Recombinant plasmid for producing oxalate decarboxylase, Escherichia coli expression system, method and application

A technology of oxalate decarboxylase and Escherichia coli, applied in the biological field, can solve the problems of inactivity, complex renaturation process and high cost, and achieve the effect of optimizing composition

Active Publication Date: 2021-07-20
WUHAN KANGFUDE BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the problems in the prior art that the expression of oxalate decarboxylase derived from eukaryotic organisms in Escherichia coli often results in inactive inclusion bodies, complex renaturation process and high cost, the purpose of the present invention is to provide a recombinant oxalate decarboxylase for producing oxalate decarboxylase Plasmid and Escherichia coli expression system comprising said recombinant plasmid, the active oxalate decarboxylase can be produced by using the plasmid / system

Method used

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  • Recombinant plasmid for producing oxalate decarboxylase, Escherichia coli expression system, method and application
  • Recombinant plasmid for producing oxalate decarboxylase, Escherichia coli expression system, method and application
  • Recombinant plasmid for producing oxalate decarboxylase, Escherichia coli expression system, method and application

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Effect test

Embodiment 1

[0043] The construction of embodiment 1 expression vector pCOLADuet-A2

[0044] Using the oxalate decarboxylase A2 gene (as shown in the sequence table SEQ ID NO.1) synthesized by the whole gene as a template, design the primer pair F1 / R1, amplify the gene, and perform gel recovery and purification on the amplified product. The method refers to According to the method described in the instructions of the commercially available DNA mini-purification kit, the A2 gene fragment was finally obtained. The PCR system is: 10×PCR Buffer 5μL, 2mM dNTP 5μL, 25mM MgSO 4 5 μL, 1.5 μL each of 10 μM primer F / R, 0.5 μL template DNA, 1 μL KOD-Plus-Neo, ddH 2 O32.5μL; PCR reaction conditions are as follows: 94°C for 3min, 30 cycles (98°C for 10s, 60°C for 30s, 68°C for 35s), 68°C for 5min, 4°C for 10min; The descriptions are the same, and will not be repeated below. The PCR reaction conditions are slightly different, mainly due to the difference in annealing temperature and extension time. ...

Embodiment 2

[0047] The construction of embodiment 2 expression vector pgroESL-A2

[0048]Using the recombinant plasmid pCOLADuet-A2 obtained in Example 1 as a template, design primer F2 / R2 to amplify, and obtain DNA fragment 1 after product purification; (shown) fragment as a template, design primers to amplify F3 / R3, and obtain DNA fragment 2 after product purification; use molecular chaperone plasmid pGro7 plasmid as a template, design primers to amplify F4 / R4, and obtain DNA fragment 3 after purification ; Prepare the following reaction system in an ice-water bath according to the instructions of the seamless cloning kit, connect the above DNA fragments 1, 2 and 3, and transform Escherichia coli DH5α.

[0049] wxya 2 o

Up to 20μl 5×Buffer 4μl DNA Fragment 1 80ng DNA Fragment 2 40ng DNA fragment 3 60ng Recombinase 2μl

[0050] Use the Inoue method to prepare DH5α supercompetent, the method refers to "Molecular Cloning Experiment Gu...

Embodiment 3

[0057] Example 3 Construction of expression vector pOESL-A2

[0058] Using the genomic DNA of Escherichia coli K12MG1655 bacterial strain as template, design primer pair F5 / R5 to amplify OxyR gene and its terminator DNA fragment (as shown in sequence table SEQ ID NO.3); Extract recombinant plasmid pgroESL-A2, use restriction Endonucleases NcoI and EcoRI respectively double-digested the DNA fragments of the OxyR gene and terminator and the pgroESL-A2 plasmid, ligated the above-mentioned double-digested fragments according to the method of the DNA ligase instruction manual, and added 2 μl of the ligated recombinant plasmid to Add 100 μl of DH5α competent cells, place in an ice-water bath for 30 minutes, heat shock in a 42°C water bath for 90 seconds, place in ice for 3 minutes, add 800 μl of antibiotic-free medium, and incubate on a constant temperature shaking shaker at 37°C for 40 minutes. Take 100 μl of the resistant LB solid medium plate containing 50 μg / ml kanamycin for scr...

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Abstract

The invention discloses a recombinant plasmid for producing oxalate decarboxylase, an Escherichia coli expression system, a method and an application. The recombinant plasmid used for producing oxalate decarboxylase comprises: oxalate decarboxylase gene, molecular chaperone gene and gene regulating intracellular manganese ion concentration. In the present invention, the recombinant plasmid is introduced into Escherichia coli expression strain and MntP gene deletion Escherichia coli, and soluble expression and active oxalate decarboxylase can be obtained. The culture temperature and induction temperature of the recombinant strain were optimized, and it was found that the expression of oxalate decarboxylase was higher when the culture temperature was 37°C and the induction temperature was 25°C. The specific activity of the oxalate decarboxylase can reach 45.6U / mg, and the purity exceeds 95% through two simple steps of organic solvent precipitation and ammonium sulfate precipitation. The technical solution of the invention has the advantages of simple production and purification process, high expression level and specific activity of oxalate decarboxylase, easy industrial scale-up, low cost, and is beneficial to industrial production and application of oxalate decarboxylase.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a recombinant plasmid for producing oxalate decarboxylase, an Escherichia coli expression system, a method and an application. Background technique [0002] Oxalic acid is a metabolite of organisms, also known as oxalic acid (Ethanedioic acid), widely distributed in plants, animals and fungi, and exerts different functions in different organisms. Current research has found that at least 100 kinds of plants are rich in oxalic acid, especially in the leaves and seeds of plants such as spinach, amaranth, beet, purslane, taro, tea, cocoa, sweet potato and rhubarb. The bioavailability of elements is easy to form calcium oxalate with calcium ions in the human body and cause kidney stones, so oxalic acid is often considered as an antagonist of the absorption and utilization of mineral elements. Oxalic acid is not easy to be oxidized and decomposed in the human body. The product ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N9/88C12N1/21A23L29/00A61K38/51A61P19/06A61P13/12C12R1/19
CPCA61K38/51A23L29/06A61P13/12A61P19/06C12N9/88C12N15/70C12Y401/01002
Inventor 汪小锋吴玉峰汪卫刘艳红陈火晴
Owner WUHAN KANGFUDE BIOTECH CO LTD
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