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A method for expressing and preparing sucrose phosphorylase

A technology of sucrose phosphorylase and phosphorylase gene, which is applied in the field of genetic engineering, can solve problems such as not suitable for large-scale preparation, low protein expression, and difficulty in separation and purification, and achieve good production technology, simple and fast cultivation, The effect of high application value

Active Publication Date: 2021-12-17
SHENYANG AGRI UNIV
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  • 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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  • A method for expressing and preparing sucrose phosphorylase
  • A method for expressing and preparing sucrose phosphorylase
  • A method for expressing and preparing sucrose phosphorylase

Examples

Experimental program
Comparison scheme
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 efficiently preparing sucrose phosphorylase. The recombinant expression vector is constructed by combining the sucrose phosphorylase SP gene and the 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 invention has high yield of sucrose phosphorylase, relatively pure protein, easy recovery and purification, simple production operation, provides convenience for large-scale industrial production of SP, improves yield, saves time and effort, and saves 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 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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Patent Type & Authority Patents(China)
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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