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Method for efficiently expressing preparation of UDP-glucose-4-epimerase

An epimerase, high-efficiency expression technology, applied in the field of genetic engineering, can solve the problems of unsuitable for large-scale preparation, low protein expression, difficult separation and purification, etc., and achieves low cost, simple and rapid cultivation, and high application. effect of value

Active Publication Date: 2019-03-01
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the prior art, the yield of UDP-glucose 4-epimerase when extracted from wild bacteria is very low, separation and purification are difficult, and it is not suitable for large-scale preparation
UDP-glucose-4-epimerase is expressed in Escherichia coli, but it is expressed intracellularly in Escherichia coli, and it is easy to form inclusion bodies. At the same time, the acquisition of the enzyme protein requires the crushing of cells, and the content of foreign proteins is high. , difficult to separate and refine, cumbersome and time-consuming to operate, and the protein expression level is not high

Method used

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  • Method for efficiently expressing preparation of UDP-glucose-4-epimerase
  • Method for efficiently expressing preparation of UDP-glucose-4-epimerase
  • Method for efficiently expressing preparation of UDP-glucose-4-epimerase

Examples

Experimental program
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Effect test

Embodiment 1

[0023] (1) Construction of recombinant expression vector pHT43-GalE

[0024] The UDP-glucose-4-epimerase (GalE) gene (sequence shown in SEQ ID NO: 1) was derived from Bifidobacterium longum JCM 1217. After PCR amplification and purification, it was connected to the cloning vector pMD19 to construct the recombinant plasmid pMD19 -GalE.

[0025] The recombinant plasmid pMD19-GalE and the expression vector pHT43 were double-digested with XbaI and SmaI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-GalE.

[0026] The expression vector pHT43-GalE was transformed into Bacillus subtilis WB800N, spread on LB plates containing chloramphenicol (5ug / mL) resistance, cultured overnight at 37°C, picked transformants, extracted recombinant plasmids and verified by double enzyme digestion. Such as figure 2 As shown, there are two fragments after enzyme digestion, the sizes are about 8000bp (expression vector pHT43) and 6611bp (UDP-glucose-4-ep...

Embodiment 2

[0043] (1) Construction of recombinant expression vector pHT43-GalE

[0044] The recombinant plasmid pMD19-GalE prepared in Example 1 and the expression vector pHT43 were double-digested with XbaI and SmaI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-GalE.

[0045] Transform the recombinant expression vector pHT43-GalE into Bacillus subtilis WB800, smear the LB plate containing chloramphenicol (5ug / mL) resistance, culture overnight at 37°C, pick the transformant, extract the recombinant plasmid and double enzyme digestion verification . Such as figure 2 As shown, there are two fragments after enzyme digestion, the sizes are about 8000bp (expression vector pHT43) and 6611bp (UDP-glucose-4-epimerase) respectively, that is, the connection is successful.

[0046] (2) Recombinant engineering bacteria

[0047] The constructed recombinant expression vector pHT43-GalE was transformed by electric shock, and 60ul of Bacillus subtilis ...

Embodiment 3

[0053] (1) Construction of recombinant expression vector pMA5-GalE

[0054] The recombinant plasmid pMD19-GalE and the shuttle vector pMA5 prepared in Example 1 were double-digested with XbaI and SmaI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pMA5-GalE.

[0055] The recombinant expression vector pMA5-GalE was transformed into Bacillus subtilis 168, coated with LB plates containing ampicillin (100ug / mL) resistance, cultured overnight at 37°C, picked transformants, extracted recombinant plasmids and verified by double enzyme digestion. The build was successful.

[0056] (2) Recombinant engineering bacteria

[0057] The constructed recombinant expression vector pMA5-GalE was transformed by electric shock. Bacillus subtilis 168 electrotransformed competent cells were mixed with the recombinant expression vector pMA5-GalE plasmid, and then placed in an electric shock cup for ice bath for 5 minutes before electric shock. Electric shock ...

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Abstract

The invention relates to a method for efficiently expressing preparation of UDP-glucose-4-epimerase. The method comprises the steps that a recombinant expression vector is constructed by using a UDP-glucose-4-epimerase (GalE) gene and a carrier of bacillus subtilis; the recombinant expression vector is then transformed into the bacillus subtilis to construct recombinant engineered bacteria; and the recombinant engineering bacteria are induced to culture in a liquid medium, bacterial liquid is centrifuged, and liquid supernatant is taken. The method has a high yield of the UDP-glucose-4-epimerase, relatively pure proteins, easy recovery and purification and simple production operation, and provides convenience for industrial large-scale production of GalE, output is improved, time and laborare saved, and the cost is saved. In particular, security assurance is provided for the application of the UDP-glucose-4-epimerase in the food industry, and great significance is achieved.

Description

technical field [0001] The invention relates to a recombinant engineering bacterium capable of efficiently expressing UDP-glucose-4-epimerase and a method for preparing UDP-glucose-4-epimerase by improving high-efficiency expression, belonging to the technical field of genetic engineering. Background technique [0002] UDP-glucose-4-epimerase (UDP-glucose 4-epimerase, GalE) is a key enzyme involved in the metabolism of galactose in organisms, and galactokinase (galactokinase), galacturidine 1-phosphate Acyltransferase (galac-tose-1-p-uridylyltransferase) is jointly involved in the metabolism of galactose, and this process is called the Leloir pathway. UDP-glucose 4-epimerase catalyzed galactose metabolism is the last and key step of Leloir, through the conversion between UDP-galactose and UDP-glucose, coupling glycolysis and TCA cycle to complete the metabolism process. In addition, Bacillus subtilis is considered to be a probiotic in the intestinal tract of animals, which...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N15/75C12N1/21C12N9/90C12R1/125
CPCC12N9/90C12N15/75C12Y501/03002
Inventor 李拖平李苏红孙玥佟超男
Owner SHENYANG AGRI UNIV
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