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Engineering bacteria based on beta-glucosidase and implementation method thereof

A technology of glucosidase and engineering bacteria, which is applied to engineering bacteria based on β-glucosidase and its realization field, and can solve problems such as insufficient development and utilization

Inactive Publication Date: 2014-06-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As an actinomycete, previous research on Streptomyces grisea has 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 relatively insufficient

Method used

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  • Engineering bacteria based on beta-glucosidase and implementation method thereof
  • Engineering bacteria based on beta-glucosidase and implementation method thereof
  • Engineering bacteria based on beta-glucosidase and implementation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Cultivation of Streptomyces griseorubens JSD‐1

[0033] Streptomyces griseorubens 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)

[0034] 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.

[0035] 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

[0037] Cloning of the β-glucosidase (Sg-Gcd) Gene of Streptomyces grisea

[0038] Through the analysis of the β-glucosidase gene sequence of Streptomyces grisea, the primers containing NdeI and EcoRI restriction sites were designed as follows:

[0039] Forward primer Gcd-NdeI-F, as shown in SEQ ID No.3:

[0040] 5'-GGAATTCCATATGATCCGGGCCATGGCGACGACGAACCC-3'

[0041] Reverse primer Gcd-EcoRI-R, as shown in SEQ ID No.4:

[0042] 5'-GGAATTCTCAGTCCCGCTGCTCCCGCAGCAGCGCGCGGT-3'

[0043] 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

[0045] Construction of cloning vector containing β-glucosidase (Sg-Gcd) gene

[0046] The PCR amplification product is carried out electrophoresis detection, and the result shows that the obtained fragment is about 1.3Kb (see figure 1 ); 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.

[0047] Pick clones with corresponding resistance and identify them by colony PCR until positive clones are 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 provides engineering bacteria based on beta-glucosidase in the technical field of bioengineering and an implementation method thereof. A beta-glucosidase gene is cloned from Streptomyces griseorubens (Streptomyces griseorubens) JSD-1, and then is connected to an expression vector to obtain a recombinant expression vector, and the recombinant expression vector is further introduced into escherichia coli and is induced to synthesize beta-glucosidase. The engineering bacteria provided by the invention overcomes the defects that the beta-glucosidase in the prior art is generally endoenzyme and the expression quantity is very low, and the like, the beta-glucosidase gene of the invention is expressed by genetically engineered bacteria to prepare beta-glucosidase, and a new method is provided for producing beta-glucosidase by 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 β-glucosidase (Sg-Gcd) 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 2×1018kJ, which is equivalent to 10 times the annual energy consumption in the world. 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...

Claims

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Application Information

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