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

Method for improving activity of dye decolorizing peroxidase

A technology for decolorization of peroxidase and dyes, applied in the field of enzyme engineering, can solve the problems of inconvenient operation, increased cost, etc., and achieve the effect of improving the specific enzyme activity

Pending Publication Date: 2020-08-28
JIANGNAN UNIV
View PDF11 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Previous studies mainly increased the supply of prosthetic groups by adding heme or 5-aminolevulinic acid (ALA) from an external source, but this method would cause increased costs and inconvenience in operation

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
  • Method for improving activity of dye decolorizing peroxidase
  • Method for improving activity of dye decolorizing peroxidase
  • Method for improving activity of dye decolorizing peroxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction and Growth Detection of Knockout Bacteria WT△eamA

[0025] Using Escherichia coli BL21 (DE3) as the starting strain WT, the upstream and downstream homology arms of 500 bp were respectively amplified in the eamA gene (reference GenBank: AM946981.2: 1569528-1570427). Using the pKD4 plasmid as a template, the Kan resistance sequence was amplified. Linearize the pET-22b plasmid with double enzyme digestion and use Novizyme The Multis One Step Cloning Kit connects the 500bp homology arms of the upstream and downstream and the three fragments of the Kan resistance sequence to the vector pET-22b, and the ligated products are transferred to the competent state of E.coliJM109, and screened on the LB plate added with Amp and Kan The positive bacteria obtained the plasmid pEUKD. Using pEUKD as a template, PCR amplification was performed to obtain a targeting fragment containing upstream and downstream homology arms of the eamA gene and Kan resistance.

...

Embodiment 2

[0027] Example 2 Construction of Engineering Bacteria WT△eamA / pA and Determination of Heme Content

[0028] The glutamyl-tRNA reductase hemA gene (GenBank: AM946981.2: 1251856-1253112) was connected to the pET28a vector and introduced into the knockout bacteria WT△eamA. The specific import method was to transfer the connected product to Into the competent cell WT△eamA: Add the ligation product to the competent cell melted after ice bath, mix well, and let it stand on ice for 30 minutes; heat shock in 42°C water bath for 90 seconds, quickly put it on ice for 5 minutes, and then add 800 μL of LB liquid medium; 37°C shaker 200r min -1 , recovered for 45 minutes, centrifuged, and the centrifuged bacterial solution was spread on the LB plate added with Kan, and the engineered bacteria WT△eamA / pA was screened, and the hemoglobin concentration of WT and WT△eamA / pA was detected by fluorescence method, specifically: : Cultivate WT and WT△eamA / pA on LB medium at 37°C and 200r / min for 8...

Embodiment 3

[0029] Example 3 Construction, expression and recombinase enzyme activity determination of Escherichia coli genetically engineered bacteria WT△eamA / pAD

[0030] The hemA gene encoding glutamyl-tRNA reductase (GenBank: AM946981.2:1251856-1253112) was combined with the gene encoding dye decolorization peroxidase DyP (GenBank: AAZ57111.1) to obtain the recombinant vector pAD. Introduce the recombinant vector pAD into WT and WT△eamA respectively to obtain Escherichia coli genetically engineered bacteria WT / pAD and WT△eamA / pAD, and activate and culture the above-mentioned Escherichia coli genetically engineered bacteria at 30-37°C and 200r / min for 8-14h Afterwards, according to the 1-4% inoculum size, transfer to culture in the LB liquid medium containing 50 μ g / mL Kan antibiotic, when the OD of the bacterium 600 =0.6-0.8, add IPTG at a final concentration of 0.1-0.5mmol / L, and induce at 30-37°C for 8h. Suspend the cells with phosphate buffer (20mmol / L, pH=7.4), sonicate the cells...

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

No PUM Login to View More

Abstract

The invention discloses a method for improving the activity of dye decolorizing peroxidase, and belongs to the field of enzyme engineering. The method comprises the following steps: jointly connectinga gene for coding dye decolorizing peroxidase (DyP) and a gene hemA for coding escherichia coli endogenous glutamyl-tRNA reductase to a vector pET28a to obtain a recombinant vector, and transferringthe recombinant vector into escherichia coli from which coding escherichia coli endogenous channel protein gene eamA is knocked out for expression. A result shows that the specific enzyme activity ofthe expressed recombinant DyP is remarkably improved. The method is simple, convenient and economical, and has application value.

Description

technical field [0001] The invention relates to a method for improving the activity of dye decolorizing peroxidase, which belongs to the technical field of enzyme engineering. Background technique [0002] Dye decolorization peroxidase (DyP) is a newly discovered peroxidase in recent years. It uses heme as a prosthetic group in organisms and participates in the regulation of oxidative stress and matrix degradation in organisms. Because the enzyme can degrade anthraquinone dyes and lignin, it can be used in wastewater treatment and reuse of biological waste, so it has become a research hotspot. [0003] In the heterologous expression of DyP, the lack of prosthetic heme often leads to a low level of enzyme activity. Escherichia coli (E.coli), as an important genetically engineered host bacterium, mainly uses glutamic acid as a precursor to synthesize heme through the C5 pathway ( figure 1 ). Glutamate synthesizes 5-aminolevulinic acid (ALA) under the catalysis of glutamyl-t...

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): C12N15/70C12N15/53C12N1/21C12N9/08C12R1/19
CPCC12N15/70C12N9/0065C12N9/0008C12Y111/01019C12Y102/0107
Inventor 唐蕾潘梅郑珂
Owner JIANGNAN UNIV
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