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Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine

A technology of uridine diphosphate and acetamido, applied in the field of biosynthesis, can solve the problems of inability to reuse enzymes, difficult to control intermediate products, low product yield, etc., and achieves improved pH tolerance and thermal stability, The effect of environmentally friendly process and simple preparation method

Pending Publication Date: 2022-07-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the patent uses the yeast cell's own UDP-GlcNAc metabolic pathway to carry out whole-cell catalytic synthesis by adding glutamine.

Method used

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  • Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine
  • Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine
  • Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Example 1. Recombinant Escherichia coli PbChi70, Z basic2 -Vf.ChbP and Z basic2 -Expression of AGX1 with immobilization alone

[0088] 1. Using the chitinase gene PbChi70 and the phosphorylase gene Z respectively basic2 -Vf.ChbP and UDP-GalNAc pyrophosphorylase gene Z basic2 -AGX1 is the target gene, and pET-21a(+) is used as the vector plasmid to construct the recombinant plasmids PbChi70, Z basic2 -Vf.ChbP and Z basic2 -AGX1, then the recombinant plasmids PbChi70, Z basic2 -Vf.ChbP and Z basic2-AGX1 was transformed into Escherichia coli E.coliBL21(DE3) to obtain recombinant Escherichia coli containing chitinase gene PbChi70 and phosphorylase gene Z basic2 -Vf.ChbP recombinant E. coli and containing UDP-GalNAc pyrophosphorylase gene Z basic2 - AGX1 recombinant E. coli. The construction of the above recombinant bacteria was completed by Nanjing GenScript Biotechnology Co., Ltd.

[0089] The GenBank accession number of the chitinase gene PbChi70 is KJ634701.1. ...

Embodiment 2

[0098] Example 2. Catalytic reaction of chitinase PbChi70

[0099] Preparation of colloidal chitin: Weigh 4 g of chitin powder, slowly add it to 60 mL of concentrated hydrochloric acid, stir on ice, stir evenly, and place in a 4°C refrigerator overnight; add ice-cold 95% ethanol to the mixture, while Stir rapidly, at this time crystals are precipitated, centrifuge at 5000g for 20min at 4°C to collect the precipitate; rinse the precipitate repeatedly with double distilled water until the pH is 7.0; finally add 100 mL of double distilled water to prepare a 4% colloidal chitin solution.

[0100] Catalytic reaction: 250 μL of citric acid buffer (pH=5.5) and 500 μL of chitinase Pbchi70 purified in Example 1 were added to 250 μL of 4% (w / v) colloidal chitin, and reacted in a water bath at 55° C. for 1 h. After the reaction, the reaction solution was taken out, boiled for 5 min to terminate the reaction, centrifuged at 6000 rpm / min for 2 min, and the centrifuged supernatant was analy...

Embodiment 3

[0103] Example 3. Individually immobilized Z basic2 -Vf.ChbP immobilized preparation and Z basic2 - Catalytic reaction of AGX1 immobilized preparations

[0104] 1) Z basic2 - Determination of catalytic activity of Vf.ChbP immobilized preparations

[0105] Z prepared in Example 1 basic2 -Vf.ChbP immobilized preparation was added to the reaction system. The reaction system was shown in Table 3. After 1 h of reaction at 37°C and 1000 rpm / min, the reaction was terminated by boiling for 5 min, and centrifuged at 6000 rpm / min for 2 min at room temperature. TLC analysis was performed after filtration through a 0.45 μm filter.

[0106] Table 3 Individually immobilized enzyme Z basic2 - Catalytic reaction system of Vf.ChbP

[0107]

[0108] TLC results such as Figure 5 As shown, compared with the negative control, GlcNAc-1-P was produced in the reaction solution, indicating that Z basic2 -Vf.ChbP immobilized preparation has the activity of converting N,N-diacetylated chitob...

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Abstract

The invention relates to a double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine. Comprising the following steps: carrying out biosynthesis by taking colloidal chitin as a substrate and chitinase as a catalyst to obtain N, N-diacetylated chitobiose; the method comprises the following steps: adding uridine triphosphate, phosphate radical ions, inorganic ions, a Tris-HCl buffer solution and inorganic pyrophosphatase into N, N-diacetylated chitobiose, and carrying out biosynthesis by taking a phosphorylase and UDP-GalNAc pyrophosphorylase co-immobilized enzyme preparation as a catalyst to obtain the uridine diphosphate-N-acetylglucosamine. According to the biosynthesis method disclosed by the invention, chitin is used as a raw material, and then chitinase PbChi70, phosphorylase and UDP-GalNAc pyrophosphorylase are used for carrying out two-step catalysis, so that the mass production of uridine diphosphate-N-acetylglucosamine is realized. And addition of an expensive auxiliary material ATP is not needed, so that the production cost is greatly reduced.

Description

technical field [0001] The invention relates to a dual-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine, which belongs to the technical field of biosynthesis. Background technique [0002] Sugar nucleotides, as functional glycoconjugates and natural glycosyl donors in carbohydrate biosynthesis, are involved in processes critical to the function and survival of organisms, and are also used to synthesize carbohydrates in vitro using Leloir-type glycosyltransferases An integral component of a compound. Glyconucleotides can also regulate the overall distribution of glycoproteins on the cell surface. The synthesis of oligosaccharides and polysaccharides using sugar nucleotides as substrates has been a research hotspot in recent years. For example, heparin in polysaccharide products has anticoagulant, anti-inflammatory, blood lipid regulation, anti-allergic and other effects, and is used in the treatment of asthma and chronic obstructive pulmon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N15/56C12N15/54C12N15/70C12N11/04C12N11/089C12N9/12C12N9/42
CPCC12P19/305C12N15/70C12N11/04C12N11/089C12N9/1241C12N9/2442C12Y302/01014C12Y207/07009C12Y207/07023
Inventor 生举正宫雪艳王凤山侯进
Owner SHANDONG UNIV
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