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Method for preparing sucrose phosphorylase by high-efficiency expression

A sucrose phosphorylase, high-efficiency expression technology, applied in microorganism-based methods, biochemical equipment and methods, glycosyltransferases, etc. and other problems, to achieve the effect of good production technology, simple and fast cultivation, and high application value

Active Publication Date: 2019-02-05
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 sucrose phosphorylase when extracted from wild bacteria is very low, separation and purification are difficult, and it is not suitable for large-scale preparation
Some express sucrose phosphorylase through 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, the content of foreign proteins is high, separation and purification are difficult, and the operation is cumbersome Time-consuming and low protein expression

Method used

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  • Method for preparing sucrose phosphorylase by high-efficiency expression
  • Method for preparing sucrose phosphorylase by high-efficiency expression
  • Method for preparing sucrose phosphorylase by high-efficiency expression

Examples

Experimental program
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Effect test

Embodiment 1

[0026] (1) Construction of recombinant expression vector pHT43-SP

[0027] The sucrose phosphorylase (SP) 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-SP.

[0028] The recombinant plasmid pMD19-SP and the expression vector pHT43 were double-digested with XbaI and BamHI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-SP.

[0029] The recombinant expression vector pHT43-SP was transformed into Bacillus subtilis WB800N, spread on LB plates containing chloramphenicol (5ug / mL) resistance, cultured 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 1819bp (sucrose acidase), respectivel...

Embodiment 2

[0046] (1) Construction of recombinant expression vector pHT43-SP

[0047]The recombinant plasmid pMD19-SP prepared in Example 1 and the expression vector pHT43 were double digested with XbaI and BamHI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pHT43-SP.

[0048] The recombinant expression vector pHT43-SP was transformed into Bacillus subtilis WB800, spread on LB plates containing chloramphenicol (5ug / mL) resistance, cultured 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 1819bp (sucrose acidase), respectively, indicating that the connection is successful.

[0049] (2) Recombinant engineering bacteria

[0050] The constructed recombinant expression vector pHT43-SP was transformed by electric shock, and 60ul of Bacillus subtilis WB800 electropora...

Embodiment 3

[0056] (1) Construction of recombinant expression vector pMA5-SP

[0057] The recombinant plasmid pMD19-SP and the shuttle vector pMA5 prepared in Example 1 were double-digested with XbaI and BamHI respectively, and ligated overnight at 16°C to obtain the recombinant expression vector pMA5-SP.

[0058] The recombinant expression vector pMA5-SP was transformed into Bacillus subtilis 168, coated with LB plates containing ampicillin (100ug / mL) resistance, cultured overnight at 37°C, the transformants were picked, the recombinant plasmid was extracted and verified by double enzyme digestion. The build was successful.

[0059] (2) Recombinant engineering bacteria

[0060] The constructed recombinant expression vector pMA5-SP was transformed by electric shock. Bacillus subtilis 168 electroporated competent cells were mixed with the recombinant expression vector pMA5-SP 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 preparing sucrose phosphorylase by high-efficiency expression. The method comprises the following steps: enabling a sucrose phosphorylase SP gene and bacillus subtilis to construct a recombinant expression vector by using a vector; converting the recombinant expression vector into the bacillus subtilis to construct recombinant engineering bacteria; and performing induction culture on the recombinant engineering bacteria in a fluid medium, centrifuging a bacteria solution, and taking liquid supernatant. The method is high in yield of sucrose phosphorylase,purer in protein, easy to be recovered and purified, simple in production operation, and capable of providing convenience for industrial mass production of SP, improving the yield, saving both time and labor, and saving cost, especially providing safety guarantee for the application of the sucrose phosphorylase in food industry, and has important significance.

Description

technical field [0001] The invention relates to a recombinant engineering bacterium capable of highly expressing sucrose phosphorylase and a method for preparing sucrose phosphorylase by improving high-efficiency expression, belonging to the technical field of genetic engineering. Background technique [0002] Sucrose phosphorylase (EC2.4.1.7, Sucrose phosphorylase, SPase) belongs to the 13th family of glycosyl hydrolases. It is a specific enzyme that catalyzes the transfer of glucosidic bonds. It can reversibly catalyze the phosphorylation of sucrose to generate 1-phosphate Glucose and D-fructose. The enzyme has wide substrate specificity and has important application value in industrial production such as food and cosmetics. However, in the prior art, the yield of sucrose phosphorylase when extracted from wild bacteria is very low, separation and purification are difficult, and it is not suitable for large-scale preparation. Some express sucrose phosphorylase through Esc...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12N9/10C12R1/125
CPCC12N9/1051C12N15/75C12Y204/01007
Inventor 李拖平李苏红佟超男孙玥
Owner SHENYANG AGRI UNIV
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