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Zearalenone hydrolase ZHD101 mutant and method for hydrolyzing zearalenone by using same

A mutant and hydrolase technology, applied in the field of hydrolysis of zearalenone, can solve the problems of unsatisfactory catalytic efficiency, irreversible deactivation of the enzyme, etc., and achieve the effects of improved catalytic activity, high expression activity, and improved enzyme activity

Active Publication Date: 2019-05-31
JILIN COFCO BIOCHEM +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Current research shows that ZHD101 has a good catalytic efficiency in an environment of 35-45°C and pH>6, but its catalytic efficiency is not ideal in an acidic system, and pH conditions lower than 4.5 may even cause irreversible inactivation of the enzyme (Takahashi-Ando N et al., Metabolism of Zearalenone by Genetically Modified Organisms Expressing the Detoxification Gene from Clonostachys rosea. Applied and Environmental Microbiology 2004; 70:3239-3245)

Method used

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  • Zearalenone hydrolase ZHD101 mutant and method for hydrolyzing zearalenone by using same
  • Zearalenone hydrolase ZHD101 mutant and method for hydrolyzing zearalenone by using same
  • Zearalenone hydrolase ZHD101 mutant and method for hydrolyzing zearalenone by using same

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Experimental program
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Embodiment 1

[0064] Embodiment 1, preparation and purification of ZHD101 mutant

[0065] 1. Construction of ZHD101 mutant coding gene and expression vector

[0066] The coding sequence (SEQ ID NO.2) of wild-type zearalenic ketohydrolase was replaced with the DNA sequence (SEQ ID NO.17) composed of Escherichia coli preferred codons, and corresponding mutations were introduced to obtain SEQ ID NO. 4. The nucleotide sequences shown in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14 and SEQ ID NO.16, at the 5' of the above sequence The Nde I restriction site CATATG was added to the end, and the DNA sequence CACCATCACCACCATCAC (SEQ ID NO.18) encoding the hexahistidine tag, the stop codon TAA and the Xho I restriction site CTCGAG were sequentially added to the 3' end, chemically synthesized The resulting DNA sequence (Invitrogen, USA). A histidine tag 6×His was added to the C-terminal end of the encoded protein sequence to facilitate subsequent purification steps. The resul...

Embodiment 2

[0080] Embodiment 2, the detection of the catalytic activity of ZHD101 mutant

[0081] The target protein obtained in Step 3 of Example 1 was analyzed by HPLC (LC-20AT, Shimadzu Corporation), so as to determine the catalytic activity and kinetic parameters of the enzyme mutant. HPLC profile such as image 3 Shown (exemplarily shows the experimental results of ZHD101M3).

[0082] Specifically, the selected chromatographic column is a Hypersil C18 reverse-phase column (Elite, 5 μm, 4.6 mM×250 mM). The proportion of the reaction system is: 4850 μL sodium acetate (pH 5.5), 50 μL substrate (dissolved in acetonitrile, the concentration changes in a gradient, the highest concentration is 2 mg / mL), 100 μL enzyme solution (stored in 100 mM Tris buffer, pH 8.0 , concentration 100 μg / mL), and reacted for 10 min in a constant temperature water bath at 37°C. After the reaction, boil in boiling water for 1 min to terminate the reaction.

[0083] The mobile phase ratio (v / v) of HPLC is: ...

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Abstract

The invention relates to a zearalenone hydrolase ZHD101 mutant and a method for hydrolyzing zearalenone by using the mutant. The mutant is obtained by introducing mutations at three key sites of the wild-type zearalenone hydrolase ZHD101. Compared with wild-type ZHD101, the ZHD101 mutant has the advantages that the specific activity for ZEN hydrolysis is improved by at least 75% under the acidic condition (pH 5.5); also, the catalytic efficiency (kcat / KM) for ZEN hydrolysis under acidic conditions (pH 5.5) can also be increased by as much as 40% compared with a wild-type ZHD101. Therefore, a foundation is laid for further improving the catalytic activity of the enzyme and realizing the industrial application under the acidic condition.

Description

technical field [0001] The invention relates to a mutant of zearalenone hydrolase ZHD101 and a method for hydrolyzing zearalenone by using the mutant. Specifically, the present invention relates to a zearalenone hydrolase ZHD101 mutant obtained through genetic modification and protein engineering that still has excellent activity under acidic conditions and a method for using the mutant to hydrolyze zearalenone . Background technique [0002] Mycotoxins are secondary metabolites of fungi. Many mycotoxins have teratogenic, carcinogenic, neurotoxic, and immunosuppressive effects. The negative impact on global food safety cannot be ignored (Kabak B, Dobson ADW, VarI.Strategies to Prevent Mycotoxin Contamination of Food and Animal Feed: AReview. Critical Reviews in Food Science and Nutrition 2006; 46:593-619). Zearalenone (Zearalenone, ZEN) is an estrogen-toxic non-steroidal toxin produced by Fusarium fungi, which has a dihydroxybenzoic acid lactone structure (Zinedine A, Sori...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55
Inventor 佟毅陈博朱玉山林敏苏会波王靖唐堂杨鑫吴延东赵雪松
Owner JILIN COFCO BIOCHEM
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