Acetaldehyde dehydrogenase and application thereof in aspect of anti-alcoholic health-care products

An acetaldehyde dehydrogenase and product technology, applied in the field of enzyme engineering, can solve problems such as unsuitable food preparation, and achieve the effect of increasing yield

Active Publication Date: 2018-06-01
ZIBO VOCATIONAL INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, there have been cases of successful expression of acetaldehyde dehydrogenase in Escherichia coli, but due to food safety issues in Escherichia coli, it is not suitable for food preparation

Method used

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  • Acetaldehyde dehydrogenase and application thereof in aspect of anti-alcoholic health-care products
  • Acetaldehyde dehydrogenase and application thereof in aspect of anti-alcoholic health-care products
  • Acetaldehyde dehydrogenase and application thereof in aspect of anti-alcoholic health-care products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Synthesize the ALDH2 gene shown in SEQ ID NO.1; activate and culture Bacillus subtilis 168, inoculate in LB medium, and extract plasmid pMA5 after culturing for 18-24 hours. The ALDH2 gene and pMA5 were digested and ligated to construct the recombinant plasmid pMA5-aldh2; the recombinant plasmid pMA5-aldh2 was transformed into Bacillus subtilis 168 competent cells, cultured in LB medium, spread on LB plates, and challenged Take positive clones, extract plasmids and verify by sequencing. The strains with correct sequencing are recombinant bacteria.

Embodiment 2

[0018] The recombinant Bacillus subtilis prepared in Example 1 was inoculated in LB medium, activated at 37°C, and then the two bacterial solutions were simultaneously transferred to fresh LB medium for cultivation. The specific operation steps are as follows:

[0019] (1) Activation of bacteria

[0020] Take 30 μL of the original bacteria and integrated bacteria stored in glycerol tubes and add them into 10 mL test tubes containing LB medium, and culture them at 37°C and 200 rpm for 14-20 hours to obtain seed liquid.

[0021] (2) Strain fermentation

[0022] Take 2 mL of the activated seed solution and add it to 200 mL of LB medium, and ferment for a period of time under the conditions of 37 ° C and 200 rpm, and take 5 mL of fermentation liquid for the determination of specific activity at 24 h, 30 h, 34 h and 48 h of fermentation.

[0023] (3) Treatment of fermentation broth

[0024] Centrifuge 5mL of the fermentation broth at 8000rpm for 5min, wash the cells once with ste...

Embodiment 3

[0031] The bacterial strain was cultivated in the same manner as in Example 2, except that 0.4 mmol / L metal salt (Table 2) was added to the culture medium. Enzyme activity was measured after 24 h of fermentation. The enzyme activity of fermenting 24h under the identical conditions of embodiment 2 is 100%, and the result is as shown in table 2, adding Ca 2+ Salt and Cu 2+ Salt can increase the production of enzymes in the fermentation broth to a certain extent. Due to Ca 2+ Salt and Cu 2+ The metal ion is harmless or even beneficial to the human body, and has important application significance for preparing products containing acetaldehyde dehydrogenase in the food field.

[0032] Table 2 Changes of enzyme activity in a unit of fermentation broth containing different metal ions

[0033]

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PUM

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Abstract

The invention discloses acetaldehyde dehydrogenase and an application thereof in an aspect of anti-alcoholic health-care products, and belongs to the technical field of enzyme engineering. The invention provides an engineering bacterium for high-efficiency expression of a human acetaldehyde dehydrogenase gene and a construction method of the engineering bacterium; the expression of the humanized acetaldehyde dehydrogenase gene, which is shown as SEQ ID NO.1, in bacillus subtilis is achieved; and an enzyme yield by fermenting the recombinant bacterium for 24h can reach 24.52U/L or above. By improving a fermentation medium, the yield of the acetaldehyde dehydrogenase is improved, and an enzyme yield of a culture medium which is added with CaCl2 salt and CuSO4 is improved to 140-180% in comparison with an original enzyme yield; therefore, a new way and a theoretical basis are provided for research and development of anti-alcoholic drugs.

Description

technical field [0001] The invention relates to an acetaldehyde dehydrogenase and its application in anti-alcohol health products, belonging to the technical field of enzyme engineering. Background technique [0002] Alcohol dehydrogenase (Alcohol dehydrogenase) and acetaldehyde dehydrogenase (EC 1.2.1.10, Aldehyde dehydrogenase) in the human body play an important role in the metabolism of ethanol. Ethanol is oxidized into acetaldehyde under the action of alcohol dehydrogenase, and then oxidized into acetic acid under the action of acetaldehyde dehydrogenase. When the two enzymes in the human body can be fully expressed, ethanol will be fully metabolized in a short time, reducing alcohol consumption. Effects on the central nervous system. However, under normal circumstances, alcohol dehydrogenase can be fully expressed, and there are more individuals lacking acetaldehyde dehydrogenase, resulting in a large amount of acetaldehyde accumulation in the body, resulting in drunk...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N1/21C12N9/02A23L33/135C12R1/125
CPCA23L33/135A23V2002/00C12N9/0008C12Y102/0101A23V2200/334
Inventor 马伟辰
Owner ZIBO VOCATIONAL INST
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