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Engineering bacteria based on manganese peroxidase and implementation method of engineering bacteria

A technology of manganese peroxidase and engineering bacteria, which is applied in the field of genes and engineering strains in the field of biogenetic engineering technology, can solve the problems of limited application range and low yield, and achieve convenient purification, stable enzymatic characteristics, and improved Effects of Efficiency and Purity

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

[0007] After searching the prior art, it was found that Chinese Patent Document No. CN102703344A was published (announced) on 2012.10.03, disclosing a strain of straw-degrading actinomycetes and its application, but the manganese peroxidase produced by this technology is an intracellular enzyme , the yield is low so the application range is limited

Method used

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  • Engineering bacteria based on manganese peroxidase and implementation method of engineering bacteria
  • Engineering bacteria based on manganese peroxidase and implementation method of engineering bacteria
  • Engineering bacteria based on manganese peroxidase and implementation method of engineering bacteria

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Embodiment 1

[0034] This embodiment includes the following steps:

[0035] Step 1) Isolation and cultivation of Streptomyces grisea

[0036] 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.

[0037] 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.

[0038] Step 2) Streptomyces grisea genomic DNA extraction

[0039]Collect 2.0 mL of bacterial liquid and centrifuge at 12000 rpm for 2 min. Discard the supernatant, collect the bacterial pellet, add 180 μL lysozyme (20 mg / mL) and 20 μL EDTA solution (0.5M, pH 8.0), treat at 37 °C for 45 min, add 4 μL RNase A (100 mg / mL), shake and mix for 15 s, Leave it at room temperature for 5 minutes, and then complete the remaining operations according to the operati...

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Abstract

The invention provides engineering bacteria based on manganese peroxidase in the field of the genetic engineering and an implementation method of the engineering bacteria. The genome DNA of the streptomyces griseorubens is taken as a template, and PCR amplification is performed on the template and a primer containing an enzyme digestion site and a polyhistidine label so that the nucleotide sequence coding the manganese peroxidase can be obtained; next, the gene sequence obtained by amplification is connected to an expression vector, and then the obtained connection product is transferred into an escherichia coli expressed strain, and finally, the over-expressed recombinant strain of the manganese peroxidase is obtained. The engineering bacteria based on the manganese peroxidase are used for overcoming the shortage of seriously restricted application range and application effect because the manganese peroxidase mostly is an endoenzyme in vivo and is low in expression amount, and in-vitro mass expression and synthesis of the manganese peroxidase are realized by use of a genetic engineering method; besides, the purity of the purified manganese peroxidase is also guaranteed while the protein activity is improved by use of the method of adding the polyhistidine label to the C end of the expressed recombinant protein; in addition, the periplasmic space expression of the target protein is realized by use of a vector signal peptide and the activity of the manganese peroxidase is improved.

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 manganese peroxidase-based engineering bacterium of Streptomyces grisea and its realization method. Background technique [0002] Lignocellulose is the most abundant natural polymer compound in the plant kingdom, and it is the main dry matter produced by plants through photosynthesis, mainly including cellulose, hemicellulose and lignin. The biodegradation and depolymerization of lignocellulose is a highly complex process involving the participation of numerous enzyme systems. Lignin in lignocellulose 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 poly...

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Application Information

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