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Eextracellular-polysaccharide-hydrolase-based engineering bacteria and implementation method thereof

A technology of exopolysaccharide and hydrolase, applied in the field of Escherichia coli engineering bacteria and its realization, can solve the problems of insufficient development and utilization

Inactive Publication Date: 2014-06-04
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous research on Streptomyces griseus focused on how to modify its metabolic pathways to increase the fermentation yield of antibiotics, especially streptomycin, and the development and utilization of other functional genes in its genome are far from enough

Method used

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  • Eextracellular-polysaccharide-hydrolase-based engineering bacteria and implementation method thereof
  • Eextracellular-polysaccharide-hydrolase-based engineering bacteria and implementation method thereof
  • Eextracellular-polysaccharide-hydrolase-based engineering bacteria and implementation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cultivation of Streptomyces griseus JSD-1

[0034] Streptomyces griseus was isolated from the rotting soil of Pujiang Town, Shanghai, and the preservation number is CGMCC No.5706; the strain was preserved in the Chinese Academy of Microbiology Research Center of General Microbiology, Chinese Academy of Sciences on January 9, 2012 Office (Address: No. 3, No. 1 Yard, Beichen West Road, Chaoyang District, Beijing 100101)

[0035] The bacteria were inoculated in LB liquid medium, cultured at 32°C for 60 hours, the bacteria were collected by centrifugation and the genomic DNA of the bacteria was extracted.

[0036] The components of the LB liquid medium are: 10 g of peptone, 5 g of yeast extract, 5 g of NaCl, 1 L of deionized water, and pH 6.8-7.2.

Embodiment 2

[0038] Cloning of Polysaccharide Hydrolase (Sg-Hyd) Gene of Streptomyces griseus

[0039] By analyzing the signal peptide sequence of Streptomyces griseus polysaccharide hydrolase gene sequence (such as figure 1 ), the primers containing NdeI and EcoRI restriction sites were designed at both ends of the coding gene sequence of the mature protein as follows:

[0040] Forward primer Hyd-NdeⅠ-F:

[0041] 5'-GGAATTCCATATGGCCGTCTGGAACTCCTGCGACCAGTGG-3'

[0042] Reverse primer Hyd-EcoRI-R:

[0043] 5'-GGAATTCTCAGCCGCCCGAGACGGTCAGGCTGTC-3'

[0044] Genomic DNA of Streptomyces griseus JSD-1 was used as a template and PrimeSTAR GXL high-fidelity enzyme (TaKaRa) was used for amplification. The PCR amplification conditions were: pre-denaturation at 98°C for 3 min; deformation at 98°C for 10 s, extension at 68°C for 1 min; and final extension at 68°C for 3 min after 30 cycles.

Embodiment 3

[0046] Construction of Cloning Vector Containing Polysaccharide Hydrolase (Sg-Hyd) Gene

[0047] PCR amplified product is carried out electrophoresis detection, and the result shows that obtaining fragment is about 700bp (see figure 2 ); then the PCR product was gel-cut and recovered and connected to pEASY-Blunt Simple Cloning Vector (Beijing Quanshijin), and then introduced into DH5α Escherichia coli competent cells.

[0048] Clones with corresponding resistance were picked and identified by colony PCR until positive clones were obtained. Pick positive clones, shake the bacteria to extract their plasmids, and send them to Shanghai Sonny Biological Co., Ltd. for sequencing.

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Abstract

The invention discloses extracellular-polysaccharide-hydrolase-based engineering bacteria and an implementation method thereof in the technical field of biological engineering. A polysaccharide hydrolase gene is cloned from streptomyces griseus JSD-1; the streptomyces griseus JSD-1 is connected to an expression vector to obtain a recombinant expression vector; the recombinant expression vector is further introduced into escherichia coli and induced to synthesize polysaccharide hydrolase. According to the extracellular-polysaccharide-hydrolase-based engineering bacteria, the defects of low expression quantity and the like caused by inducible expression of most of polysaccharide hydrolases in the prior art are overcome, the polysaccharide hydrolase is prepared by using the polysaccharide hydrolase gene expressed by genetic engineering bacteria, and a new method for producing the polysaccharide hydrolase can be provided by virtue of industrial fermentation.

Description

technical field [0001] The invention relates to a gene in the technical field of biogenetic engineering and its realization method, in particular to an Escherichia coli engineering bacterium capable of exogenously expressing extracellular polysaccharide hydrolase (Sg-Hyd) and its realization method. Background technique [0002] Polysaccharide hydrolase is a class of enzymes that can hydrolyze glycosidic bonds in polysaccharide molecules. Common ones include amylase, xylanase, chitinase, cellulase, lysozyme, pectinase, and β-glucan Carbohydrases and β-galactosidases, etc., polysaccharide hydrolases are found in almost every organism. [0003] In recent years, a large number of studies on polysaccharide hydrolases have been carried out. At the same time, various polysaccharide hydrolases are also widely used in feed, food, medicine, catering, papermaking and textile industries and play an important role. Therefore, the development of new high-efficiency polysaccharide hydrol...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N9/42C12N15/56C12N15/70C12R1/19
Inventor 周培冯海玮支月娥初少华罗艳青孙玉静
Owner SHANGHAI JIAO TONG UNIV
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