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A method for expressing and preparing udp-glucose-4-epimerase

An epimerase, UDP-technology, applied in the direction of racemase/epimerase, isomerase, microbial-based methods, etc., can solve the problem of not suitable for large-scale preparation and low protein expression , cumbersome and time-consuming operations, and achieve the effects of low cost, simple and fast cultivation, and high application value

Active Publication Date: 2021-08-13
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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  • A method for expressing and preparing udp-glucose-4-epimerase
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  • A method for expressing and preparing 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] UDP-glucose-4-differentially isomerase (Gale) gene (sequence, such as SEQ ID NO: 1) is derived from Bifidobacterium Longum JCM 1217, amplified by PCR, after purification, is connected to the cloned carrier PMD19, and construct recombinant plasmid PMD19 -Gale.

[0025] The recombinant plasmid PMD19-Gale and the expression vector PHT 43 were sub-excnabusted in Xbai and SMAI, respectively, and were attached to the night at 16 ° C to obtain recombinant expression vectors PHT43-Gale.

[0026] The expression vector pHT43-GALE was converted to Bacillus chlorobacillus, which was coated with LB plate containing chloramphenicol (5 ug / ml) resistance, and cultured overnight at 37 ° C, and the transformant, extract recombinant plasmid and double enzyme digested. like figure 2 As shown, there are two fragments after the enzyme cutting, and the size of 8000 bp (expression vector pHt43) and 6611 bp (UDP-glucose-4-diffe...

Embodiment 2

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

[0044] The recombinant plasmid PMD19-Gale and expression vector PHT 43 prepared in Example 1 were subcapaseed in XBAI and SMAI, respectively, and were attached to the night at 16 ° C to obtain recombinant expression vectors pHT43-Gale.

[0045] The recombinant expression vector pHT43-Gale is converted to the Bacillus subtilis WB800, which is coated with chloramphenicol (5 ug / ml) resistance, 37 ° C for culture overnight, to pick the transformant, extract recombinant plasmid and double enzyme digestive verification . like figure 2 As shown, there are two fragments after the enzyme cutting, and the size of 8000 bp (expression vector pHt43) and 6611 bp (UDP-glucose-4-differentially isomer enzyme) are successful.

[0046] (2) Recombinant engineering

[0047] The recombinant expression vector PHT43-Gale was used to use electric shock conversion, and 60 ul of Bacillus WB800 electrically converted sensitic cells were ...

Embodiment 3

[0053] (1) Recombinant expression vector PMA5-Gale

[0054] The recombinant plasmid PMD19-Gale and the shuttle carrier PMA5 prepared in Example 1 were digested in XBAI and SMAI, respectively, and were attached to the night at 16 ° C to obtain recombinant expression vectors PMA5-Gale.

[0055] The recombinant expression vector PMA5-Gale was converted to Bacillus 168, coating LB plate containing ampicillin (100 ug / ml) resistance, 37 ° C culture overnight, to pick the transformant, extract recombinant plasmid and double enzyme digestion, verified Construct success.

[0056] (2) Recombinant engineering

[0057] The recombinant expression vector PMA5-Gale constructed is a method of electric shock conversion, and the brown sporettylobacterium 168 is mixed with recombinant expression vector PMA5-Gale plasmid, adding an electric shock cup for 5 minutes after electric shock, electric shock condition: 22kV / CM got recombinant engineering containing recombinant expression vectors PMA5-Gal...

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Abstract

The invention relates to a method for efficiently expressing and preparing UDP-glucose-4-epimerase, which is to construct a recombinant expression vector by combining UDP-glucose-4-epimerase (GalE) gene and Bacillus subtilis vector. Then the recombinant expression vector was transformed into Bacillus subtilis to construct recombinant engineering bacteria. The recombinant engineered bacteria were induced and cultivated in the liquid medium, the bacterial liquid was centrifuged, and the supernatant was taken. The method of the present invention has high yield of UDP-glucose-4-epimerase, relatively pure protein, easy recovery and purification, and simple production operation, which provides convenience for the industrialized large-scale production of GalE, improves the yield, saves time and effort, and saves energy. cost. In particular, the application of the enzyme in the food industry provides a safety guarantee, which is of great significance.

Description

Technical field [0001] The present invention relates to a method of efficiently expressing a recombinant engineering of UDP-glucose-4-differentially isomerase and a method of preparing UDP-glucose-4-differentially heterogeneous enzymes with improved express expression, belonging to the field of genetic engineering technology. Background technique [0002] UDP-Glucose-4-Differentially isomerase (UDP-GLucose 4-Epimerase, Gale) is a key enzyme involved in galactoglyciton metabolism in the biological body, and galactokinase, 1-phosphate galactoside The acyltransferase (Galac-Tose-1-P-Uridyltransferase) is involved in galactoglyciton metabolism, which is called the Leloir pathway. UDP-glucose 4-decomposition isomerase catalytic galactogue metabolism is the last step of Leloir, is also a key step, transforming between UDP-galactose and UDP-glucose, coupled to the TCA cycle to complete metabolism Process. In addition, Bacillus Bacillus were considered to be probiotics in animal intestin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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