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Bacillus subtilis glycosyltransferase and application thereof

A technology of glycosyltransferase and glycosyl, applied in the field of glycosyltransferase, can solve the problems of many by-products, high cost and low yield of chemical synthesis methods

Active Publication Date: 2018-11-02
INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chemical synthesis method has the problems of many by-products, low yield, and high cost, and a large amount of pollutants will be generated during the synthesis process, causing harm to the environment.

Method used

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  • Bacillus subtilis glycosyltransferase and application thereof
  • Bacillus subtilis glycosyltransferase and application thereof
  • Bacillus subtilis glycosyltransferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1. Cloning of Glycosyltransferase Gene BsUGT1

[0096] Bacillus subtilis (Bacillus subtilis CTCC63501) genomic DNA was extracted using a bacterial genomic DNA extraction kit ( figure 1 A). Using Bacillus subtilis genomic DNA as a template, PCR amplification was performed using primers BsUGT1-F (SEQ ID NO: 3) and BsUGT1-R (SEQ ID NO: 4) to obtain the glycosyltransferase gene BsUGT1 (SEQ ID NO: 2) . Wherein, the sequences of the primers BsUGT1-F (SEQ ID NO: 3) and BsUGT1-R (SEQ ID NO: 4) used are listed in Table 1. PCR products were detected in 1.0% agarose gel electrophoresis ( figure 1 B).

[0097] Table 1 Primer Sequence

[0098]

[0099] PCR reaction system and reaction conditions:

[0100] PCR reaction system (50μL)

[0101]

[0102] PCR reaction conditions

[0103] 98℃, 2min

[0104] 98℃, 15s; 55-60℃, 30s; 72℃, 1kb / 15-30s; 30 cycles

[0105] 72℃, 10min

[0106] 4°C, ∞

[0107] After the PCR reaction, 4 μL of the PCR product was connected to...

Embodiment 2

[0109] Example 2. Prokaryotic expression of glycosyltransferase BsUGT1

[0110] According to the sequence information obtained from the sequencing of plasmid pEASY-Blunt-BsUGT1, primers BsUGT1-F1 (SEQ ID NO: 5) and BsUGT1-R1 (SEQ ID NO: 6) were designed to amplify the same sequence as the prokaryotic expression plasmid pET-32a(+). The PCR fragment BsUGT1 of the source arm was simultaneously introduced with restriction enzyme cutting sites BamH I and Sal I, and the primer sequences are shown in Table 2.

[0111] Table 2 Primer Sequence

[0112]

[0113] PCR reaction system and reaction conditions:

[0114] PCR reaction system (50μL)

[0115]

[0116] PCR reaction conditions

[0117] 98℃, 2min

[0118] 98℃, 15s; 55-60℃, 30s; 72℃, 1kb / 15-30s; 30 cycles

[0119] 72℃, 10min

[0120] 4°C, ∞

[0121] The expression vector pET-32a(+) was double digested with restriction endonucleases BamH I and Sal I. The PCR fragment and the double-enzyme-digested vector were gel-cut an...

Embodiment 3

[0126] Example 3. Detection of recombinant BsUGT1 catalyzed DM glycosylation activity

[0127] Using Transetta-32a cells as negative control group, Transetta-32a cells and Transetta-BsUGT1 cells were sonicated. The crushed supernatant was used as the crude enzyme solution, UDP-glucose was used as the glycosyl donor, and DM was used as the substrate to carry out the enzymatic reaction in vitro.

[0128] Glycosyltransferase catalytic activity identification system: 100μL, 20mM Tris-HCl (pH 8.0)

[0129] Crude enzyme solution: 88μL

[0130] 50mM substrate: 2μL

[0131] 50mM UDPG: 10μL

[0132] The reaction system was mixed well, and left to react at 37°C for 24 hours, then 200 μL of ice methanol was added to terminate the reaction, mixed well, centrifuged at 12,000 rpm for 10 minutes, the supernatant was passed through a 0.45 μm filter membrane, and the glycosylation product of DM catalyzed by BsUGT1 was detected by HPLC.

[0133] HPLC detection conditions: Cosmosil C18 rever...

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Abstract

The invention relates to novel glycosyltransferase derived from bacillus subtilis and application thereof. Specifically, the invention provides novel glycosyltransferase BsUGT1 derived from bacillus subtilis, a polynucleotide coding the glycosyltransferase, a vector containing the polynucleotide, and recombinant cells containing the vector or integrated with the polynucleotide. The BsUGT1 is capable of catalyzing hydroxyglycosylation on sites C3, C12 and / or C20 of a dammarane type tetracyclic triterpenoid compound. The invention further provides a method for performing glycosylation by using the glycosyltransferase, and a method for producing rare ginsenosides by using the glycosyltransferase. The glycosyltransferase is capable of catalyzing to produce multiple rare ginsenosides and novelrare ginsenosides (3beta, 12beta-Di-O-G1c-PPT), particularly a rare ginsenoside subjected to C12 glycosylation.

Description

technical field [0001] The present invention relates to a novel glycosyltransferase and its application. Background technique [0002] Ginseng (Panax ginseng C.A. Meyer) is a traditional precious medicinal material, first recorded in my country's first herbal monograph "Shen Nong's Materia Medica", listed as the top grade, has the effects of nourishing the spleen and lungs, promoting body fluid and quenching thirst, calming the nerves and improving intelligence, and prolonging life. Because of its miraculous and extensive effects, ginseng enjoys the reputation of "the king of all herbs". Modern medical research has proved that in addition to nourishing and strengthening the body, ginseng has significant effects on anti-tumor, anti-aging, anti-oxidation, immune regulation and memory enhancement. [0003] Pharmacological studies have proved that the main active ingredient of ginseng is ginsenoside. Ginsenosides belong to triterpenoids and are important secondary metabolites ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12P33/02
CPCC12N9/1048C12P33/02
Inventor 杨金玲朱平梁会超胡宗风梁兰张婷婷巩婷陈晶晶
Owner INST OF MATERIA MEDICA AN INST OF THE CHINESE ACAD OF MEDICAL SCI
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