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Separation method of components of non-peroxide enzyme capable of degrading zearalenone toxins

A zearalenone, non-peroxide technology, applied in the field of microbial applications, can solve the problems of inability to purify non-peroxidase enzymes

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

AI Technical Summary

Problems solved by technology

The method of isolating peroxidase only yields pure peroxidase and cannot be applied to the purification of non-peroxidase

Method used

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  • Separation method of components of non-peroxide enzyme capable of degrading zearalenone toxins
  • Separation method of components of non-peroxide enzyme capable of degrading zearalenone toxins
  • Separation method of components of non-peroxide enzyme capable of degrading zearalenone toxins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] (1) Culture: Inoculate with 1% (v / v) Acinetobacter sp.SM04 suspension (OD 600 =0.6) culture (M2 medium) was cultured in an air-bath shaker at 28°C (150r / min) for 24h, the liquid culture was centrifuged for 10min (8000×g, 4°C), and the supernatant after centrifugation was used 0.22μm membrane filtration, and the filtrate was concentrated 8 times into a crude enzyme solution at 45°C using a vacuum rotary evaporator;

[0073] The formulation of the medium used is as follows:

[0074] M2 medium: 12.5g sodium acetate, 2.5g NH 4 NO 3 , 1.2g K 2 HPO 4 ·3H 2 O, 1g KCl, 0.4g MgSO 4 ·7H 2 O, add 10mL trace element stock solution, add distilled water to make up to 1000mL, adjust the pH to 7.0 after mixing;

[0075] The formula of described trace element stock solution is as follows: 2g / L FeSO 4 ·7H 2 O, 0.5g / L MnSO 4 4H 2 O, 0.4g / L CuSO 4 ·5H 2 O, 0.5g / L CoCl 6 ·6H 2 O and 0.4g / L ZnCl 2 .

[0076] (2) Add the crude enzyme solution in step (1) to an anionic Sephad...

Embodiment 2

[0084] (1) Culture: inoculate with 2% (v / v) Acinetobacter sp.SM04 suspension (OD 600 =0.8) culture (M2 medium) was cultured in an air-bath shaker at 30°C (180r / min) for 36h to the end of logarithmic growth, and the liquid culture was centrifuged for 12min (9000×g, 5°C). The supernatant was filtered with a 0.22 μm filter membrane, and the filtrate was concentrated 9 times at 50°C using a vacuum rotary evaporator to become a crude enzyme solution;

[0085] The formulation of the medium used is as follows:

[0086] 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;

[0087] The formula of the trace element stock solution is the same as that in Example 1-(1).

[0088] (2) Add the crude enzyme solution in step (1) to an anionic Sephadex column DEAE Sephadex A-50 column (2.0cm×15cm), and use phosphoric aci...

Embodiment 3

[0094] (1) Culture: Inoculate with 3% (v / v) Acinetobacter sp.SM04 suspension (OD 600 =1.0) culture (M2 medium) was cultured in an air bath shaker at 28°C (200r / min) for 48h, the liquid culture was centrifuged for 15min (10000×g, 6°C), and the supernatant after centrifugation was used 0.22μm membrane filtration, and the filtrate was concentrated 10 times by a vacuum rotary evaporator at 55°C to form a crude enzyme solution;

[0095] The formulation of the medium used is as follows:

[0096] M2 medium: 17.5g sodium acetate, 3g NH 4 NO 3 , 1.5g K 2 HPO 4 ·3H 2 O, 1.5g KCl, 0.6g MgSO 4 ·7H 2 O, add 10mL trace element stock solution, add distilled water to make up to 1000mL, adjust the pH to 7.5 after mixing;

[0097] The formula of the trace element stock solution is the same as that in Example 1-(1).

[0098] (2) Add the crude enzyme solution in step (1) to an anionic Sephadex column DEAE Sephadex A-50 column (2.0cm×15cm), and use phosphoric acid with pH 7.0, 6.5, 6.0, 5...

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Abstract

The invention discloses a separation method of components of a non-peroxide enzyme capable of degrading zearalenone toxins. The separation method comprises the following steps: firstly purifying acinetobacter crude enzyme by using an anionic sephadex column, and carrying out gradient elution by using 15-25mM of sodium phosphate buffer liquid; then purifying the acinetobacter crude enzyme by using a cationic sephadex column, and eluting by using 10-30mM of Tris-HCl buffer liquid; and purifying the acinetobacter crude enzyme by using the anionic sephadex column again, and carrying out gradient elution by using 15-25mM of sodium phosphate buffer liquid, thus obtaining the components of the non-peroxide enzyme capable of degrading the zearalenone toxins in the end. The obtained components of the non-peroxide enzyme capable of degrading the zearalenone toxins have relatively strong fungal zearalenone toxin degradation capability, are capable of degrading above 60% of ZEN within 12 hours without the synergistic effect of hydrogen peroxide and lay the foundation for the future research of the zearalenone toxin degradation property of non-peroxide enzyme and the application of the non-peroxide enzyme.

Description

technical field [0001] The invention belongs to the field of microbial application, and in particular relates to a method for separating non-peroxidase enzyme components capable of degrading zearalenone (ZEA, ZEN) toxins. 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. 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. ZEN can cause premature maturation in breeding pigs or pou...

Claims

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Application Information

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IPC IPC(8): C12N9/18A23L1/015A23K1/165C12R1/01A23L5/20
CPCA23K20/189A23L5/25C12N9/18
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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