Preparation of lactic acid bacteria multi-copper oxidase and application of lactic acid bacteria multi-copper oxidase in biogenic amine degradation

A technology of multi-copper oxidase and biogenic amine, which is applied in the field of bioengineering to achieve the effect of good effect, wide application range and good amine reducing effect.

Pending Publication Date: 2022-02-25
DALIAN POLYTECHNIC UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still few studies on bacterial multicopper oxidases that can degrade biogenic amines. Therefore, screening for multicopper oxidases that can degrade biogenic amines, especially histamine and tyramine, which are more toxic, has a very wide application. foreground

Method used

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  • Preparation of lactic acid bacteria multi-copper oxidase and application of lactic acid bacteria multi-copper oxidase in biogenic amine degradation
  • Preparation of lactic acid bacteria multi-copper oxidase and application of lactic acid bacteria multi-copper oxidase in biogenic amine degradation
  • Preparation of lactic acid bacteria multi-copper oxidase and application of lactic acid bacteria multi-copper oxidase in biogenic amine degradation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1: Degradation of biogenic amine by cell disruption liquid of Lactobacillus sake

[0048]The Lactobacillus sakei preserved in our laboratory was inoculated in liquid MRS medium and cultured overnight at 37°C and 200r / min. Centrifuge the bacterial solution at 4°C and 10000r / min for 7min, collect the bacterial sediment, wash the bacterial cells with 20mmol / L pH 7.0 phosphate buffer solution, and then resuspend the bacterial cells with phosphate buffer saline. Add lysozyme at a final concentration of 0.5 mg / ml, place on ice for 30 min, and then freeze and thaw repeatedly 3-5 times in liquid nitrogen to lyse it. The lysate was centrifuged at 4°C and 10,000 r / min for 15 minutes to collect the supernatant, and then the collected solution was passed through a 0.45 μm water filter to obtain the desired crude enzyme solution. Using ABTS as the substrate, the activity level of multi-copper oxidase was measured to be 4.63U / L. Add 2U / L crude enzyme solution to biogenic am...

Embodiment 2

[0049] Example 2: Cloning, expression and purification of multi-copper oxidase gene

[0050] (1) Cloning and expression of multi-copper oxidase gene

[0051] Using the genomic DNA of Lactobacillus sakei preserved in our laboratory as a template, the target gene was amplified, and the amplified product fragments were amplified, as shown in the electrophoresis figure 1 As shown, the band size is about 1500bp, and the nucleotide sequence is shown in SEQ ID NO.2.

[0052] The amplified fragment was connected between the Sac I enzyme and the Xho I enzyme cutting site of the plasmid pET-28a to construct a recombinant vector. The recombinant vector was transformed into E.coli DH5α, cultivated, and the plasmid was extracted for screening and sequencing, and it was confirmed that the recombinant vector with the correct coding frame was obtained.

[0053] Extract the recombinant plasmid with correct sequencing and transform it into Escherichia coli BL21(DE3) competent cells. The trans...

Embodiment 3

[0057] Example 3: Degradation effect of recombinant multi-copper oxidase on histamine under different conditions

[0058] (1) Effect of the amount of recombinant multi-copper oxidase added on the degradation rate of histamine

[0059] The reaction system is a phosphate buffer solution with pH 7.0, and the final content of histamine is 100 mg / L; the purified recombinant enzyme Ls1b is added in different amounts (7U / L, 17U / L, 33U / L, 66U / L, 132U / L, L) adding to the reaction system; reacting at 25° C. for 24 hours; measuring the content of biogenic amine by ultra-high performance liquid chromatography-mass spectrometry. Histamine degradation results as figure 2 As shown, with the increase of the recombinant enzyme Ls1b content, the degradation rate of histamine increased from 12.16% to 65.5% within 24 hours.

[0060] (2) Effect of salinity and pH on the degradation rate of recombinant multi-copper oxidase on histamine

[0061] The reaction system is a phosphate buffer (pH 4.0 / ...

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Abstract

The invention discloses preparation of lactic acid bacteria multi-copper oxidase and application of the lactic acid bacteria multi-copper oxidase in biogenic amine degradation, and belongs to the technical field of bioengineering. The multi-copper oxidase provided by the invention is derived from Lactobacillus sakei, and is produced by carrying out heterologous expression on the Lactobacillus sakei in escherichia coli. The enzyme has good biogenic amine degradation performance, and can degrade 40.90% of histamine and 41.24% of tyramine at a low concentration. Under the high-salt condition, the amine reduction performance can be further improved, and the histamine degradation rate and the tyramine degradation rate are 75.95% and 66.81% respectively. And partial amine reduction capability can be maintained under a high ethanol condition. The enzyme has a good amine reduction effect when applied to simulation systems of fermented foods such as salted fish and grape wine, and can be widely applied to industrial production.

Description

technical field [0001] The invention relates to the preparation of a lactic acid bacteria multi-copper oxidase and its application in degrading biogenic amines, in particular to the gene and preparation method of multi-copper oxidase Ls1b derived from Lactobacillus sake and its application in degrading biogenic amines , belonging to the field of bioengineering technology. Background technique [0002] Biogenic amines are a class of biologically active small molecule nitrogen-containing organic compounds, which widely exist in food, especially fermented food with high protein content. It is mainly formed by the corresponding amino acids through the decarboxylation of microorganisms, or produced by aldehydes and ketones under the action of amino acid transaminases. An appropriate amount of biogenic amines can promote the normal physiological activities of the human body, but excessive intake of exogenous biogenic amines will cause many adverse reactions in the human body, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23L5/20A23L11/30A23L11/50A23L17/00A23L27/50C12N9/02
CPCA23L5/25A23L11/33A23L11/50A23L17/00A23L27/50C12N9/0057
Inventor 纪超凡赵云松韩静梁会朋张素芳林心萍陈映羲
Owner DALIAN POLYTECHNIC UNIVERSITY
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