Escherichia coli expression system for producing oxalate oxidase, production method and application of oxalate oxidase

A technology of oxalate oxidase and Escherichia coli, which is applied in the direction of biochemical equipment and methods, oxidoreductase, chemical instruments and methods, etc., can solve the problems of complex renaturation process, high cost, inactivity, etc., and achieve the production and purification process Simple, low-cost, easy-to-industrial scale-up effect

Active Publication Date: 2021-07-20
WUHAN KANGFUDE BIOTECH CO LTD
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  • Description
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Problems solved by technology

[0008] Aiming at the problems in the prior art that expressing oxalate oxidase 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 new Escherichia coli producing oxalate oxidase An expression system comprising an Escherichia coli strain and corresponding plasmids, which can be used to produce soluble and active oxalate oxidase

Method used

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  • Escherichia coli expression system for producing oxalate oxidase, production method and application of oxalate oxidase
  • Escherichia coli expression system for producing oxalate oxidase, production method and application of oxalate oxidase
  • Escherichia coli expression system for producing oxalate oxidase, production method and application of oxalate oxidase

Examples

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Embodiment 1

[0055] Example 1 Construction of expression vector pGEL-MntH-B10

[0056] Using the B10 gene (sequence shown in SEQ ID NO.1) synthesized from the whole gene as a template, design a primer pair F1 / R1, perform PCR amplification on the gene, and perform gel recovery and purification on the amplified product. The method refers to commercially available DNA According to the method in the instruction manual of the mini-purification kit, DNA fragment 1 (ie, the B10 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, template DNA 0.5 μL, KOD-Plus-Neo 1 μL, ddH2O 32.5 μL; PCR reaction conditions are as follows: 94 °C for 3 min, 30 cycles (98 °C for 10 s, 60 °C for 30 s, 68 °C 35s), 68°C for 5 minutes, and 4°C for 10 minutes; the PCR system in the description of the following vector construction is consistent with the above description, and will not be repeated below. The PCR reaction conditions are slight...

Embodiment 2

[0077] Example 2 Construction of expression vectors pGEL-MntS-B10 and pGEL-OxyR-B10

[0078]Using the genomic DNA of the Escherichia coli K12MG1655 strain as a template, the primer pair F9 / R9 was designed to amplify the DNA fragment of the MntS gene and the terminator (sequence shown in SEQ ID NO.8), and the amplified product was purified by the method of a DNA purification kit Finally, DNA fragment 9 was obtained; using the pGEL-MntH-B10 plasmid extracted by the plasmid mini-extraction kit as a template, primers were designed to amplify F8 / R10, and the product was purified to obtain DNA fragment 10; The above DNA fragments 9 and 10 were ligated, transformed into Escherichia coli DH5α, spread on the resistant LB solid medium plate containing 50 μg / ml karamycin for screening, verified positive clones by PCR and sequencing, and named the correct carrier after sequencing is pGEL-MntS-B10. The system and method of PCR amplification and seamless cloning used in this example are si...

Embodiment 3

[0088] The expression test of embodiment 3 different manganese ion channel proteins

[0089] The plasmid DNA of pET28a-B10, pGEL-B10, pGEL-MntH-B10, pGEL-MntS-B10 and pGEL-OxyR-B10 was extracted by the method of plasmid mini-extraction kit, transformed into Origami2 (DE3) strain, and coated to contain 50 μg / ml karamycin-resistant LB solid medium plate, and positive clones were verified by PCR to obtain recombinant strains pET28a-B10 / Origami2(DE3), pGEL-B10 / Origami2(DE3), pGEL-MntH-B10 / Origami2 (DE3), pGEL-MntS-B10 / Origami2(DE3) and pGEL-OxyR-B10 / Origami2(DE3).

[0090] Inoculate the single clone on the resistant plate into LB liquid seed medium (yeast extract 0.5% (w / v), tryptone 1% (w / v), NaCl 1% (w / v), 20 μg / ml chloride Mycin, 50μg / ml kalamycin) to OD600=1.0-1.2, inoculated to JL medium (yeast extract 0.5% (w / v), tryptone 1.0 %(w / v), KH 2 PO 4 10mM, (NH 4 ) 2 SO 4 20mM, Mannitol 1.2% (w / v), Sodium Succinate 10mM, MgSO 4 0.15mM, initial pH 6.0-7.5), culture temper...

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Abstract

The invention discloses an Escherichia coli expression system for producing oxalate oxidase, a production method of oxalate oxidase and application thereof. The recombinant expression plasmid in the Escherichia coli expression system comprises: an oxalate oxidase gene, a molecular chaperone gene and a gene for promoting manganese ion pumping into Escherichia coli cell-related proteins. The present invention obtains the optimal expression of oxalate oxidase by screening and testing the genes and copy numbers of different manganese ion-related proteins, the type of promoters controlling the expression of molecular chaperones, and the type of medium used in the production process of oxalate oxidase Combined, the expression activity of oxalate oxidase in Escherichia coli exceeds 80U / mL. The specific activity of the oxalate oxidase preliminarily purified from the supernatant of the cell disruption liquid is greater than 20 U / mg, and the molecular weight of the monomer is about 25 kDa. The technical scheme of the invention has the advantages of simple production and purification process, high expression amount and specific activity, easy industrial scale-up, low cost, and favorable industrial production and application of this type of enzyme.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an Escherichia coli expression system for producing oxalate oxidase, a production method of oxalate oxidase and an application thereof. Background technique [0002] Oxalate oxidase (OxOx, EC1.2.3.4) catalyzes the oxidation of oxalate to CO 2 and H 2 o 2 , mainly exists in plants (Hu Yihong et al., Plant Oxalate Oxidase. Chemistry of Life, 2009, 29(2): 295-297), and has great applications in the diagnosis of oxalic acid accumulation-related diseases and the removal of oxalic acid in the body potential. Oxalate oxidase activity has been detected in various plant tissues and microorganisms (Kunal Kumar&Prasanna.D.Belur.A new extracellularthermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6:Purification and biochemical characterization.Preparative Biochemistry and Biotechnology,2016,46(7): 734-739), such as barley, wheat, sugar beet, corn, so...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N9/02C12Q1/26A61K38/44A61P19/06A61P13/12A61P13/04C02F3/34A23L29/00C02F101/34C02F103/28
CPCA61K38/44A23L29/06A61P13/04A61P13/12A61P19/06C02F3/342C02F2101/34C02F2103/28C12N9/0008C12N15/70C12Q1/26C12Y102/03004G01N2333/90203
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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