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Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method

A technology of aminolevulinic acid, engineering bacteria, applied in the directions of microorganism-based methods, genetic engineering, biochemical equipment and methods, etc.

Inactive Publication Date: 2007-06-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no report of using the 5-aminolevulinic acid synthase gene of Agrobacterium radiatus to produce ALA

Method used

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  • Engineering bacteria for producing 5-amino acetyl propionic acid and its constructing method

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Experimental program
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Effect test

Embodiment 1

[0020] The method for constructing an engineered strain for producing 5-aminolevulinic acid of the present invention includes the following steps:

[0021] 1. Extraction of Genomic DNA of Agrobacterium radiata

[0022] 1) Place the overnight cultured bacterial solution in a 1.5ml centrifuge tube, centrifuge at 13,000×g for 1 min, and then remove the supernatant;

[0023] 2) After dissolving with 475μl TE buffer, add lysozyme to a final concentration of 100μg / ml, and keep it at 37°C for 20min;

[0024] 3) Add 10% (w / v) sodium dodecyl sulfonate (SDS) solution to a final concentration of 2% (w / v), and add 20 mg / ml proteinase K to a final concentration of 100 μg / ml. After mixing, Incubate at 37°C for 1h;

[0025] 4) Add 1 / 5 volume of 5mol / L NaCl and mix well, then add 1 / 5 volume of 2×hexadecyltrimethylammonium bromide (CTAB), and keep it at 65°C for 30min;

[0026] 5) Add an equal volume of a mixture of phenol, chloroform and isoamyl alcohol, the volume ratio of phenol: chloroform: is...

Embodiment 2

[0047] The expression of 5-aminolevulinic acid synthase under the action of the inducer isopropyl-β-D-thiogalactoside (IPTG)

[0048] 1) Cultivate the engineered bacteria BL21(DE3)pET28a-AR-hemA of the present invention in a 250ml shake flask containing 50ml of LB medium, first incubate at 37℃, 200rpm for 1.5h; then cool to 28℃, use 4μl1mol / L IPTG induction, after continuing to culture for 4h, take 30ml of bacterial solution and centrifuge at 8000×g for 10min to remove the supernatant;

[0049] 2) Resuspend in 3ml of 50mmol / L Tris·Cl (pH 7.5), sonicate the cells, and the conditions are 400w working for 5min, intermittent 5min, repeating 30 times, 13,000×g centrifugation for 10min;

[0050] 3) Take the supernatant for 10% SDS-polyacrylamide gel electrophoresis, the electrophoresis conditions are 200v, 500mA, and the time is 1h10min;

[0051] 4) According to the results of electrophoresis, there is an expression of 23.7% of the total protein at about 44.0kDa (see Figure 2). This si...

Embodiment 3

[0053] Determination of Specific Activity of 5-Aminolevulinic Acid Synthase

[0054] 1) 500μl containing 50mmol / L Tris-HCl (pH7.5), 20mmol / L MgCl 2 , 0.1mol / L glycine, 0.1mmol / L pyridoxal phosphate, 0.2mmol / L succinate coenzyme A and the cell extract after rupture, react at 37℃ for 10min;

[0055] 2) Take 300μl of the above reaction solution and 150μl of 10% trichloroacetic acid, mix in a 1.5mL centrifuge tube, and centrifuge at 13,000×g for 5 minutes;

[0056] 3) Take 300μl of supernatant and mix 400μl of 1mol / L acetate buffer (pH 4.6) and 35μl of acetylacetone, and bath in boiling water for 15min;

[0057] 4) After cooling, add 700μl of newly prepared Ehrlich reagent (in a 50ml graduated cylinder, add 30ml of glacial acetic acid, 1g of p-dimethylaminobenzaldehyde, 5ml of 70% perchloric acid, 5ml of water, and dissolve it with Dilute with glacial acetic acid to 50ml), measure the absorbance at 554nm after 5min reaction, the concentration of ALA (mg / L)=26.35×OD 554 -0.09(R 2 =0.99...

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Abstract

An engineered bacterium able to generate 5-aminolevulic acid is disclosed, which contains the 5-aminolevulic acid, synthetase gene of radial Agrobacterium. Its configuring process includes such steps as extracting genom DNA from Agrobacterium liquid, PCR amplification of 5-aminoevulic acid synthetase gene, linking it with carrier pGEM-T, sequencing linking sequenced target gene fragment with expression carrier pET28 a to configue recombination pET28a-A.R-hemA, and transfering it in host.

Description

Technical field [0001] The invention relates to an engineered bacteria producing 5-aminolevulinic acid and a construction method thereof. Background technique [0002] 5-aminolevμlinic acid (ALA) is widely present in organisms. It is the first complex in the biosynthetic pathway of porphyrin and also forms heme, cytochrome, and vitamin B. 12 The common precursor of tetrapyrrole compounds. In addition, as a photodynamic agent (PDT), ALA has a wide range of uses in the fields of agricultural chemicals and medicine. In the field of agriculture, ALA has multiple functions such as weeding, killing insects, increasing plant resistance and promoting plant growth. It is easily degraded and has no residues and is non-toxic to humans and animals. It has become a pollution-free green agricultural chemical with great development prospects. In the medical field, ALA has the effect of selectively killing cancer cells. It is called the second-generation photodynamic medicine. It has low toxicit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/52C12N15/31C12N9/00C12N15/70C12N1/21C07C229/04C12R1/01
Inventor 林建平岑沛霖刘晓侠
Owner ZHEJIANG UNIV
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