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A method for expressing and preparing lactobiose phosphorylase

A technology of phosphorylase and phosphorylase gene, applied in the direction of microorganism-based methods, biochemical equipment and methods, glycosyltransferase, etc. High content and other problems, to achieve the effect of low cost, simple and fast cultivation, pure protein

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 lactobiose 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 lactobiose 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, and it is difficult to separate and refine. The operation is cumbersome and time-consuming, and the protein expression level is not high

Method used

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  • A method for expressing and preparing lactobiose phosphorylase
  • A method for expressing and preparing lactobiose phosphorylase
  • A method for expressing and preparing lactobiose phosphorylase

Examples

Experimental program
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Effect test

Embodiment 1

[0025] (1) Construction of recombinant expression vector pHT43-LNBP

[0026] Lactobiose phosphorylase (LNBP) gene (sequence shown in SEQ ID NO: 1) was derived from Bifidobacteriumlongum JCM 1217, amplified by PCR, purified and ligated with cloning vector pMD19 to construct recombinant plasmid pMD19-LNBP.

[0027] The recombinant plasmid pMD19-LNBP 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-LNBP.

[0028] The recombinant expression vector pHT43-LNBP 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 2335bp (lactobiose phosphorylase) respectiv...

Embodiment 2

[0045] (1) Construction of recombinant expression vector pHT43-LNBP

[0046] The recombinant plasmid pMD19-LNBP 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-LNBP.

[0047] The recombinant expression vector pHT43-LNBP was transformed into Bacillus subtilis WB800, 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, the sizes are about 8000bp (expression vector PHT43) and 2335bp (lactobiose phosphorylase), respectively, indicating that the connection is successful.

[0048] (2) Recombinant engineering bacteria

[0049] The constructed recombinant expression vector pHT43-LNBP was transformed by electric shock. Take 60 ul of Bacillus subtilis WB800 electroporation competent c...

Embodiment 3

[0055] (1) Construction of recombinant expression vector pMA5-LNBP

[0056] The recombinant plasmid pMD19-LNBP 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-LNBP.

[0057] The recombinant expression vector pMA5-LNBP 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.

[0058] (2) Recombinant engineering bacteria

[0059] The constructed recombinant expression vector pMA5-LNBP was transformed by electric shock. Bacillus subtilis 168 electroporation competent cells were mixed with the recombinant expression vector pMA5-LNBP plasmid, and then electric shock was performed after adding to the electric shock cup and ice-bathed for 5 minutes. E...

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Abstract

The invention relates to a method for high-efficiency expression and preparation of lactobiose phosphorylase, which is to construct a recombinant expression vector by using a lactobiose phosphorylase LNBP gene and a 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 lactobiose phosphorylase, relatively pure protein, easy recovery and purification, simple production operation, provides convenience for large-scale industrial production of LNBP, improves production, saves time and effort, and saves costs. 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 efficiently expressing lactobiose phosphorylase and a method for preparing lactobiose phosphorylase by improving high-efficiency expression, belonging to the technical field of genetic engineering. Background technique [0002] LNBP (EC2.4.1.211, lacto-N-biose phosphorylase, LNBP) is a reversible phosphorylase that can catalyze LNB and GNB (galacto-N-biose) to produce N-acetylglucosamine (GlcNAc), N - Acetylgalactosamine (GalNAc) and β-D-galactose 1-phosphate (Gal 1-P). It was confirmed that there is a gene cluster encoding the metabolism of LNB in ​​Bifidobacteria, where LNB is transported to the cytoplasm by specific ABC-type transporters, passes through LNBP and is finally metabolized in the glycolysis and amino sugar metabolism pathways. Bifidobacterium is an important dominant bacterium in the intestinal tract of infants and one of the most important beneficial bacteria in th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/75C12N1/21C12N9/10C12R1/125
CPCC12N9/1051C12N15/75C12Y204/01021
Inventor 李拖平李苏红佟超男孙玥
Owner SHENYANG AGRI UNIV
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