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Engineering bacterium based on extracellular laccase and implementation method thereof

A technology for engineering bacteria and laccase, which is applied in the field of engineering bacteria based on extracellular laccase, can solve the problems of low yield and difficulty in meeting production requirements, and achieve the effects of stabilizing enzymatic properties and improving efficiency and purity.

Inactive Publication Date: 2014-12-24
SHANGHAI JIAO TONG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the output of this technology is low and it is difficult to meet the production demand

Method used

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  • Engineering bacterium based on extracellular laccase and implementation method thereof
  • Engineering bacterium based on extracellular laccase and implementation method thereof
  • Engineering bacterium based on extracellular laccase and implementation method thereof

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Embodiment 1

[0037] This embodiment includes the following steps:

[0038] Step 1) Isolation and cultivation of Streptomyces griseorubens: Streptomyces griseorubens is isolated from rotten straw collected in Pujiang Town, Shanghai, and the preservation number is CGMCC No.5706. The strain was inoculated in LB liquid medium and cultured at 32°C for 48h.

[0039] 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.

[0040] Step 2) Genomic DNA extraction of Streptomyces griseorubens: 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 oper...

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Abstract

The invention relates to an engineering bacterium based on extracellular laccase. The implementation method comprises the following steps: by using DNA of a streptomyces griseorubens genome as a template, carrying out PCR (polymerase chain reaction) amplification on the template and a primer containing an enzyme cutting site and a polyhistidine label to obtain a nucleotide sequence which encodes the extracellular laccase; and then, connecting the gene sequence obtained by amplification to an expression vector, and then transferring the obtained connecting product into an escherichia coli expression strain to obtain an extracellular laccase over-expressed recombinant strain. Aiming at the defects in the prior art that the application range and effect are severely limited because most in-vivo extracellular laccase is intracellular enzyme and has a relatively low expression quantity, massive in-vitro expression and synthesis are achieved by applying genetic engineering means. In addition, by virtue of a method of adding the polyhistidine label to the terminal N of an expression recombinant protein, the purity of purified extracellular laccase is ensured while the activity of the protein is improved. Moreover, periplasmic space expression of a target protein is achieved by virtue of a vector signal peptide, so that the activity of the extracellular laccase is improved.

Description

technical field [0001] The invention relates to a gene in the technical field of biogenetic engineering and its engineering strain, in particular to an engineering bacterium based on extracellular laccase 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 polymer...

Claims

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Application Information

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