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Glycosyl transferase-bimetal organic framework composite catalytic material, preparation method thereof and application of glycosyl transferase-bimetal organic framework composite catalytic material in synthesis of disaccharide and polysaccharide

A technology of glycosyltransferase and catalytic materials, which is applied in the field of catalysts, can solve problems such as affecting activity, high catalytic cost, and high price, and achieve the effects of improving stability, accelerating reaction rate, and improving catalytic activity

Pending Publication Date: 2022-05-03
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

While improving the catalytic efficiency of the enzyme, the enzyme can be protected so that the enzyme can withstand a certain degree of denaturing conditions, such as temperature, pH and organic solvents; maintain the activity of the enzyme and realize the repeated use of the enzyme, solving the problem of catalytic reaction Among them, the enzyme is easily affected by the environment, its activity is difficult to be reused, and the catalytic cost is high, and it is the first time to realize the immobilization of multi-metal composite catalytic materials with unstable and expensive glycosyltransferases

Method used

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  • Glycosyl transferase-bimetal organic framework composite catalytic material, preparation method thereof and application of glycosyl transferase-bimetal organic framework composite catalytic material in synthesis of disaccharide and polysaccharide
  • Glycosyl transferase-bimetal organic framework composite catalytic material, preparation method thereof and application of glycosyl transferase-bimetal organic framework composite catalytic material in synthesis of disaccharide and polysaccharide
  • Glycosyl transferase-bimetal organic framework composite catalytic material, preparation method thereof and application of glycosyl transferase-bimetal organic framework composite catalytic material in synthesis of disaccharide and polysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] 1. Glycosyltransferase-metal organic framework composite catalytic material HS2@ZIF-90

[0057] In 2mL Zn(NO 3 ) 2 ·6H 2 Add 2 mL of glycosyltransferase (0.33 mg / ml) to the O (40 mM) solution and stir evenly, then add 2 mL of 2-imidazole-carboxaldehyde (HICA, 160 mM), and add ultrapure water to make up to 10 mL. React at 37° C. and 500 rpm for 24 hours. After the reaction, centrifuge at 11,000rpm for 4min, recover the precipitate and wash it three times with ultrapure water to remove the glycosyltransferase not wrapped by the MOFs material, and dry it in the air.

[0058] 2. Preparation of glycosyltransferase-bimetallic organic framework composite catalytic material HS2@ZIF-90(Mg)

[0059] In 2mL Zn(NO 3 ) 2 ·6H 2 Add 2 mL of glycosyltransferase (0.33 mg / ml) to the O (40 mM) solution and stir evenly, then add 2 mL of MgCl 2 4H 2O (40mM), then add 2mL of 2-imidazole-carboxaldehyde (HICA, 160mM), and add ultrapure water to make the volume to 10mL. React at 37° C...

Embodiment 2

[0061] Application of glycosyltransferase-metal-organic framework composite catalytic materials and glycosyltransferase-bimetallic-organic framework composite catalytic materials in the synthesis of disaccharides

[0062] 1. HS2@ZIF-90 catalyzes glycosylation of monosaccharides to generate heparin disaccharides

[0063] In Tris-HCl buffer (50mM pH 7.4), add uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl-β-D-glucuronic acid (final concentration 10mM), magnesium chloride ( The final concentration is 10mM) and shake and mix in a water bath at 37°C at a speed of 150rpm for 5min. After mixing evenly, add the HS2@ZIF-90 composite catalyst prepared in Example 1 with a mass of 5% of the above reaction solution and shake at 150rpm in a water bath at 37°C Rotational speed, after fully reacting for 12h, the reaction was terminated. The reaction solution was centrifuged, the supernatant was collected for TLC separation, and the separated composite catal...

Embodiment 3

[0079] Real-time monitoring of glycosyltransferase free enzyme, glycosyltransferase-metal organic framework composite catalytic material and glycosyltransferase-bimetallic organic framework composite catalytic material for disaccharide synthesis

[0080] 1. Real-time monitoring of the ability of HS2@ZIF-90 to catalyze the glycosylation of monosaccharides to generate heparin disaccharides

[0081] In Tris-HCl buffer (50mM pH 7.4), add uridine diphosphate-N-acetylglucosamine (final concentration 10mM), p-nitrophenyl-β-D-glucuronic acid (final concentration 10mM), magnesium chloride ( The final concentration is 10mM) shake and mix in a water bath at 37°C, the speed is 150rpm, and the time is 5min. After mixing evenly, add 5% of the mass of the reaction solution to the HS2@ZIF-90 composite catalyst prepared in Example 1, and shake in a water bath at 37°C at 150rpm Rotational speed, the reaction solution was centrifuged at the 1st, 2nd, 3rd, 6th, 9th, 12th, 24th, and 36th hour of t...

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Abstract

The invention discloses a glycosyl transferase-bimetal organic framework composite catalytic material, a preparation method thereof and application of the glycosyl transferase-bimetal organic framework composite catalytic material in synthesis of disaccharide and polysaccharide, and the composite catalytic material is formed by taking a Zn-based MOFs material as a main body, doping non-Zn metal ions and wrapping glycosyl transferase in the Zn-based MOFs material. According to the composite catalytic material, a metal organic framework doped with bimetal ions serves as a novel immobilized enzyme material, glycosyl transferase is wrapped in the metal organic framework, and the glycosyl transferase-bimetal organic framework composite catalytic material is prepared and added into a glycosyl donor and a glycosyl acceptor, so that the glycosyl transferase-bimetal organic framework composite catalytic material is obtained. And catalyzing glycosylation reaction to obtain heparin disaccharide and polysaccharide. The composite catalytic material disclosed by the invention can efficiently catalyze the glycosylation reaction of monosaccharide, not only has good thermal stability and chemical stability, but also can improve the enzymatic reaction rate of immobilized enzyme compared with a common metal organic framework material, and can be recycled for multiple times while keeping higher catalytic activity.

Description

technical field [0001] The invention relates to the technical field of catalysts, in particular to a glycosyltransferase-bimetallic organic framework composite catalytic material, a preparation method thereof and an application in the synthesis of disaccharides and polysaccharides. Background technique [0002] Heparan sulfate (HS) and heparin (Heparin) are polydisperse disaccharide repeating units composed of hexuronic acid (HexA) and glucosamine (GlcN) connected by 1-4 glycosidic bonds. A linear sulfated acidic polysaccharide. Modifications such as N-sulfation, N-acetylation and O-sulfation can occur on the disaccharide constituent units, making heparin a complex variable sequence. HS is widely distributed on animal cell surface and extracellular matrix, and has physiological and pharmacological effects in anticoagulation, embryonic development, inflammatory response and bacterial / viral infection. Heparin is widely used in clinical application of anticoagulation and trea...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N11/14C12N11/089C12P19/12C12P19/04C12P19/18
CPCC12N9/1048C12N11/14C12N11/089C12P19/12C12P19/04C12P19/18
Inventor 张幸乔梦纪元李乐沈宝星
Owner NANJING NORMAL UNIVERSITY
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