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A novel glycosyltransferase derived from dolwoe and use thereof

A technology of glycosyltransferase and glucose, applied in the direction of glycosyltransferase, transferase, enzyme, etc.

Inactive Publication Date: 2016-03-30
KOREA ADVANCED INST OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] On the other hand, only a part of the ginsenoside biosynthesis pathway is known

Method used

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  • A novel glycosyltransferase derived from dolwoe and use thereof
  • A novel glycosyltransferase derived from dolwoe and use thereof
  • A novel glycosyltransferase derived from dolwoe and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1: Cloning and purification of UDP-glycosyltransferase GpUGT23 derived from Gynostemma pentaphyllum

[0058] Gene amplification from Gynostemmapentaphyllum cDNA by PCR using GpUGT23-F (5'-GATCGGATCCATGAAGAAAATTTTGATGTTTCC-3'; Sequence 3), GpUGT23-R (5'-GATCCTCGAGTATTTTTGCTTGACAAAGC-3'; Sequence 4) primers and polymerase , each end of the gene was cut with BamHI and XhoI as restriction enzymes. Then, it was cloned into pET50 (Ohetal. PlantCell, 16, 3045-3058.2004) vector to prepare an expression vector, and the expression vector was transformed into Escherichia coli BL21(DE3)-RIL strain to prepare a GpUGT23 expression strain. The protein was obtained after the expression of the protein was induced by using IPTG, and the GpUGT23 enzyme was purified by Ni-NTAHis-binding resin.

Embodiment 2

[0059] Embodiment 2: Invitro enzyme experiment

[0060] Glycosyltransferase experiments were performed in a reaction buffer solution (10 mM PBS buffer, pH 7) containing purified GpUGT23 (30 μg), ginsenoside compound (5 mM) and UDP-glucose (50 mM). For this purpose, 4 different kinds of ginsenosides, ie, CK (compound K), F2, Rd and F1 were used to react with the enzyme of the present invention.

[0061] After the reaction mixture was incubated at 37° C. for 12 hours, the product was analyzed by TLC (thin-layer chromatography) or HPLC (high performance liquid chromatography).

[0062] Use 60F with mobile phase (acetone:methanol:DDW=65:35:10vol / vol) 254 TLC analysis was performed on silica gel plates (Merck, Germany). Using 10% (vol / vol) sulfuric acid (H 2 SO 4 ) to detect by spraying the dissolved product (resolved product) on the TLC plate, and then heating at 110° C. for 5 minutes ( figure 2 ).

[0063] HPLC analysis was performed using an ODS(2) C18 column (Phenomenex, ...

experiment example 1

[0065] Experimental Example 1: GpUGT23 is linked to the 20th carbon of PPD and PPT ginsenosides through a glycosidic bond Confirmation of specific glycosyltransferase activity on glucose

[0066] By the method described in Example 2, it was confirmed whether or not GpUGT23 has substrate specificity and regioselectivity.

[0067] First, using 9 kinds of ginsenosides (PPD, Rh 2 , Rg 3 , CK, F2, Rd, PPT, F1 and Rh 1 ), in the presence of UDP-glucose, when GpUGT23 as the recombinant GpUGT of Example 1 was reacted, it was confirmed by TLC (thin-layer chromatography, thin-layer chromatography) that in CK, F2, Rd and F1 reaction.

[0068] In order to confirm this again, four different ginsenosides (CK, F2, Rd, and F1) were co-cultured with GpUGT23 to react, and the product transformed by the recombinant GpUGT23 was analyzed by TLC analysis. show the result in figure 2 . CK, Gyp75 (Gypenoside LXXV), F2, Gyp17 (Gypenoside XVII), Rd, Rb used as standard samples for migrating s...

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Abstract

Provided are a novel UDP-glycosyltransferase (uridine diphosphate glycosyltransferase) protein from Dolwoe (Gynostemma pentaphyllum) having glycosyltransfer activity for glucose linked by a glycosidic bond at the C-20 position of PPD (protopanaxadiol)-type or PPT (protopanaxatriol)-type ginsenoside, and use thereof.

Description

technical field [0001] The present invention relates to a novel UDP-glucose transfer activity to glucose linked to the 20th carbon of ginsenosides of PPD (protopanaxadiol) or PPT (protopanaxatriol) via a glycosidic bond. Glycosyltransferase (uridine diphosphate glucosyltransferase) proteins and uses thereof. Background technique [0002] Ginsenosides are glycosylated dammarene-type tetracyclic triterpenes (tetracyclic triterpenes), and can be classified into protopanaxadiols (protopanaxadiol-type , PPD class) ginsenosides, protopanaxatriols (protopanaxatriol-type, PPT class) ginsenosides, and oleanolic acids (oleanolic acid class) ginsenosides. These three categories are based on the position and quantity of the sugar moieties (sugarmoieties (aglycones)) attached to the 3rd carbon, 6th carbon, and 20th carbon of the ring in the compound structure through glycosidic bonds. And classification. In addition, PPD and PPT have different hydroxylation patterns. PPD has -OH grou...

Claims

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Application Information

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IPC IPC(8): C12P33/20C12N9/10C12N15/54C12N15/63
CPCC12P19/56C12N9/1051C12P33/00C12Y204/01C12P33/20
Inventor 金善昌崔吉柱郑硕采金佑炫林修焕林完泽
Owner KOREA ADVANCED INST OF SCI & TECH
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