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ZEN (zearalenone) toxin degrading enzyme Oxa of acinetobacter as well as coding gene and application thereof

A technology of encoding genes and degrading enzymes, applied in the direction of applications, enzymes, enzymes, etc., to achieve the effect of strong degradation ability

Active Publication Date: 2014-06-25
河南亿万中元生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

No information about non-peroxidase (Oxa) has been reported so far

Method used

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  • ZEN (zearalenone) toxin degrading enzyme Oxa of acinetobacter as well as coding gene and application thereof
  • ZEN (zearalenone) toxin degrading enzyme Oxa of acinetobacter as well as coding gene and application thereof
  • ZEN (zearalenone) toxin degrading enzyme Oxa of acinetobacter as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A kind of preparation of the non-peroxidase enzyme component of acinetobacter degrading ZEN comprises the following steps:

[0039] 1. Cultivation: Cultivate the culture (M2 medium) inoculated with 2% (v / v) Acinetobacter sp.SM04 suspension (OD600=0.8) in an air bath shaker at 30°C (180r / min) for 36h At the end of logarithmic growth, centrifuge the liquid culture for 12min (9000×g, 5°C), filter the centrifuged supernatant with a 0.22 μm filter membrane, and concentrate the filtrate 9 times at 50°C using a vacuum rotary evaporator to obtain crude enzyme liquid;

[0040] The formulation of the medium used is as follows:

[0041] M2 medium: 15g sodium acetate, 2.75g NH 4 NO 3 , 1.35g K 2 HPO 4 ·3H 2 O, 1.25g KCl, 0.5g MgSO 4 ·7H 2 O, add 10mL trace element stock solution, add distilled water to make up to 1000mL, adjust the pH to 7.3 after mixing;

[0042] Formula of trace element stock solution: 2g / L FeSO 4 ·7H 2 O, 0.5g / L MnSO 4 4H 2 O, 0.4g / LCuSO 4 ·5H 2 O...

Embodiment 2

[0051] The analysis and identification of the ZEN toxin degrading enzyme Oxa enzyme component comprises the following steps:

[0052]1. The ZEN toxin-degrading enzyme Oxa enzyme component finally obtained in Example 1 is analyzed by 12.5% ​​(w / v) SDS-PAGE gel electrophoresis, and after Coomassie brilliant blue staining, the result is as follows image 3 .

[0053] SDS-PAGE protein electrophoresis solution: 10% (w / v) sodium dodecylbenzenesulfonate (SDS) solution; 1% (v / v) tetramethylethylenediamine (TEMED); 10% (w / v) ) ammonium persulfate (AP);

[0054] Sample solution: 2% (w / v) SDS, 5% (v / v) β-mercaptoethanol, 10% (v / v) glycerol, 0.02% (w / v) bromophenol blue, 10mM pH8.0Tris- HCl buffer;

[0055] Gel storage solution: 30% separating gel storage solution, 10% concentrating gel storage solution;

[0056] Gel formulation: 4.9mL monomer, 5.0mL buffer, 3.5mL distilled water, 2.0mL glycerol, 50μL 10% (w / v) AP, 10μL TEMED.

[0057] Stacking gel formula: 1.62mL monomer, 3.10mL buf...

Embodiment 3

[0068] Cloning of ZEN Toxin Degrading Enzyme Oxa Gene

[0069] (1) Extract the total DNA of Acinetobacter sp.SM04

[0070] Take 1 mL of Acinetobacter sp.SM04 cells cultured to the logarithmic phase, first add 567 μL of TE buffer, then add 30 μL of 10% SDS and 15 μL of proteinase K, mix well, and incubate at 37 °C for 1 h. Add 100 μL 5mol / L NaCl, mix well, then add 80 μL CTAB (cetyltrimethylammonium bromide) / NaCl solution, mix well, and then incubate at 65°C for 10 minutes.

[0071] Add an equal volume of phenol / chloroform / isoamyl alcohol and mix well, centrifuge for 4-5 minutes, transfer the supernatant to a new tube, add 0.6-0.8 times the volume of isopropanol, and gently mix to precipitate DNA. After washing with 1 mL of 70% ethanol by volume, centrifuge, discard the ethanol, and air dry. Suspend the DNA in sterile water or 50 μL TE buffer to dissolve, and store at -20°C for later use.

[0072] (2) Acquisition of gene encoding ZEN toxin degrading enzyme Oxa

[0073] 1. P...

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PUM

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Abstract

The invention discloses a ZEN (zearalenone) toxin degrading enzyme Oxa of acinetobacter as well as a coding gene and application thereof. An amino acid sequence of the ZEN toxin degrading enzyme Oxa of acinetobacter is as shown in SEQ ID NO.6. A nucleotide sequence of the coding gene A4-Oxa of the ZEN toxin degrading enzyme Oxa of acinetobacter is as shown in SEQ ID NO.5. The ZEN toxin degrading enzyme Oxa of acinetobacter disclosed by the invention has stronger ability of degradation on mycotoxin zearalenone; the Oxa enzyme for prokaryotic recombinant expression can degrade over 30% of ZEN toxins within 12 hours. According to the invention, a non-peroxidase coding sequence, i.e., an A4-Oxa gene of Acinetobacter sp. SMO4-degreaded zearalenone toxin is determined, so that a foundation is laid for researching an active site of the enzyme and site-specific mutagenesis Oxa enzyme by a specific site in future.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a zearalenone (ZEN, ZEA) toxin-degrading enzyme non-peroxidase (named Oxa) of Acinetobacter sp.SM04 and its coding gene and application . Background technique [0002] Zearalenone (ZEARALENONE, ZEN, ZEA) is an estrogenic mycotoxin produced by fungi such as Gibberella zeara, Fusarium graminearum, Fusarium three-line, etc. It mainly contaminates wheat, barley, oats, corn and other crops and Animal food. Samples were taken from warehouses and feed factories across the country, and it was found that the detection rate of ZEN in corn and other crops and feed was as high as 100%, and the detected concentration exceeded the standard seriously. Xie Liangmin et al.’s survey of various foods in large supermarkets in Shanghai showed that the detection rate of ZEN is extremely high, and the seasonings all exceed the national standard (ZEN content in food ≤60μg / kg), and the ...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52A23L1/015C12R1/01A23L5/20
CPCA23L5/25C12N9/00
Inventor 唐语谦钟凤陈艺吴晖
Owner 河南亿万中元生物技术有限公司
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