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

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

Example Embodiment

[0039] Example 1 Cloning of Glucosyltransferase Gene

[0040] 1. Extraction of genomic DNA

[0041] Inoculate Marine Bacillus B-9987 in 10 mL LB liquid medium, cultivate overnight at 37°C, collect the bacteria by centrifugation, discard the supernatant; wash twice with 1 mL of STE buffer; add 500 μL of STE buffer 3~5mg / mL lysozyme solution, carefully and fully suspend the bacteria, bath in 37℃ water for 30min until the cells become translucent; add 250 μL of 3% SDS, mix up and down gently, continue to 37℃ water bath until clear; add After 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, centrifuge at 12,000 rpm for 10 minutes; Take the supernatant and repeatedly extract 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 the volume of absolute ethanol), and mix upside down until Wh...

Example Embodiment

[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 μ1

[0050] 0.1mM MgCl 2 : 1 μ1

[0051] 15 mM Macrolactin A: 2 μ1

[0052] 20 mM UDP-Glu: 5 μ1

[0053] 0.2 mM MlnGT1 protein: 5 μ1

[0054] ddH 2 O: 86 μ1

[0055] Reaction conditions: 37℃, 30min. After the reaction is over, add 100 μl methanol to stop the reaction, centrifuge at 13,000 rpm for 20 minutes, discard the precipitate, and remove the protein in the reaction solution. The obtained supernatant was tested by HPLC.

[0056] HPLC detection: use reversed phase C18 column (specification: 150 × 4. 6 mm, 5μ); column temperature is 30 ℃; elution conditions: 0-5 min equilibrium: 65% phase A (ddH 2 O+0.1% formic acid) and 35% B phase (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%; detection wavelength is 260 nm; flow ...

Example Embodiment

[0059] Example 3: Transglycosylation of MlnGT1 glycosyltransferase on macrolide compounds

[0060] When TDP-Glc is used as the glycosyl donor, the UDP-Glu in the enzyme reaction system in Example 2 is replaced with TDP-Glu, incubated at 37°C for 30 minutes, and the reaction is terminated and then subjected to HPLC detection and HRMS analysis. The results are the same Obtain glycosylated Macrolactin A ( Figure 4 B). This proves that the glycosyltransferase of the present invention has good application prospects.

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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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