A kind of unnatural ginsenoside rd12 and its preparation method and application

A ginsenoside, non-natural technology, applied in the fields of biotechnology and botany, can solve problems such as high price

Active Publication Date: 2021-02-26
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The in vitro glycosylation reaction has the characteristics of high catalytic efficiency, good substrate tolerance, and simple product purification. However, the in vitro glycosylation reaction requires the use of expensive uridine diphosphate glucose (UDP-glucose, UDPG) as the glycosyl donor

Method used

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  • A kind of unnatural ginsenoside rd12 and its preparation method and application
  • A kind of unnatural ginsenoside rd12 and its preparation method and application
  • A kind of unnatural ginsenoside rd12 and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Cloning, expression and purification of embodiment 1 glycosyltransferase and sucrose synthase

[0061] 本发明专利中的糖基转移酶Bs-YjiC筛选自枯草芽孢杆菌(Bacillus subtilis168),其氨基酸序列如SEQ ID NO.1所示,其核酸序列(SEQ ID NO.7)如下:ATGAAAAAGTACCATATTTCGATGATCAATATCCCGGCGTACGGACATGTCAATCCTACGCTTGCTTTAGTAGAGAAGCTTTGTGAGAAAGGGCACCGTGTCACGTACGCGACGACTGAGGAGTTTGCGCCCGCTGTTCAGCAAGCCGGTGGAGAAGCATTGATCTATCATACATCCTTGAATATTGATCCTAAGCAAATCAGGGAGATGATGGAAAAGAATGACGCGCCCCTCAGCCTTTTGAAAGAATCACTCAGCATTCTGCCGCAGCTTGAGGAGTTATATAAGGATGATCAGCCTGATCTGATCATCTATGACTTTGTTGCGCTGGCTGGTAAATTGTTTGCTGAAAAGCTTAATGTTCCGGTCATTAAGCTCTGTTCGTCATATGCCCAAAATGAATCCTTTCAGTTAGGAAATGAAGACATGCTGAAAAAAATAAGAGAAGCAGAGGCTGAATTTAAAGCCTACTTGGAGCAAGAGAAGTTGCCGGCTGTTTCATTTGAACAGTTAGCTGTGCCGGAAGCATTAAATATTGTCTTTATGCCGAAGTCTTTTCAGATTCAGCATGAGACGTTCGATGACCGTTTCTGTTTTGTCGGCCCCTCTCTCGGAGAACGGAAGGAAAAAGAAAGCCTGTTGATTGACAAGGATGATCGCCCGCTTATGCTGATTTCTTTGGGTACGGCGTTTAACGCATGGCCGGAATTTTACAAGATGTGCATCAAGGCATTTCGGGATTCTTCATGGCAAGTGATCATGTCGGTTGGGAAAACGATTGATCCAGAAAG...

Embodiment 2

[0073] Example 2 Glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy are coupled to catalyze the synthesis of unnatural ginsenoside Rd12 from ginsenoside Rg3

[0074] Using ginsenoside Rg3 (3-O-[β-D-glucopyranosyl(1-2)-β-D-glucopyranosyl]-20(S)-protopanaxadiol) as substrate and cheap sucrose as glycosyl donor, by Glycosyltransferase-sucrose synthase double-enzyme coupling reaction can catalyze the C12-OH glycosylation of ginsenoside Rg3 to generate unnatural ginsenoside Rd12 (3-O-[β-D-glucopyranosyl(1-2)-β -D-glucopyranosyl]-12-β-D-glucopyranosyl-protopanaxadiol), the specific reaction is as figure 2 As shown, the details are as follows:

[0075]

[0076] The enzyme reaction system includes: 2mM Rg3, 0.5mM UDP, 300mM sucrose, 160U / mL glycosyltransferase Bs-YjiC and 200U / mL sucrose synthase AtSuSy, react at 35°C for 0.5h.

[0077] The enzyme reaction product was identified by high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS...

Embodiment 3

[0079] Example 3 Fed-batch Synthesis of Unnatural Ginsenoside Rd12

[0080] In order to avoid the inhibition of glycosyltransferase activity by adding too much ginsenoside Rg3 at one time, and in view of the coupling reaction of glycosyltransferase Bs-YjiC and sucrose synthase AtSuSy within 0.5h, 2mM ginsenoside Rg3 sugar Kylation produces unnatural ginsenoside Rd12. Therefore, by feeding batches, 2mM ginsenoside Rg3 was supplemented at time points 1h, 2h, 4h and 8h to synthesize higher concentrations of non-natural ginsenoside Rd12.

[0081] From Figure 4 It can be seen that after 4 batches of supplementing ginsenoside Rg3 substrates and reacting for 18 hours, 9.8mM ginsenoside Rd12 (9.3g / L) can be finally obtained, the conversion rate of ginsenoside Rg3 reaches 98%, and the synthesis of ginsenoside Rd12 The rate reaches 0.5g / L / h.

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Abstract

The invention relates to a new unnatural ginsenoside Rd12 (3-O-β-D-glucopyranosyl (1-2)-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-protopanaxadiol) and a preparation method thereof and application, the present invention uses cheap sucrose as glucose glycosyl donor, utilizes glycosyltransferase and sucrose synthase as coupling catalyst, uses ginsenoside Rg3 as glycosyl acceptor, and uses cheap sucrose as glycosyl donor, Efficiently catalyze the C12 hydroxyl glycosylation of ginsenoside Rg3 to produce unnatural ginsenoside Rd12, which has the characteristics of good specificity, high catalytic efficiency, and simple product purification. In addition, the unnatural ginsenoside Rd12 prepared by the invention can be used as an antitumor drug, and the unnatural ginsenoside Rd12 has significant inhibitory effects on colon cancer cells, liver cancer cells, lung cancer cells and gastric cancer cells.

Description

technical field [0001] The invention belongs to the technical fields of biotechnology and botany, and relates to a non-natural ginsenoside Rd12 and its preparation method and application, in particular to a method of catalyzing ginsenoside Rg3 to generate non-natural ginseng by the coupling reaction of glycosyltransferase and sucrose synthase Methods and uses of saponin Rd12. Background technique [0002] Ginseng (Panax ginseng C.A.Mayer) is an important medicinal plant in the genus Panax of Araliaceae. It is mainly distributed in the Changbai Mountains in the northeast of my country and East Asian countries such as Japan, South Korea, and North Korea. As a traditional Chinese medicine, it has been used for several years in China. For thousands of years, "Shen Nong's Materia Medica" records that ginseng has many functions such as "mainly nourishing the five internal organs, calming the nerves, calming the soul, relieving panic, eliminating evil spirits, and name." Therefore,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07J17/00A61K31/704A61P35/00C12P33/20C12N15/54
CPCC07J17/00C12N9/1048C12N9/1062C12P33/00C12P33/20C12Y204/00C12Y204/01013
Inventor 孙媛霞戴隆海李娇杨建刚门燕
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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