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Method for producing acetaldehyde dehydrogenase of recombinant basophilic salt-tolerant bacillus halodurans (XJU-1) by using genetic engineering technique

A technology of halotolerant bacillus and acetaldehyde dehydrogenase, applied in the field of bioengineering, can solve problems such as high cost, difficulty in large-scale production, and complicated extraction process

Inactive Publication Date: 2011-01-12
SICHUAN UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Acetaldehyde dehydrogenase can be extracted from the liver, pancreas and other viscera of some animals, but the extraction process is relatively complicated, the cost is high, and it is not easy to produce on a large scale

Method used

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  • Method for producing acetaldehyde dehydrogenase of recombinant basophilic salt-tolerant bacillus halodurans (XJU-1) by using genetic engineering technique
  • Method for producing acetaldehyde dehydrogenase of recombinant basophilic salt-tolerant bacillus halodurans (XJU-1) by using genetic engineering technique
  • Method for producing acetaldehyde dehydrogenase of recombinant basophilic salt-tolerant bacillus halodurans (XJU-1) by using genetic engineering technique

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Effect test

Embodiment 1

[0046] This embodiment is to clone the alkalophilic bacillus halodurans XJU-1 acetaldehyde dehydrogenase aldA and aldB gene fragments, the cloned alkalophilic halodurans XJU-1 acetaldehyde dehydrogenase aldA and aldB gene fragments were sequenced and identified to be 1521bp and 1359bp in length, respectively.

Embodiment 2

[0048] This example is the recombination of the acetaldehyde dehydrogenase aldA and aldB gene fragments of Bacillus halodurans XJU-1 with the pET30b(+) expression vector to form pET30b(+)-aldA and pET30b(+)- The steps of aldB recombinant vector are as follows:

[0049] A. Bacillus halodurans XJU-1, the size of the original clone of its acetaldehyde dehydrogenase gene aldA is 1521bp, digested with Nde I and HindIII, and its enzyme digestion reaction system is as follows:

[0050]

[0051] After mixing, enzyme digestion was carried out at 37°C for 4 hours, detected by electrophoresis on 1% agarose gel, and the target fragment was recovered by cutting the gel.

[0052] The size of the original clone of the acetaldehyde dehydrogenase gene aldB of Bacillus halodurans XJU-1 is 1359 bp, which is digested with NdeI and BamH I. The enzyme digestion reaction system is as follows:

[0053]

[0054] B. Double digestion of expression vector pET30b(+)

[0055] The expression vector ...

Embodiment 3

[0065] This example is an experiment in which the pET30b(+)-aldA and pET30b(+)-aldB recombinants are transformed into Escherichia coli BL21(DE3), and then screened with kanamycin to obtain the BL21(DE3) engineered bacteria with acetaldehyde dehydrogenase Proceed as follows:

[0066] Take the single colony formed in the culture medium of Escherichia coli BL21 (DE3) after screening with kanamycin and transforming with pET30b(+)-aldA and pET30b(+)-aldB recombinant vectors, and inoculate them in culture medium containing 50 μg / ml kanamycin respectively. Mycin 5ml LB culture solution, shake culture overnight at 37°C; take 1ml of the culture solution, and extract the plasmid according to the instructions of the small amount of plasmid DNA extraction kit from OMEGA company; take 5μl of plasmid DNA, add 12μl of distilled water, 1μl of buffer and Mix 1 μl Nde I, 1 μl HindIII enzyme and place in a 37°C water bath for 2 hours; at the same time, take 5 μl plasmid DNA, add 12 μl distilled...

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Abstract

The invention discloses a method for producing acetaldehyde dehydrogenase of recombinant basophilic salt-tolerant bacillus halodurans (XJU-1) by using a genetic engineering technique. The method comprises the following steps of: cloning an aldA and aldB genetic fragments of the acetaldehyde dehydrogenase of the basophilic salt-tolerant bacillus halodurans (XJU-1); recombining the genetic fragments with a pET30b(+) expression vector respectively, and constructing pET30b(+)-aldA and pET30b(+)-aldB recombinant vectors; converting colibacillus BL21(DE3) by using the recombinant vectors respectively, and then performing screening by using kanamycin to respectively obtain BL21(DE3) engineering bacteria of the aldA and the aldB for expressing the acetaldehyde dehydrogenase; and performing amplification culture on the BL21(DE3) engineering bacteria of the acetaldehyde dehydrogenase in an LB culture medium, and extracting and separating acetaldehyde dehydrogenase crude enzyme solution from fermentation liquor.

Description

technical field [0001] The invention relates to a method for producing recombinant alkalophilic halodurans XJU-1 acetaldehyde dehydrogenase by using genetic engineering technology, which belongs to the field of bioengineering. Background technique [0002] Alcohol is harmful to the human body, especially the liver, and can also cause varying degrees of damage to the gastrointestinal tract, pancreas, heart, and kidneys. The World Health Organization stated in a report: "Alcoholism is the number one public hazard in the world today. Its toxicity can affect the main organs of the whole body, and it has the greatest impact on the liver. In Western countries, 80% of liver cirrhosis (liver cirrhosis) is associated with alcohol. It is related to poisoning. Alcoholism has an important impact on the occurrence, development and prognosis of viral hepatitis and liver cancer. [0003] The metabolism of alcohol in the human body mainly depends on two enzymes in the body: one is alcohol ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/70C12N9/02C12N1/21C12R1/19
Inventor 高秀峰赵绪光王国庆李永生
Owner SICHUAN UNIV
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