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

A technology of glucose derivatives and uridine diphosphate, which is applied in the field of biosynthesis, can solve the problems of inability to reuse enzymes, difficult control of intermediate products, and low yield of products, so as to improve pH tolerance and thermal stability , process environmental protection, the effect of simple steps

Pending Publication Date: 2022-07-05
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 and derivatives thereof
  • Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine and derivatives thereof
  • Double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine and derivatives thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1. Recombinant E. coli Z basic2 -NahK and Z basic2 -Expression of AGX1 with immobilization alone

[0103] 1. N-acetylhexosamine-1-kinase gene Z basic2 -NahK 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 plasmid Z basic2 -NahK and Z basic2 -AGX1 and then recombinant plasmid Z respectively basic2 -NahK and Z basic2 -AGX1 was transformed into Escherichia coli E.coliBL21(DE3) to obtain the gene Z containing N-acetylhexosamine-1-kinase basic2 -NahK 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.

[0104] The N-acetylhexosamine-1-kinase gene Z basic2 - The GenBank accession number of NahK is 69578838, and the GenBank accession number of the UDP-GalNAc pyrophosphorylase gene AGX1 is 6675.

[01...

Embodiment 3

[0112] Example 3. Individually immobilized Z basic2 -NahK immobilization preparation and Z basic2 - Catalytic reaction of AGX1 immobilized preparations

[0113] 1) Z basic2 - Determination of catalytic reaction activity of NahK immobilized preparations

[0114] Z prepared in Example 1 basic2 -NahK immobilized preparation was added to the reaction system. The reaction system was shown in Table 3. After reacting at 37°C and 1000 rpm / min for 1 h, the reaction was terminated by boiling for 5 min, and centrifuged at 6000 rpm / min for 2 min at room temperature. After membrane filtration, TLC analysis was performed.

[0115] Table 3 Z basic2 - Catalytic reaction system of NahK immobilized preparation

[0116]

[0117] TLC results such as Figure 4 As shown, compared with the negative control, GlcNAc-1-P was produced in the reaction solution, indicating that Z basic2 -NahK immobilized preparation has the activity of converting N-acetylglucosamine to GlcNAc-1-P.

[0118] 2) ...

Embodiment 4

[0138] Example 4. N-Acetylhexosamine-1-kinase Z basic2 -NahK and UDP-GalNAc pyrophosphorylase Z basic2 -AGX1 co-immobilized enzyme preparation

[0139] 1. Z basic2 -NahK and Z basic2 - Preparation of AGX1 co-immobilized enzyme preparation

[0140] Take the gene Z containing N-acetylhexosamine-1-kinase in Example 1 basic2 -NahK recombinant E. coli and containing UDP-GalNAc pyrophosphorylase gene Z basic2 -The crushed supernatant of AGX1 recombinant Escherichia coli was mixed and added to the cation exchange resin purolite chromalite MS / C, and incubated at 4°C and 1000rpm / min for 3h for co-immobilization. After the incubation, the supernatant was removed, the resin was washed with Tris-HCl, and the immobilized enzyme was analyzed by SDS-PAGE.

[0141] The result is as Figure 14 As shown, two proteins were bound on the solid support, which were consistent with the molecular weights of the corresponding proteins, indicating that Z basic2 -NahK and Z basic2 -AGX1 was co-i...

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Abstract

The invention relates to a double-enzyme co-immobilization synthesis method of uridine diphosphate-N-acetylglucosamine and a derivative thereof. According to the method, N-acetylglucosamine or an N-acetylglucosamine derivative is taken as a substrate, an N-acetylhexosamine-1-kinase and UDP-GalNAc pyrophosphorylase co-immobilized enzyme preparation is taken as a catalyst, and the uridine diphosphate-N-acetylglucosamine and the derivative thereof are obtained through biosynthesis. According to the biosynthesis method disclosed by the invention, N-acetylhexosamine-1-kinase and UDP-GalNAc pyrophosphorylase are taken as catalysts, and mass production of uridine diphosphate-N-acetylglucosamine and the derivatives thereof is realized according to different carbohydrate substrates. 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 and derivatives thereof, belonging 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. 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, and anti-allergic effects. It is used in the treatment of asthma and chronic obstructi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30
CPCC12P19/305Y02P20/55
Inventor 生举正宫雪艳侯进
Owner SHANDONG UNIV
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