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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  • 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 Escheri

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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