Zearalenone (ZEN) toxin degrading enzyme for acinetobacter and coding gene and applications of ZEN toxin degrading enzyme

A technology of coding genes and degrading enzymes, which is applied in the field of zearalenone toxin degrading enzymes and their coding genes and applications, can solve the problems of Acinetobacter ZEN degrading enzyme gene sequence has not been reported and applied for patents, and achieve strong degradation effect of ability

Inactive Publication Date: 2012-08-01
SOUTH CHINA UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The above literature reports provide a basis for the primer design of Acinetobacter ZEN degrading enzyme gene cloning and the homology

Method used

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  • Zearalenone (ZEN) toxin degrading enzyme for acinetobacter and coding gene and applications of ZEN toxin degrading enzyme
  • Zearalenone (ZEN) toxin degrading enzyme for acinetobacter and coding gene and applications of ZEN toxin degrading enzyme
  • Zearalenone (ZEN) toxin degrading enzyme for acinetobacter and coding gene and applications of ZEN toxin degrading enzyme

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation of Acinetobacter ZEN degrading enzyme comprises the following steps:

[0035] (1) Purify ZEN-degrading enzyme from Acinetobacter sp.SM04 culture supernatant

[0036] 1. The culture (M1 medium) inoculated with 1% (v / v) Acinetobacter sp.SM04 bacterial suspension (OD600=0.8) was cultivated (200r / min) in an air bath shaker at 30°C for 36h to the right At the end of growth, centrifuge the liquid culture for 5 min (12000×g, 4°C), filter the centrifuged supernatant with a 0.22 μm filter membrane, and concentrate the filtrate 5 times at 50°C using a vacuum rotary evaporator;

[0037] The formulation of the medium used is as follows:

[0038] Basic inorganic salt medium: 3g NH 4 NO 3 , 1.5g K 2 HPO 4 ·3H 2 O, 1g KCl, 0.5gMgSO4·7H 2 O, 0.1g CaCl 2 After mixing with 10mL trace element stock solution, add deionized water to make up to 1000mL, pH7.3.

[0039] Trace element stock solution: 2g / L FeSO 4 ·7H 2 O, 0.4g / L MnSO 4 4H 2 O, 0.4g / LCuSO 4 ·5H 2 O,...

Embodiment 2

[0053] Efficiency detection of ZEN degrading enzyme degrading ZEN

[0054] Based on the ZEN degrading enzyme activity assay method in the embodiment 1 step (1), get the ZEN degrading enzyme finally obtained in the embodiment 1 step (1), measure the content change of ZEN in the treatment solution under different treatment times, the results are shown in figure 2 .

[0055] from figure 2 It can be seen that the purified ZEN-degrading enzyme utilizes H 2 o 2 The degradation rate of oxidative degradation of ZEN is relatively slow, and more than 90% of ZEN can be degraded within 12 hours. According to the slope, the rate of oxidative degradation of ZEN is about 1.6μgmL -1 h -1 .

Embodiment 3

[0057] Detection of estrogenic activity of products degraded by ZEN degrading enzymes

[0058] With 0.40mL of the ZEN degrading enzyme finally obtained in step (1) of Example 1, 0.10mL of 0.25mol / L Tris-HCl (pH8.5) buffer and 20μL of 0.5mol / L H 2 o 2 The solution was mixed evenly, 10 μL of ZEN methanol stock solution (1 mg / mL) was added, incubated in a constant temperature water bath at 40° C. for 12 hours, and 0.5 mL of methanol was added to terminate the reaction.

[0059] The above reaction products (ie, the experimental group) were used to test the proliferation rate of MCF-7 cells. With the treatment group without ZEN and ZEN solution (10 μL 1mg / mL ZEN methanol stock solution mixed with 0.14mL 0.25mol / LTris-HCl (pH8.5) buffer and 20μL 0.5mol / L H 2 o 2 solution mixed uniformly) was used as the control group.

[0060] MCF-7 human breast cancer cells were purchased from the Cell Bank of the Chinese Academy of Sciences. For the experimental method of MCF-7 human breast c...

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Abstract

The invention discloses a zearalenone (ZEN) toxin degrading enzyme for acinetobacter and a coding gene and applications of the ZEN toxin degrading enzyme. The amino acid sequence of the ZEN toxin degrading enzyme for the acinetobacter is shown by SEQ ID NO.6. The sequence of the coding gene of the ZEN toxin degrading enzyme for the acinetobacter is shown by SEQ ID NO.5. The ZEN toxin degrading enzyme for the acinetobacter performs stronger degrading capacity on mycotoxin ZEN, is capable of degrading at least 90 percent of ZEN into a low-estrogen active product in 12h without generating high-estrogen active analogues, such as ZEN, zeranol and the like, and has a real detoxification effect. According to the invention, the coding sequence of the ZEN toxin degrading enzyme for acinetobacter sp.SM04 is determined, i.e. a Prx gene, and thereby, a foundation is laid for researching the active site of the enzyme and changing the Prx enzyme activity through site-specific mutagenesis in the future.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a zearalenone toxin-degrading enzyme of Acinetobacter sp.SM04, its coding gene and its application. Background technique [0002] Zearalenone (ZEA, ZEN) is an estrogenic mycotoxin with a dihydroxybenzoic acid lactone structure, which is produced by Gibberella zeara, Fusarium graminearum, and Fusarium tritina, and mainly pollutes wheat , barley, oats, corn and other crops. About 25% of the crops in the world are polluted by mycotoxins every year, and my country's food and feed industry loses more than 10% of its total output due to mildew every year on average. Sampling and testing from national warehouses and feed factories found that the detection rate of ZEN in corn and other crops and feed was as high as 100%, and the detection concentration exceeded the standard seriously. ZEN can cause premature maturation in breeding pigs or poultry, disordered reproductive cycle,...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55A23L1/015A23K1/00C12R1/01A23L5/20
Inventor 唐语谦吴晖余元善肖性龙肖俊梅陈艺
Owner SOUTH CHINA UNIV OF TECH
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