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An engineering bacterium with high l-aspartate α-carboxylase activity and its application in the production of β-alanine

A technology of engineering bacteria and alanine, applied in the field of engineering bacteria, can solve the problems of side reactions, environmental pollution, harsh reaction conditions, etc., and achieve the effects of mild conditions, environmental friendliness and simple operation.

Active Publication Date: 2017-06-06
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The succinimide method refers to degrading succinimide in alkaline sodium chlorate solution to obtain β-alanine. The process conditions of this method are harsh, the cost of raw materials is high and other side reactions are prone to occur, so it is not suitable industrialization
In short, the raw materials and intermediates of the chemical synthesis method are poisonous, and the reaction conditions are relatively harsh, requiring high temperature and high pressure, which seriously pollutes the environment.
[0005] Biological synthesis of β-alanine, there are mainly two domestic related reports, one is to use Sarcina luteus to convert acrylic acid to β-alanine (conversion rate is 54%), and the other is to use large intestine Bacillus BL21(DE3) is used as a host to express L-aspartate α carboxylase derived from Escherichia coli, and finally produce 2.94g / L of β-alanine, which is far below the level of industrialization

Method used

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  • An engineering bacterium with high l-aspartate α-carboxylase activity and its application in the production of β-alanine
  • An engineering bacterium with high l-aspartate α-carboxylase activity and its application in the production of β-alanine
  • An engineering bacterium with high l-aspartate α-carboxylase activity and its application in the production of β-alanine

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Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1, the construction of recombinant plasmid and engineering bacterium

[0021] The double-stranded DNA molecule shown in Sequence 1 of the sequence listing is the gene encoding L-aspartic acid α-carboxylase in Escherichia coli BL21 (DE3). The double-stranded DNA molecule shown in sequence 2 of the sequence listing is the coding gene of L-aspartic acid α-carboxylase in Corynebacterium glutamicum. The double-stranded DNA molecule shown in sequence 3 of the sequence listing is the coding gene of L-aspartic acid α-carboxylase in Mycobacterium tuberculosis. The double-stranded DNA molecule shown in sequence 4 of the sequence listing is the coding gene of L-aspartic acid α-carboxylase in Bacillus subtilis.

[0022] 1. Construction of engineering fungus armor

[0023] 1. Recombinant plasmid pET30-panD E build

[0024] (1) Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0025] (2) Using the double-stranded DNA molecule s...

Embodiment 2

[0070] Embodiment 2, detection of enzyme activity of genetically engineered bacteria

[0071] The engineering bacterium A, engineering bacterium B, engineering bacterium C and engineering bacterium D prepared in embodiment 1 are carried out as follows respectively:

[0072] 1. Pick a single colony of engineering bacteria, inoculate it in 10 mL of LB liquid medium containing 50 μg / ml kanamycin, and culture it with shaking at 37°C and 200 rpm for 12 hours.

[0073] 2. Take the entire culture system obtained in step 1, inoculate it in 100 mL of LB liquid medium containing 50 μg / ml kanamycin, and incubate with shaking at 30° C. and 200 rpm for 12 hours.

[0074] 3. At the end of step 2, take 2 mL of the culture system (i.e. OD 600nm =3 bacteria solution), add 1mL pH8.0, 0.01mol / L phosphate buffer, mix well, then perform ultrasonic crushing (power 200W, work for 3 seconds and stop for 3 seconds, the total time is 4min), centrifuge at 10000rpm for 1min, Take the supernatant.

[0...

Embodiment 3

[0084] Embodiment 3, whole cell transformation

[0085] 1. Pick a single colony of the engineering bacterium prepared in Example 1, inoculate it in 100 mL of LB liquid medium, and culture it with shaking at 37° C. and 200 rpm for 12 hours.

[0086] 2. Take the entire culture system obtained in step 1, inoculate it into 1000 mL of LB liquid medium containing 50 μg / ml kanamycin, and incubate with shaking at 30° C. and 200 rpm for 12 hours.

[0087] 3. Take the entire culture system obtained in step 2, centrifuge at 9000rpm for 10min, and collect the bacteria.

[0088] 4. Take all the bacterial cells obtained in step 3, suspend them with 300mL distilled water, add L-aspartic acid, and conduct biotransformation at 37°C and 450rpm for 15 hours. The concentration of β-alanine was detected by HPLC.

[0089] see results image 3 . When the fermentation is completed, the concentration of β-alanine in the fermentation system reaches 178g / L, and the conversion rate reaches 99%.

[0...

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Abstract

The invention discloses an engineering bacterium and its application in the production of beta-alanine. A construction method of the engineering bacterium comprises the following steps: 1, inserting the coding gene of a target protein the multiple cloning site of a vector pET-30a(+) to obtain a recombinant plasmid pET30-panDB, wherein the target protein is represented by a sequence 6 in a sequence table; and 2, introducing the recombinant plasmid pET30-panDB obtained in step 1 to Escherichia coli BL21(DE3) in order to obtain the engineering bacterium. The invention also discloses a method for producing beta-alanine. The engineering bacterium is biologically converted by using L-aspartic acid as a substrate to obtain beta-alanine. The method for preparing beta-alanine has the advantages of no need of an inducer, no need of high temperature or high pressure in the whole process, mild conditions, simple operation and environmental protection, and reaches the industrial application level.

Description

technical field [0001] The invention relates to an engineering bacterium with high L-aspartic acid alpha carboxylase activity and its application in producing beta-alanine. Background technique [0002] β-alanine, namely 3-aminopropanoic acid (3-aminopropanoic), is the only β-type non-protein amino acid that exists in nature. β-alanine is an important biochemical raw material that can be used It is widely used in the synthesis of pantothenic acid, carnosine, sodium pamidronate, balsalazide, etc. in the fields of medicine, feed and food. [0003] Currently, there are mainly chemical and biological methods for the synthesis of β-alanine. [0004] Chemical synthesis of β-alanine mainly includes acrylonitrile method, acrylic acid method and succinimide degradation method. The acrylonitrile method uses the ammoniation reaction of acrylonitrile and ammonia water to generate β-aminopropionitrile, which is then hydrolyzed under acidic or alkaline conditions. This method has easy-t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12P13/06C12R1/19
Inventor 蔡真张君丽邓思颖李寅
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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