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Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof

A technology of aminolevulinic acid and its construction method, which is applied in the field of engineering bacteria for producing 5-aminolevulinic acid and its construction, and can solve the problems of high cost, long cycle, low enzyme activity and low yield, etc.

Inactive Publication Date: 2007-10-31
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

One is the biological mutagenesis method, which optimizes the culture conditions of the mutant strain CR 720 (Rhodobactersphaeroides CR720) of Rhodobacter sphaeroides. The output of ALA can reach 27mmol / L, but the culture conditions are complicated, the cycle is long and the cost is high
The second is the genetic engineering method. Mariet J.Vander Werf et al. (1996) and Choi et al. (1999) respectively constructed 5-aminolevulinic acid containing Rhodobacter sphaeroides and Bradyrhizobium japonic μm. Synthetase gene (hemA) engineered bacteria for the production of ALA, but the enzyme activity and yield are low

Method used

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  • Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof
  • Engineering bacterium producing 5-glycyl ethylformic acid and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0020] The construction method of the engineering bacteria producing 5-aminolevulinic acid of the present invention comprises the following steps:

[0021] 1. Extraction of Rhodobacter sphaeroides genomic DNA

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

[0023] 2) After dissolving with 500 μl TE buffer solution, add 30 μl 10% (w / v) sodium dodecyl sulfate (SDS) solution, and at the same time add 3 μl 20 mg / ml proteinase K, mix well, and incubate at 37°C for 1 hour;

[0024] 3) Add 1 / 5 volume of 5mol / L NaCl solution, plus about 1 / 5 volume of CTAB / NaCl solution (4.1g NaCl dissolved in 80ml H 2 O, slowly add 10g CTAB, add water to 100ml), keep warm at 65°C for 30min;

[0025] 4) Add an equal volume of a mixture of phenol, chloroform and isoamyl alcohol (the volume ratio of phenol: chloroform: isoamyl alcohol is 25:24:1), and centrifuge at 13,000×g for 5 min;

[0026] 5) Take the super...

Embodiment 2

[0045] Expression of 5-aminolevulinic acid synthase under the action of inducer isopropyl-β-D-thiogalactoside (IPTG)

[0046] 1) Cultivate the engineering bacterium Rosetta(DE3)-pET28a-R.S.hemA of the present invention in a 250ml shake flask containing 50ml LB medium, first culture at 37°C and 200rpm for 2.0h; then cool down to 28°C and use 2.5μl Induced by 1mol / L IPTG, after continuing to cultivate for 6 hours, take 30ml of bacterial liquid and centrifuge at 10,000×rpm for 10min to remove the supernatant;

[0047] 2) Wash once with 9ml 50mmol / L Tris Cl (pH 7.5), resuspend with the same amount of buffer, and disrupt the cells by ultrasonication. The conditions are 300w for 5s, 7s for 30 repetitions, and centrifugation at 10,000×rpm 10min;

[0048] 3) Take the supernatant and carry out 10% SDS-polyacrylamide gel electrophoresis;

[0049] 4) According to the electrophoresis results, there is a soluble protein expression accounting for 25% of the total protein amount at about 5...

Embodiment 3

[0051] Determination of specific activity of 5-aminolevulinic acid synthase

[0052] 1) 500μl containing 50mmol / L Tris-HCl (pH7.5), 20mmol / L MgCl 2 , 0.1mol / L glycine, 0.27mmol / L pyridoxal phosphate, 0.2mmol / L succinyl-CoA and the mixed solution of the cell supernatant after cell destruction, react at 37°C for 10min;

[0053] 2) Add 150 μl of 10% trichloroacetic acid to the above reaction solution, mix in a 1.5 mL centrifuge tube, and centrifuge at 13,000×g for 5 min;

[0054] 3) Mix 300 μl supernatant with 400 μl 1mol / L acetate buffer (pH 4.6) and 35 μl acetylacetone, and bathe in boiling water for 15 minutes;

[0055] 4) After cooling to room temperature for 15 min, add 700 μl of freshly prepared Ehrlich reagent (add 30 ml of glacial acetic acid, 1 g of p-dimethylaminobenzaldehyde, 5 ml of 70% perchloric acid, 5 ml of water in turn, dissolve and fix with glacial acetic acid to 50ml), after reacting for 30min, measure its absorbance at 554nm, the concentration of ALA (mg / L)...

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Abstract

The invention discloses a method to prepare engineering bacteria of 5-amino levulinic acid, which comprises the following steps: 1) extracting total genom DNA from bacterial liquid of red ball bacterium; 2)augmenting 5- glycyl ethylformic acid synthase gene with polyose chain reaction; 3) connecting with colonic carrier pMD-18T simple; proceeding DNA testing sequence; 4) connecting the goal gene segment with expressing carrier pET28a; constructing recon pET28a-R. S. hemA; 5) transforming the recon pET28a-R. S. hemA to host bacteria; getting the product. This invention possesses higher enzyme activity, which output of external cell ALA can reach 6. 6g / L.

Description

technical field [0001] The invention relates to an engineering bacterium for producing 5-aminolevulinic acid and a construction method thereof. Background technique [0002] 5-Aminolevulinic acid (5-aminolevulinic acid, ALA) is a prefix compound of tetrahydropyrrole, which is used by organisms to synthesize chlorophyll, heme, porphyrin and vitamin B 12 and other essential substances. In recent years, in addition to being a new type of photoactivated pesticide with high environmental compatibility and selectivity, ALA is also used as a photochemotherapy agent in clinical practice. As a new generation of photodynamic agents, it has significant advantages such as low toxicity and high selectivity. It is used to treat various cancers such as skin cancer, bladder cancer, colon cancer, and pancreatic cancer. It is gradually showing its great applicability in the field of photochemotherapy. . [0003] Due to the cumbersome steps and low yield of chemical synthesis of ALA, biosyn...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/52C12P13/00C12R1/19
Inventor 林建平傅维琦岑沛霖
Owner ZHEJIANG UNIV
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