Engineering bacterium based on glutamate dehydrogenase and implementation method of engineering bacterium

A technology of glutamate dehydrogenase and engineering bacteria, which is applied in the field of engineering bacteria based on glutamate dehydrogenase, can solve the problems of aggravating the eutrophication of water bodies, greenhouse gas emissions, increase the production cost of farmers, etc., and achieve the maintenance of protein activity. , to ensure the effect of protein purity

Inactive Publication Date: 2015-04-08
SHANGHAI JIAO TONG UNIV
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  • Description
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Problems solved by technology

At present, the main method to maintain or increase crop yield is to apply a large amount of fertilizer. However, the excessive application of nitroge...

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  • Engineering bacterium based on glutamate dehydrogenase and implementation method of engineering bacterium
  • Engineering bacterium based on glutamate dehydrogenase and implementation method of engineering bacterium
  • Engineering bacterium based on glutamate dehydrogenase and implementation method of engineering bacterium

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Embodiment 1

[0025] This embodiment includes the following steps:

[0026] Step 1) Isolation and cultivation of Streptomyces griseorubens: Streptomyces griseorubens 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.

[0027] 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.

[0028] Step 2) Genomic DNA extraction of Streptomyces griseorubens: 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 minutes, add 4 μl RNase A (100 mg / mL), shake and mix for 15 seconds, Leave it at room temperature for 5 minutes, and then complete the remaining operations accordin...

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Abstract

The invention relates to a streptomyces griseorubens intracellular glutamate dehydrogenase coding gene and application thereof. The glutamate dehydrogenase coding gene is constructed onto an expression vector, and the recombinant expression vector is introduced into escherichia coli, so that transgenic recombinant escherichia coli capable of producing glutamate dehydrogenase in a fermentation manner is obtained. The defect that application range and effect are seriously restricted as glutamate dehydrogenase is obtained in an intracellular expression manner and expression quantity is low in the prior art is overcome, and in-vitro high expression and synthesis of glutamate dehydrogenase are realized by applying a genetic engineering technique; besides, purification of protein in a late phase is facilitated by adopting a method of adding a histidine tag in recombinant protein. The invention provides a new method for large-scale fermentation production of glutamate dehydrogenase.

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 glutamic acid dehydrogenase-based engineering strain of Streptomyces grisea and its realization method. Background technique [0002] Nitrogen is one of the most important nutrients in the process of plant growth and development. At present, the main method to maintain or increase crop yield is to apply a large amount of fertilizer. However, the extensive application of nitrogen fertilizer not only increases the production cost of farmers, but also aggravates the eutrophication of water bodies and the emission of greenhouse gases. [0003] For crops, the ability to absorb and assimilate NO 3 － , NH 4 + And different nitrogen forms such as ammoniacal N. NO 3 － When nitrogen source is used, nitrogen assimilation can be divided into three stages: (1) inorganic assimilation of nitrogen (NO 3 － -NO 2 -NH 4 + ); ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P13/14C12R1/19
CPCC12N9/0016C12P13/14C12Y104/01002
Inventor 周培冯海玮王大欣俞明磊毛亮唐冬
Owner SHANGHAI JIAO TONG UNIV
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