Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A technology of oxalate oxidase and Escherichia coli, applied in biochemical equipment and methods, oxidoreductase, chemical instruments and methods, etc., can solve the problems of complex renaturation process, high cost, inactivity, etc., and achieve simple separation and purification process , The effect of low production cost and high specific vitality

Active Publication Date: 2018-12-18
WUHAN KANGFUDE BIOTECH CO LTD
View PDF6 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Escherichia coli expression system for production of oxalate oxidase, production method of oxalate oxidase and application thereof
  • Escherichia coli expression system for production of oxalate oxidase, production method of oxalate oxidase and application thereof
  • Escherichia coli expression system for production of oxalate oxidase, production method of oxalate oxidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Specific vitalityaaaaaaaaaa
Login to View More

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 E. coli expression system comprises: an oxalate oxidase gene, a chaperone gene, and a gene that promotes to pump manganese ions into E. coli cell-associated protein. By screening and testing the genesof different manganese ion-related proteins and their copy number, the type of a promoter for controlling expression of the molecular chaperone and the type of a culture medium used in the productionprocess of oxalate oxidase, an optimized expression combination of oxalate oxidase is obtained, and the expression activity of oxalate oxidase in E. coli exceeds 80 U / mL. The specific activity of theoxalate oxidase preliminarily purified by a supernatant of a cell disrupted 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 level and specific activity, easy industrial amplification and low cost, and is advantageous for industrial production and application of such enzymes.

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com