Engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof

A technology of furanosidase and engineering bacteria, applied in the field of biological genetic engineering, can solve the problems of insufficient development and utilization

Inactive Publication Date: 2014-06-18
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 grisea has focused on how to modify its metabolic pathways to increase the fermentation yield of antib

Method used

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  • Engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof
  • Engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof
  • Engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cultivation of Streptomyces griseus JSD-1

[0035] 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)

[0036] 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.

[0037] 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

[0039] Cloning of Arabinofuranosidase (Sg-Abl) Gene of Streptomyces grisea

[0040] Through signal peptide sequence analysis of Streptomyces grisea arabinofuranosidase 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:

[0041] Forward primer Abl-NdeⅠ-F:

[0042] 5'-GGAATTCCATATGGCGACCGTGGACACGAACGCCTCGTAC-3'

[0043] Reverse primer Abl-EcoRI-R:

[0044] 5'-GGAATTCTCAGCGCCGCAGCGTCAGCAGACC-3'

[0045] Genomic DNA of Streptomyces grisea 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

[0047] Construction of Cloning Vector Containing Arabinofuranosidase (Sg-Abl) Gene

[0048] The PCR amplification product is carried out electrophoresis detection, and the result shows that the obtained fragment is about 1.4Kb (see figure 2 ); Then, after the PCR product was cut and recovered, the A-adding reaction was performed through the A-Tailing Kit (TaKaRa) and connected to pMD TM 19-T Vector (TaKaRa), then introduced into DH5α Escherichia coli competent cells.

[0049] Clones with corresponding resistance were picked and identified by colony PCR until positive clones were obtained. Pick positive clones, extract their plasmids by shaking bacteria and send them to Shanghai Sonny Biological Co., Ltd. for sequencing.

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Abstract

The invention relates to an engineering bacteria based on extracellular Alpha-L-arabinofuranosidase and implementation method thereof, belonging to a field of bio-engineering technology. The arabinofuranosidase is prepared by cloning arabinofuranosidase gene from Streptomyces griseus JSD-1, linking the arabinofuranosidase gene with an expression vector to obtain a recombinant expression vector, further introducing the recombinant expression vector into E. coli and carrying out induced synthesis. The deficiencies of induced expression and low expression levels in prior art are overcome and a new method is provided for the industrial fermentative production of Alpha-L-Arab furan-glucosidasethe by applying gene engineering bacteria to the expression of the arabinofuranosidase prepared by the gene according to the invention.

Description

technical field [0001] The present invention relates to a gene in the technical field of biological genetic engineering and its realization method, in particular to an E. coli engineering bacterium capable of exogenously expressing extracellular α-L-arabinofuranosidase (Sg-Abl) 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 ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N9/42C12N15/56C12R1/19C12R1/465
Inventor 周培冯海玮支月娥孙玉静初少华罗艳青
Owner SHANGHAI JIAO TONG UNIV
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