 1% of its original dosage; thus, preparation cost of the rare ginsenosides is greatly saved, and large-scale commercial preparation of the ginsenosides is better facilitated.

Description

technical field [0001] The invention relates to the fields of biotechnology and plant biology; more specifically, the invention relates to a co-catalyzed reaction system of glycosyltransferase and sucrose synthase and its application. Background technique [0002] Glycosylation modification is an important step in the synthesis of many natural products. The glycosyl composition affects the physical and chemical properties, stability and selectivity of natural products, and thus plays an important role in their physiological activities. In addition, glycosylation modification also greatly increases the diversity of natural products. Many natural products have the same aglycon structure, but a variety of glycoside compounds with different biological activities can be derived through different glycosylation modifications. The research on glycosylation modification of natural products has important prospects in the fields of synthetic biology and new drug development. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/20C12N9/10C12N15/54C12N15/70C12N15/81C12N1/21C12N1/19C12R1/19C12R1/865
Inventor 周志华王平平严兴魏维魏勇军范云杨成帅
Owner SYNBIOTECH (SUZHOU) CO LTD
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