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Method for synthesizing UDP-galactose and galactosyl compound

A technology of galactose and galactokinase, which is applied in the field of bioengineering and can solve problems such as unseen galactosyl compounds

Active Publication Date: 2021-08-17
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no patent on the synthesis of galactosyl compounds by thermophilic enzymes

Method used

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  • Method for synthesizing UDP-galactose and galactosyl compound
  • Method for synthesizing UDP-galactose and galactosyl compound
  • Method for synthesizing UDP-galactose and galactosyl compound

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: TTHA0595, TTE0732 and TON_1857 Engineering The construction of bacteria and purification of proteins, including the following steps:

[0024] 1. TTHA0595, TTE0732 and TON_1857 gene amplification

[0025] The primer sequences of TTHA0595, TTE0732 and TON_1857 gene amplification are as follows:

[0026] (1) Thermophilic hemodalose kinase TTHA0595:

[0027] Upstream primers: atgaat Catatg Atgggcttccaagaggttttac, the horizontal line is the restriction NdeI identification site;

[0028] Downstream primer: aatccc Gaattc Ttagaggaccttgagg, horizontal line is restriction enzyme EcoRI identification site;

[0029] (2) Thermophilic UDP-glucose phosphorylation enzyme TTE0732:

[0030] Upstream primers: agcggaagagagggaaag Catatg AAAATAA, the horizontal line is the restriction enzyme NdeI identification site;

[0031] Downstream primer: cgccgccctctct Gaattc TTACACATCT, the horizontal line is the restriction enzyme EcoRI identification site;

[0032] (3) Iophilic preheating seq...

Embodiment 2

[0041] Example 2: Preparation of UDP-galactose

[0042] UDP-galactose synthesis includes two-step reaction, the specific process is as follows:

[0043] First step reaction: thermophilic galactose TTHA0595 catalyzed 1-p-GAL synthesis

[0044] The galactose kinase is a phosphate donor by a molecular ATP (adenosine thiophosphate) to catalyzes D-galactose to form 1-p-gal and ADP (adenosine dithonic acid), and the reaction system is shown in Table 2. The reaction buffer is 20 mM Caps (3-cycloisopropathulfonic acid) aqueous solution (pH 8.0), sequentially adding the substrate D-galactose and ATP to a final concentration of 150 mm, then add MGCL 2 The final concentration was 5 mM, 50 ° C, and the thermophilic semicondrin kinase TTHA0595 was added to a final concentration of 0.4 mg / mL, and the revolution of the revolution of 150 rpm at 50 ° C was 0 to 120 min. figure 2 ). The experimental results show that the reaction is in 80min to achieve a balance, and the amount of product in 60 t...

Embodiment 3

[0055] Example 3: Synthesis of galactoselated compounds

[0056] Example 3 UDP-galactose synthesized in Example 2 was galactose-based donor, which catalyzed the syrogl-based alkyl group using the thermophilic preheating sequetolatum-based transferase TON_1857, and realized the compound fragrant laceanol, p-nitrophenol and water. The glycosylation modification of Jobin, the reaction system is shown in Table 4.

[0057] Table 4: TON_1857 Catalyzed the reaction raw material table of synthetic galactose compound

[0058]

[0059] The reaction buffer is 20 mM Caps aqueous solution (pH 9.0), and UDP-galactose having a final concentration of 5 mm, MGCl 2 And the fragrant lanterol or the nitrophenol or water fly Bin, mixed with a mixture of 5 min at 50 ° C, and then add a final concentration of 0.4 mg / ml of thermophilic preheating sequrites Ton_1857 at 50 ° C to 150 rpm / The number of revolutions of MIN reacted for 5h.

[0060] After the end of the reaction, it was boiled for 1 min i...

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Abstract

The invention discloses a method for synthesizing UDP-galactose and a method for synthesizing a galactosyl compound, and belongs to the technical field of bioengineering. The invention relates to a method for generating UDP-galactose by catalyzing UTP, ATP and D-galactose through a thermophilic enzyme method two-step reaction. The method comprises the following two steps: i) taking D-galactose and ATP as substrates, and catalytically synthesizing 1-P-Gal (1-phosphate-galactose) by using thermophilic galactokinase TTHA0595; and (ii) by taking the 1-P-Gal and the UTP as the substrates, carrying out catalytic synthesis on the UDP-galactose by using the thermophilic UDP-glucose pyrophosphorylase TTE0732. The synthesized UDP-galactose is used as a glycosyl donor, and thermophilic galactosyl transferase TON1857 can be used for further catalyzing substrate compounds such as vanillin, p-nitrophenol or silybin to synthesize the galactosyl compound, so that the method for synthesizing the galactosyl compound by the thermophilic enzyme method is constructed.

Description

Technical field [0001] The present invention belongs to the field of bio-engineering, and more particularly to a method of synthesizing UDP-galactose and synthesis galactose compounds. Background technique [0002] The sugar complex refers to a compound which is formed by covalent bonds such as a bio molecule such as a small molecule compound and a small molecule compound and a protein, lipid and nucleic acid. Some of these small molecule sugar complexes have important application value, which is the current hotspot, such as glycoside, small molecular drug glycosylated products, glycosylated products of pigments, and fragrances, probiotics, and the like. Glycoside is a compound of hydrogen on the semi-aldehyde hydroxyl group of sugar to substituting the compound in which the compound is substituted in a substituted molecule or other small molecule compound. Galactoside is a candidate molecule in drug precursor design and synthesis, and the pre-solubility of the hyperlidose drug i...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12N15/70C12N15/54C12N9/12
CPCC12P19/305C12N15/70C12N9/1205C12N9/1241C12Y207/01006C12Y207/07009
Inventor 高仁钧李雅静邓林解桂秋
Owner JILIN UNIV
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