Recombinant plasmid for production of oxalate decarboxylase, system and method for expressing escherichia coli, and application

A technology of oxalate decarboxylase and Escherichia coli, applied in the biological field, can solve the problems of complex renaturation process, high cost, inactivity and the like, and achieve the effects of simple separation and purification process, low production cost and high specific activity

Active Publication Date: 2018-12-11
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 production of oxalate decarboxylase, system and method for expressing escherichia coli, and application
  • Recombinant plasmid for production of oxalate decarboxylase, system and method for expressing escherichia coli, and application
  • Recombinant plasmid for production of oxalate decarboxylase, system and method for expressing escherichia coli, and application

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Experimental program
Comparison scheme
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. PCR system: 10×PCR Buffer 5μL, 2mMdNTP 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; They are all the same, and will not be described in detail below. The PCR reaction conditions are slightly different, mainly due to the difference in annealing temperature and extension time. Extra...

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, obtain DNA fragment 2 after product purification; use molecular chaperone plasmid pGro7 plasmid as template, design primers to amplify F4 / R4, 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 Exp...

Embodiment 3

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

[0058] Using the genomic DNA of Escherichia coli K12MG1655 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 screening, an...

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Abstract

The invention discloses a recombinant plasmid for production of oxalate decarboxylase, a system and a method for expressing escherichia coli, and application. The recombinant plasmid for production ofoxalate decarboxylase comprises an oxalate decarboxylase gene, a molecular chaperone gene and a gene for regulating and controlling intracellular manganese ion concentration. The recombinant plasmidis guided into an escherichia coli expression bacterial strain and the escherichia coli with MntP gene deletion to respectively obtain oxalate decarboxylase with solubility expression and activity; the culture temperature and induction temperature of the recombinant bacterial strain are optimized; when the culture temperature is 37 DEG C, and the induction temperature is 25 DEG C, the expression amount of the oxalate decarboxylase is higher; by utilizing the two steps of organic solvent precipitation and ammonia sulfate precipitation to simply purify, the specific activity of the oxalate decarboxylase can reach 45.6U / mg, and the activity is higher than 95%. By adopting the technical scheme, the recombinant plasmid has the advantages that the production and purifying technologies are simple; the expression amount and specific activity of the oxalate decarboxylase are higher, the industrialized amplification is easy, the cost is low, and the recombinant plasmid is suitable for the industrialized production and application of the 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 Applications(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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