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Diaminobutyrate-2-oxoglutarate transaminase and application thereof

A technology of ketoglutarate aminotransferase and diaminobutyric acid, which is applied in the field of genetic engineering and can solve problems such as high price

Inactive Publication Date: 2012-12-12
NANJING TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, L-2,4-diaminobutyric acid has not yet been produced on a large scale. As a fine chemical, it can only be obtained through organic synthesis. It is expensive, and the price is as high as 900 yuan / gram

Method used

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  • Diaminobutyrate-2-oxoglutarate transaminase and application thereof
  • Diaminobutyrate-2-oxoglutarate transaminase and application thereof
  • Diaminobutyrate-2-oxoglutarate transaminase and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1: Extraction of PD-1 genomic DNA from Streptomyces albulus.

[0038] Genomic DNA of Streptomyces albulus PD-1 (Streptomyces albulus PD-1, CCTCC NO: M2011043) in the logarithmic growth phase was extracted with the Genomic DNA Purification Kit (Takara, Dalian) according to the instructions provided by the manufacturer, and extracted with 8g / L agarose gel electrophoresis was used to detect the obtained bacterial genome.

Embodiment 2

[0039] Example 2, the cloning of the gene (dabat) of diaminobutyric acid-2-oxoglutarate aminotransferase and the construction of recombinant bacteria.

[0040] 2.1 PCR amplification of dabat:

[0041] According to the genome sequence of Streptomyces coelicolor A3 (2) published by GeneBank, the following pair of primers were designed:

[0042] Primer 1: 5'-CCG GAATTC ATGACCATCACCCAGCCCGA-3'

[0043] Primer 2: 5'-CCC AAGCTT TCAGGCGCAGTCGCGCACCG-3'

[0044] The underlined part of the 5' end of the above primers was respectively introduced with EcoR I and Hind III restriction sites, and the total DNA of Streptomyces albulus PD-1 (Streptomyces albulus PD-1, CCTCC NO: M2011043) was used as a template for PCR amplification , PCR reaction composition is as follows (25μl system): 2.5μl 10×Ex Taq Buffer, 2μl dNTP, 2.5μl MgCl 2 , 2 μl DMSO, 2 μl template, 2 μl each of primer 1 and primer 2, 0.5 μl Ex Taq, 9.5 μl ddH 2 O, the PCR reaction program is: denaturation at 95°C for 5min, ...

Embodiment 3

[0060] Example 3: Induced expression of diaminobutyric acid-2-oxoglutarate aminotransferase.

[0061] Inoculate the recombinant Escherichia coli BL21-pET-dabat in 5 mL of LB liquid medium supplemented with 25 μg / mL kanamycin, cultivate overnight at 37°C on a shaker; then transfer to In a 500mL shake flask equipped with 100mL LB medium (containing 25μg / mL kanamycin), culture on a shaker at 37°C until the OD600 is about 0.6, add IPTG for induction (final concentration of IPTG 1mmol / L), cool down to 25~ After continuing to express for 12 hours at 30°C, the cells were collected by centrifugation.

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Abstract

The invention discloses diaminobutyrate-2-oxoglutarate transaminase and application thereof. The diaminobutyrate-2-oxoglutarate transaminase has an amino acid sequence shown in SEQ ID NO:2. The invention also discloses an encoding gene of the diaminobutyrate-2-oxoglutarate transaminase, wherein the encoding gene has a nucleotide sequence shown in SEQ ID NO:1. The invention also discloses an expression vector containing the encoding gene of the diaminobutyrate-2-oxoglutarate transaminase, a genetic engineering strain and a construction method thereof. In the invention, an expressed diaminobutyrate-2-oxoglutarate transaminase gene is extracted, sequenced and cloned from Streptomyces albulus for the first time; and the genetic engineering strain is constructed and L-2,4-diaminobutyrate is fermented and produced by utilizing the genetic engineering strain, so that a novel L-2,4-diaminobutyrate acquisition way is opened up.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a diaminobutyric acid-2-ketoglutarate transaminase and an application thereof. Background technique [0002] L-2,4-diaminobutyric acid (L-2,4-diaminobutyric acid, English abbreviation DABA), also known as 2,4-diaminobutyric acid, is a secondary non-protein amino acid, belonging to four carbon compounds, Its structural formula is (NH 2 )CH 2 CH 2 CH(NH 2 )COOH is a basic amino acid. It widely exists in animals, plants and microorganisms, and has unique physiological functions. At present, its research is in its infancy and has not yet been applied on a large scale. Diaminobutyric acid has good application prospects in agricultural and pharmaceutical industries. In terms of agriculture, studies have found that in higher plants, especially in Lathyrus (Lathyrus), the content of free diaminobutyric acid is relatively high. When the external environment i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N1/15C12N1/19C12N1/21C12P13/04
Inventor 徐虹夏军冯小海张扬李莎倪芳欧阳平凯
Owner NANJING TECH UNIV
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