Xylosidase-based engineering bacterium and realization method thereof

A technology of xylosidase and engineering bacteria, which is applied in the field of engineering bacteria based on xylosidase, can solve the problems that the environmental suitability of recombinant xylosidase is difficult to meet industrial needs, and achieve wide industrial use, strong tolerance, and improved protein active effect

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

However, the environmental adaptability of recombinant xylosidase obtained by

Method used

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  • Xylosidase-based engineering bacterium and realization method thereof
  • Xylosidase-based engineering bacterium and realization method thereof
  • Xylosidase-based engineering bacterium and realization method thereof

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Embodiment 1

[0029] This embodiment includes the following steps:

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

[0031] Streptomyces griseorubens is isolated from rotten straw collected in Pujiang Town, Shanghai, and its preservation number is 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] 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 op...

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Abstract

The invention relates to a xylosidase-based engineering bacterium. The realization method of the xylosidase-based engineering bacterium comprises the following steps: by taking streptomyces griseorubens genome DNA as a template, performing PCR amplification together with a primer containing restriction enzyme cutting sites and a poly-histidine tag to obtain a nucleotide sequence for coding xylosidase; then connecting the gene sequence obtained by amplification to an expressive carrier, and transforming the obtained connection product into an escherichia coli expression strain to obtain a xylosidase-overexpressed recombinant strain. Aiming at deficiencies that the majority of xylosidase is endoenzyme in a biological body, and low expression quantity causes seriously limited application range and effect in the prior art, a great amount of expression synthesis of the xylosidase in vitro can be realized by applying gene engineering means; in addition, by adopting a method of adding the poly-histidine tag on the C end of the expressive recombinant protein, the purity of the purified xylosidase can be guaranteed while the protein activity is improved; in addition, expression of periplasmic space of a target protein can be realized by a carrier signal peptide, and the activity of the xylosidase can be enhanced.

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 xylosidase-based engineering strain 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. [0003] The hemicellulose component of lignocellulose is a heterogeneous polymer composed of several different types of monosaccharides, namely five- and six-carbon sugars, including xylose, arabinose, mannose, and hemi- lactose etc. Hemicellulose is mainly divided into three categories: polyxylosaccharides, polyglucomannose and polygalactosylglucomannose....

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/42C12N15/56C12N15/70C12P19/14C12P19/02C12R1/19
Inventor 周培冯海玮孙玉静支月娥罗艳青
Owner SHANGHAI JIAO TONG UNIV
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