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Glucosyltransferase and application thereof

A technology of glycosyltransferase and glucosyl, which is applied in the field of new glucosyltransferase, can solve the problems of unreported research on modification steps, and achieve the effects of improving bioavailability, enhancing drug efficacy, and improving water solubility

Inactive Publication Date: 2013-09-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] As a class of drug lead compounds with great application potential, Macrolactins have attracted great attention. Macrolactins belong to polyketide compounds, and its skeleton structure Macrolactin A is catalyzed by polyketide synthases (Polyketide synthases, PKSs). However, the subsequent modification steps have not been reported

Method used

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  • Glucosyltransferase and application thereof
  • Glucosyltransferase and application thereof
  • Glucosyltransferase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Cloning of Glucosyltransferase Gene

[0040] 1. Genomic DNA extraction

[0041] Marine Bacillus B-9987 was inoculated in 10 mL of LB liquid medium, cultured overnight at 37°C, collected by centrifugation, and the supernatant was discarded; washed twice with 1 mL of STE buffer; added 500 μL of STE buffer prepared 3-5 mg / mL lysozyme solution, carefully suspend the bacteria, and bathe in 37°C water for 30 minutes until the cells become translucent; add 250 μL of 3% SDS, mix gently up and down, continue to bathe in 37°C water until clarified; add After adding 1 / 10 volume of 3 M NaAc (pH=4.8), add 200 μL of phenol: chloroform: isoamyl alcohol (25: 24: 1 ), invert the centrifuge tube several times, and centrifuge at 12,000 rpm for 10 min; pipette carefully Take the supernatant, extract repeatedly with phenol: chloroform: isoamyl alcohol, until the middle layer is free of protein impurities, transfer the supernatant, add an equal volume of isopropanol (or 2 times th...

Embodiment 2

[0047] Example 2: In vitro enzyme activity detection of MlnGT1 protein

[0048] In vitro enzyme activity reaction system: (100 μl )

[0049] 2.5M Tris-HCl buffer (pH7.5): 1 μl

[0050] 0.1mM MgCl 2 : 1 μ1

[0051] 15 mM Macrolactin A: 2 μl

[0052] 20 mM UDP-Glu: 5 μl

[0053] 0.2 mM MlnGT1 protein: 5 μl

[0054] wxya 2 O: 86μ1

[0055] Reaction conditions: 37°C, 30min. After the reaction, add 100 μl of methanol to stop the reaction, centrifuge at 13,000rpm for 20 minutes, discard the precipitate, and remove the protein in the reaction solution. The resulting supernatant was subjected to HPLC detection.

[0056] HPLC detection: use reversed phase C18 column (specification: 150 × 4.6 mm, 5μ); column temperature is 30°C; elution condition: 0-5 min equilibration: 65% phase A (ddH 2 O+0.1% formic acid) and 35% phase B (acetonitrile+0.1% formic acid); 5-20 min linear elution, 35-0% A phase and 35-100% B phase; 20-30 min isocratic elution: Phase A: 0%, Phase B: 100%; detec...

Embodiment 3

[0059]Example 3: Transglycosylation of macrolides by MlnGT1 glycosyltransferase

[0060] When TDP-Glc was used as the glycosyl donor, UDP-Glu in the enzyme reaction system in Example 2 was replaced by TDP-Glu, incubated at 37°C for 30min, and after the reaction was terminated, HPLC detection and HRMS analysis were performed, and the results were the same Obtain glycosylated Macrolactin A ( Figure 4 B). Thus it is proved that the glycosyltransferase of the present invention has a good application prospect.

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Abstract

The invention aims to provide a novel glucosyltransferase, which can be used for performing galactosylated modification on Macrolactins macrolide compounds. According to the glucosyltransferase, the water solubility of the macrolide compounds can be improved through glycosylation to the Macrolactins, further the bioavailability of the macrolide compounds can be improved, the pharmacological effect is enhanced, the toxic or side effect is reduced and a new strategy is provided for medicine research of the macrolide compounds.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and biopharmaceuticals, and specifically relates to a novel glucosyltransferase and its application. Background technique [0002] In general, compared with the corresponding unglycosylated compounds, glycosylated compounds may have improved water solubility, which can improve their bioavailability, enhance drug efficacy, and reduce toxic and side effects. Therefore, the glycosylation modification of natural compounds is a hot research topic. [0003] Macrolactins are a series of 24-membered macrolide compounds with biological and pharmacological activities such as antibacterial, antiviral, antitumor, anti-infection and anti-aging. At the earliest, the Fenical research group isolated Macrolactins A-F from a strain of deep-sea bacteria in 1989. Studies have shown that Macrolactin A can inhibit melanoma cells of B1-F10 murine animals and mammalian herpes simplex virus, and can inhibit ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/62C12R1/07
Inventor 李文利秦文刘扬
Owner OCEAN UNIV OF CHINA
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