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
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Previous studies mainly increased the supply of prosthetic groups by adding heme or 5-aminolevulinic a

Method used

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  • 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
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Example Embodiment

[0024] Example 1 Construction and growth detection of knockout bacteria WT△eamA

[0025] Using Escherichia coli BL21 (DE3) as the starting strain WT, each of the 500bp homology arms of the upstream and downstream of the eama gene (reference GenBank: AM946981.2:1569528-1570427) was amplified. Using the pKD4 plasmid as a template, the Kan resistance sequence was amplified. Linearize the pET-22b plasmid with double enzyme digestion, using Novartis Multis One Step CloningKit connects the upstream and downstream 500bp homology arms and Kan resistance sequence 3 fragments to the vector pET-22b, the ligation product is transferred to the competent E.coli JM109, and it is screened on the LB plate with the addition of Amp and Kan The positive bacteria get the plasmid pEUKD. Using pEUKD as a template, PCR amplification was performed to obtain a targeting fragment containing the upstream and downstream homology arms of the eamA gene and Kan resistance.

[0026] Using the targeting fragment...

Example Embodiment

[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 ligated to the pET28a vector and introduced into the knockout bacterium WT△eamA. The specific introduction method was to use heat shock to transfer the ligated product Into the competent cells WT△eamA: add the ligation product to the competent cells that have melted after an ice bath, mix, and let stand on ice for 30 minutes; heat shock in a 42℃ water bath for 90 seconds, quickly place on ice for 5 minutes, and then add 800 μL of LB liquid medium; 37℃ shaker 200r·min -1 , Resuscitate for 45 minutes, centrifuge, take the centrifuged bacterial solution and spread it on the LB plate with Kan, and screen the engineered bacteria WT△eamA / pA, and use the fluorescence method to detect the heme concentration of WT and WT△eamA / pA, specifically :Cultivate WT and WT△eamA / pA on LB medium at 37℃, 200r / min fo...

Example Embodiment

[0029] Example 3 Construction and expression of E. coli genetically engineered strain WT△eamA / pAD and determination of recombinase activity

[0030] The hema gene (GenBank: AM946981.2: 1251856-1253112) encoding glutamyl-tRNA reductase and the gene encoding dye decoloring peroxidase DyP (GenBank: AAZ57111.1) were used to obtain the recombinant vector pAD. The recombinant vector pAD was introduced into WT and WT△eamA respectively to obtain E. coli genetically engineered bacteria WT / pAD and WT△eamA / pAD. The above-mentioned E. coli genetically engineered bacteria were activated and cultured at 30-37℃, 200r / min for 8-14h Then, transfer to LB liquid medium containing 50μg / mL Kan antibiotics according to the inoculum amount of 1-4%. When the OD of the bacteria is 600 When =0.6-0.8, add IPTG at a final concentration of 0.1-0.5mmol / L and induce 8h at 30-37℃. The cells were suspended in phosphate buffer (20mmol / L, pH=7.4), the cells were disrupted by ultrasound for 20min, and centrifuged a...

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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

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Application Information

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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
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