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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: 2011-09-14
NANJING UNIV OF TECH
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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
Comparison scheme
Effect test

Embodiment 1

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

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

Embodiment 2

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

[0040] 2.1 Dabat PCR amplification:

[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] Introduce the EcoR I and Hind III restriction sites into the underlined part of the 5'end of the above primers, and use the total DNA of Streptomyces albulus PD-1 (Streptomyces albulus PD-1, CCTCC NO: M2011043) 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 , 2μl DMSO, 2μl template, 2μl each of primer 1 and primer 2, 0.5μl Ex Taq, 9.5μl ddH 2 O, the PCR reaction program is: denaturation at 95°C for 5 minutes, then 94°C for 30s, 58°C for 60s, 72°C for 90s,...

Embodiment 3

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

[0061] Recombinant E. coli BL21-pET-dabat was inoculated into 5 mL of LB liquid medium supplemented with 25 μg / mL kanamycin, cultured in a shaker at 37°C overnight; then transferred to 5% (v / v) inoculum In a 500mL shake flask containing 100mL LB medium (containing 25μg / mL kanamycin), culture in a shaker at 37°C until the OD600 is about 0.6, add IPTG for induction (IPTG final concentration 1mmol / L), and cool 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 specifically relates to a diaminobutyric acid-2-ketoglutarate transaminase and its application. Background technique [0002] L-2,4-diaminobutyric acid (L-2,4-diaminobutyric acid, English abbreviation DABA), also known as 2,4-butanediine, is a secondary non-protein amino acid that belongs to a four-carbon compound. Its structural formula is (NH 2 )CH 2 CH 2 CH(NH 2 ) COOH is a basic amino acid. It is widely present 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 agriculture and pharmaceutical industries. In agriculture, studies have found that in higher plants, especially Lathyrus (Lathyrus), the content of free diaminobutyric acid is relatively high. When the external environment is bad, the content of diaminobu...

Claims

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Application Information

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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 UNIV OF TECH
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