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Method for improving stability and biogenic amine degradation capacity of multicopper oxidase

A biogenic amine and catalase technology, applied in the field of bioengineering, can solve the problems of low biogenic amine degradation rate, little effect, process reduction and control, etc., and achieve the effect of improving degradation ability

Active Publication Date: 2021-02-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage is that this method may greatly affect the flavor and quality of the product
Other methods such as reducing amine-producing microorganisms in the fermentation system are generally only suitable for closed single-bacteria system fermentation, and have little effect on mixed-bacteria systems or open systems (susceptible to contamination)
2. Process control reduction
[0004] Multi-copper oxidase is a copper-containing oxidase. It has been found that some enzymes in multi-copper oxidase can degrade biogenic amines, but because of their low stability when degrading biogenic amines, the degradation rate of biogenic amines is low. However, it is limited in practical application

Method used

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  • Method for improving stability and biogenic amine degradation capacity of multicopper oxidase
  • Method for improving stability and biogenic amine degradation capacity of multicopper oxidase
  • Method for improving stability and biogenic amine degradation capacity of multicopper oxidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction and expression of MCOF and CAT fusion expression

[0041] Using the gene of multi-copper oxidase MCOF shown in SEQ ID NO.2 and the gene of catalase CAT shown in SEQ ID NO.4 as templates, two corresponding genes are fused in direct order and in the two genes Insert different linkers: GGGGS, (GGGGS) 2 , EAAAK, (EAAAK) 2 Design and construct specific primers encoding fusion enzymes MCOF and CAT respectively, then carry out PCR amplification to the genes of MCOF and CAT with specific primers. Prepare a 50 μL reaction system according to the instructions of the Primer STAR HS DNA Polymerase (TAKARA) kit. Amplification conditions: 1 cycle at 98°C for 3 minutes, 10 seconds at 98°C, 15 seconds at 55°C, 30 seconds at 72°C for 1 minute, 30 cycles at 72°C for 5 minutes. Bad. The PCR product was verified by 1% agarose gel electrophoresis, and the target fragment obtained with a single band was recovered by column, and the recovery method was carried out a...

Embodiment 2

[0045] Embodiment 2: Exogenous addition of catalase improves the stability of multi-copper oxidase

[0046] Set up the experimental group: mix different concentrations of catalase solution and multi-copper oxidase enzyme solution in equal volumes, so that the final concentration of catalase solution is 200, 400, 800, 1200, 1600, 3200U / L, and then follow the The multi-copper oxidase enzyme activity assay method measures the enzyme activity of the multi-copper oxidase. In the control group, the catalase solution was replaced with an equal volume of buffer solution, and the rest of the conditions were the same. The result is as image 3 As shown, catalase can improve the enzyme activity of multi-copper oxidase, and with the increase of catalase concentration, the enzyme activity of multi-copper oxidase also increases gradually thereupon ( image 3 ).

[0047] H produced during the degradation of biogenic amines by multicopper oxidase 2 o 2 May affect the enzyme activity of m...

Embodiment 3

[0048] Example 3: Fusion expression of CAT improves the tolerance of multi-copper oxidase MCOF to hydrogen peroxide

[0049] According to the method of Example 1, different CAT-MCOF fusion enzymes were respectively constructed, and the recombinant Escherichia coli expressing the fusion enzyme was cultured in LB medium for 12 hours at 37°C and 220 rpm, and then transferred to TB culture according to the inoculum size of 2%. culture medium at 37°C and 220rpm to OD=0.6, adding final concentrations of 0.1mmol IPTG and 1mmol Cu 2+ The expression was induced at 20°C for 20h. After induction of expression, wash twice with PBS, resuspend the cells, break the wall and centrifuge, collect the supernatant, and obtain the crude enzyme solution of MCOF and 8 kinds of MCOF-CAT fusion enzymes connected in different ways for multi-copper oxidation Determination of Enzyme Activity of Enzyme and Catalase.

[0050] Measurement results such as Figure 5 As shown in A, the eight fusion enzymes ...

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Abstract

The invention discloses a method for improving the stability and biogenic amine degradation capacity of multicopper oxidase, and belongs to the technical field of bioengineering. According to the method, catalase CAT and the multicopper oxidase MCOF are connected in sequence, and fusion expression of the multicopper oxidase and the catalase is successfully realized by utilizing an escherichia coliexpression system BL21 (DE3) / pET28a; and the obtained fusion enzyme can make the stability of the multicopper oxidase improved by 51%-68%. The fusion enzyme is used for degrading biogenic amines, sothat the degradation capacity of the multicopper oxidase on the different biogenic amines can be improved to different degrees. Within 24-48h, compared with the multicopper oxidase MCOF not fused withthe CAT, the fusion enzyme CAT and MCOF is improved by 38.9%-132% in the aspect of biogenic amine degradation capacity.

Description

technical field [0001] The invention relates to a method for improving the stability of multi-copper oxidase and the ability to degrade biogenic amine, belonging to the technical field of bioengineering. Background technique [0002] Biogenic amine is a general term for a class of low-molecular-weight nitrogen-containing organic compounds produced in organisms, and is a restrictive indicator in the hygienic standards of fermented foods. An appropriate amount of biogenic amines can promote the normal physiological activities of the human body, but excessive intake of biogenic amines through food can cause adverse reactions such as migraines, headaches, stomach and intestinal problems, and pseudoallergic reactions, which may be life-threatening in severe cases. Therefore, it is necessary to reduce its content in fermented foods to an appropriate concentration. [0003] The measures currently used to control and reduce biogenic amines in fermented foods can be mainly divided i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/96C12N15/62C12N15/70C12N1/21A23L5/20C12R1/19
CPCC12N9/96C12N9/0022C12N9/0065C12N15/70A23L5/25A23L5/28C12Y104/03006C12Y111/01006C07K2319/00
Inventor 方芳倪秀梅陈坚堵国成
Owner JIANGNAN UNIV
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