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Engineering bacterium based on lignin peroxidase and implementation method of engineering bacterium

A technology of peroxidase and lignin, which is applied in the field of genes and engineering strains in the field of biogenetic engineering technology, can solve the problems of low expression level and difficulty in meeting production needs, and achieve the effect of increasing expression level and facilitating purification

Inactive Publication Date: 2014-12-24
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

[0008] Chinese Patent Document No. CN102703344A Publication (Announcement) Date 2012.10.03 discloses a strain of straw-degrading actinomycetes and its application, but the lignin peroxidase involved in this technology is an expressed enzyme in Streptomyces grisea. In order to induce the enzyme and the expression level is low, it is difficult to meet the production needs

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  • Engineering bacterium based on lignin peroxidase and implementation method of engineering bacterium
  • Engineering bacterium based on lignin peroxidase and implementation method of engineering bacterium
  • Engineering bacterium based on lignin peroxidase and implementation method of engineering bacterium

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Embodiment 1

[0029] This embodiment includes the following steps:

[0030] Step 1) Isolation and cultivation of Streptomyces grisea

[0031] Streptomyces grisea was isolated from rotten straw collected in Pujiang Town, Shanghai, and the preservation number was CGMCC No.5706. The strain was inoculated in LB liquid medium and cultured at 32°C for 48h.

[0032] The above LB liquid medium components are: peptone 10.0g / L, yeast extract 5.0g / L, NaCl 10.0g / L, pH 6.8-7.2. Add 15.0‐20.0g / L agar to the liquid medium to obtain LB solid medium.

[0033] Step 2) Streptomyces grisea genomic DNA extraction

[0034] Genomic DNA Extraction of Streptomyces grisea

[0035] Collect 2.0 mL of bacterial liquid and centrifuge at 12000 rpm for 2 min. Discard the supernatant, collect the bacterial pellet, add 180μL lysozyme (20mg / mL) and 20μL EDTA solution (0.5M, pH8.0), treat at 37℃ for 45min, add 4μL RNase A (100mg / mL), shake and mix for 15s , placed at room temperature for 5 minutes, and then completed th...

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Abstract

The invention relates to an engineering bacterium based on lignin peroxidase and an implementation method of the engineering bacterium. The implementation method comprises the following steps: with DNA of a streptomyces griseorubens genome as a template, carrying out PCR amplification on the DNA of the streptomyces griseorubens genome and a primer containing a restriction site and a glutathione label to obtain a nucleotide sequence for coding the lignin peroxidase; and then connecting the gene sequence obtained by virtue of amplification to an expression vector, and then transferring an obtained ligation product to an escherichia coli expression strain to obtain a lignin peroxidase expression recombination strain. With the adoption of the implementation method, by virtue of methods of constructing a heterogeneous expression vector, optimizing conditions and fusion tags of protein inducing expression and the like, extracellular over expression of the lignin peroxidase is realized.

Description

technical field [0001] The invention relates to a gene in the technical field of biological genetic engineering and its engineering strain, in particular to a lignin peroxidase-based engineering bacterium of Streptomyces grisea and its realization method. Background technique [0002] Lignin is a complex phenolic polymer formed from four alcohol monomers (p-coumaryl alcohol, coniferyl alcohol, 5‐hydroxy coniferyl alcohol, sinapyl alcohol). Lignin is one of the components that make up the plant cell wall and has the function of connecting and strengthening cells. In addition, lignin is also a polycyclic polymer organic compound containing many negatively charged groups, which has a strong affinity for high-valent metal ions in the soil. [0003] According to different monomers, lignin can be divided into 3 types: syringyl lignin (S‐lignin) polymerized from syringylpropane structural monomer, polymerized from guaiacylpropane structural monomer Guaiac-based lignin (G-lignin) ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/02C12N15/53C12N15/70C12R1/19
Inventor 周培冯海玮支月娥孙玉静
Owner SHANGHAI JIAO TONG UNIV
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