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Phage resistance L-aspartic acid transformed bacterium and application thereof

A technology of aspartic acid and phage, applied in the direction of bacteria, biochemical equipment and methods, microorganisms, etc., can solve the problems of prolonged fermentation period, slow consumption of fermentation substrate products, phage contamination, etc., and achieve good genetic stability, The effect of good cell growth rate and improved economic efficiency

Active Publication Date: 2015-08-19
CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Bacteriophage contamination in the production of l-aspartic acid on an industrial scale by microorganisms is a common phenomenon, which can easily cause the extension of the fermentation cycle, slow down the consumption of fermentation substrates and the formation of products, affect the yield and quality of fermentation products, and even cause the suspension of fermentation. Fermentation failure caused huge losses to production and became one of the key factors restricting the stability of product production capacity and quality improvement (Sun Wenjing, et al. Food Science and Technology. 2013, 38:323–327; Zhou Haiyan, et al. Food and Fermentation Industry. 2013, 39:88–93)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Isolation and Purification of Bacteriophage CICC 80001

[0028] Bacteria: Escherichia coli HY-05C, an l-aspartic acid transformation production strain screened earlier (Patent No. CN201110382584.X).

[0029] Take 10 ml of the Escherichia coli transformation solution contaminated by phage, centrifuge at 6,000 rpm for 10 minutes, take the supernatant and filter it through a 0.22 μm microporous membrane to sterilize, take 200 μl and put it into the medium containing 5 ml of the upper layer of LB medium. In the test tube, add 200 μl of HY-05C bacterial suspension at the same time, mix well, pour it on the solidified LB plate, spread it evenly, and place it at 37 o C incubator for 18 – 24 hours. Single phage plaques with different shapes and sizes were picked by inoculation needle puncture culture method, and were respectively inserted into liquid LB medium containing HY-05C, 37 o C, 200 rpm for 18-24 hours. Repeat the above operation to check the shape and size of the pl...

Embodiment 2

[0032] Under the condition of adding the lysate of phage CICC 80001, the comparative verification of the ability of Escherichia coli CICC 11021S and the control group of Escherichia coli HY-05C without phage resistance to convert fumaric acid to produce l-aspartic acid.

[0033] The preferred medium composition is:

[0034] (1) Seed medium

[0035] LB medium: yeast powder 5.0 g / L, peptone 10.0 g / L, sodium chloride 10.0 g / L, pH 7.0 – 7.2, solid medium with 2% agar powder added.

[0036] (2) Fermentation medium

[0037] Beef extract peptone medium (g / L): corn steep liquor dry powder 13.0, magnesium sulfate 0.2, potassium dihydrogen phosphate 1.0, sodium chloride 1.5, fumaric acid 5.0, glucose 5.0, adjust the pH to 6.5 – 7.0 with ammonia water.

[0038] The method of strain activation is: use an inoculation loop to scrape a small amount of strains from the slope of Escherichia coli strain HY-05C or CICC 11021S, put them into a 500 mL Erlenmeyer flask containing 100 mL of LB med...

Embodiment 3

[0045] Example three: large-scale production of l-aspartic acid

[0046] Bacteria: Escherichia coli CICC 11021S

[0047]Fermentation medium: Beef extract peptone medium (g / L): corn steep liquor dry powder 13.0, magnesium sulfate 0.2, potassium dihydrogen phosphate 1.0, sodium chloride 1.5, fumaric acid 5.0, glucose 5.0, adjust the pH to 6.5 with ammonia water – 7.0.

[0048] The method of strain activation is: use an inoculation loop to scrape a small amount of strains from the slope of Escherichia coli strain CICC 11021S, put them into a 5000 mL Erlenmeyer flask containing 1000 mL of fermentation medium, 37 o C, cultivated for 8-16 hours under the condition of 100-200 rpm to obtain seed liquid.

[0049] The fermenter culture method is as follows: the seed liquid is inserted into 20 m 3 In the fermenter, the volume of the fermentation medium is 12 – 16 m 3 , stirring at 80 – 200 rpm, and incubate for 8 – 16 hours.

[0050] The l-aspartic acid conversion production method ...

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Abstract

The invention relates to Escherichia coli CICC 11021S having phage CICC 80001 resistance and l-aspartase activity and capable of transforming fumaric acid to produce l-aspartic acid. The Escherichia coli CICC 11021S is capable of effectively resisting the pollution of phage CICC 80001, excellent in cell growth rate and therefore capable of effectively improving the economic benefit of l-aspartic acid production enterprises.

Description

technical field [0001] The invention relates to an L-aspartic acid producing bacterium with phage resistance, which is a new strain variety and belongs to the technical field of bioengineering. Background technique [0002] L-aspartic acid is an important amino acid, and it is the main raw material for the production of compounds such as aspartame, polyaspartic acid, and l-alanine. The global annual demand is about 300,000 tons, and the market demand is huge ( Lee KM, et al. J Org Chem. 1989, 54:3195–3198). The microbial transformation method uses l-aspartase-producing strains to convert ammonium fumarate to produce l-aspartic acid. Compared with physical or chemical production methods, it has mild reaction conditions, high conversion rate, and less by-products. The advantage is that it is a widely used large-scale production method (Sheng Xiaoyan, et al. Food Science and Technology. 2010, 35:23?26.). [0003] Bacteriophage contamination in the production of l-aspartic aci...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P13/20C12R1/19
CPCC12P13/20C12N1/205C12R2001/19
Inventor 程池马玉岳徐友强刘勇裴疆森姚粟李金霞姜增妍张京涛
Owner CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD
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