Engineering bacterium based on exoglucanase and realization method thereof

A technology of exoglucanase 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 that the enzymatic properties are difficult to meet industrial needs, and achieve the effect of convenient purification and increased expression

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 enzymatic properties of the engineered bacteria under alkaline conditions are difficult to meet the needs of existing industries

Method used

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  • Engineering bacterium based on exoglucanase and realization method thereof
  • Engineering bacterium based on exoglucanase and realization method thereof
  • Engineering bacterium based on exoglucanase and realization method thereof

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Embodiment 1

[0027] This embodiment includes the following steps:

[0028] Step 1) Isolation and cultivation of Streptomyces grisea

[0029] 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.

[0030] 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.

[0031] Step 2) Streptomyces grisea genomic DNA extraction

[0032] Genomic DNA Extraction of Streptomyces grisea

[0033] 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 c...

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Abstract

The invention provides an engineering bacterium based on exoglucanase and a realization method thereof in the field of gene engineering. The method comprises the following steps: carrying out PCR amplification on DNA of a streptomyces griseorubens genome serving as a template and primers containing cleavage sites and glutathione tags to obtain a nucleotide coding exoglucanase; then, connecting the gene sequence obtained by amplification to an expression vector; and transferring the obtained connecting product to an escherichia coli expressed strain to obtain over-expressed recombinant strain of exoglucanase. According to the method, extracellular overexpression of exoglucanase is realized by methods of constructing a heterogenous expression vector, optimizing protein induced expression conditions and fusing tag and the like.

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 of Streptomyces grisea based on extracellular exoglucanase 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. It is estimated that the total dry weight of lignocellulose produced by photosynthesis of green plants in the world is 173 billion tons every year, and the total energy contained can reach 2×10 18 kJ, which is equivalent to 10 times the energy consumed by the whole world every year. The biodegradation and depolymerization of lignocellulose is a highly complex process involving the participation of numerous enzyme systems. [0003] The cellulose component in lignocellulose is a macr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12N9/42C12R1/19
Inventor 周培冯海玮支月娥孙玉静罗艳青
Owner SHANGHAI JIAO TONG UNIV
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