Zearalenone hydrolase with improved trypsin resistance

A technology of zearalenone and trypsin, applied in hydrolytic enzymes, microbial-based methods, animal feed, etc.

Active Publication Date: 2021-12-07
JINAN UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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  • Zearalenone hydrolase with improved trypsin resistance
  • Zearalenone hydrolase with improved trypsin resistance
  • Zearalenone hydrolase with improved trypsin resistance

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Embodiment 1

[0025] Example 1: Synthesis of Zearalenone Hydrolase Gene

[0026] The present invention adopts the gene of wild-type zearalenone hydrolase derived from Clonostachys rosea (GenBank registration number is KR363960.1), which is synthesized by Shanghai Jierui Gene Company (other commercial companies with full gene synthesis can also complete it).

Embodiment 2

[0027] Embodiment 2: the connection of zearalenone hydrolase gene (ZHD101) and cloning vector Taox+PgHT+BBPB

[0028] 1. The pGH plasmid containing the ZHD101 target gene synthesized by the whole gene and the cloning vector Taox+PgHT+BBPB were respectively digested with restriction endonucleases EcoRI and SpeI / XbaI at 37°C for 30min, and the digestion conditions were as follows:

[0029]

[0030] 2. The digested products were subjected to 1% agarose gel electrophoresis and the two target fragments were respectively recovered, and T 4 DNA ligase connection, the connection system is as follows:

[0031] ZHD101 digestion product 7.0 μL Cloning Vector Restriction Products 1.0 μL T 4 DNA ligase

[0032] Use DNA ligase to ligate at 16°C for 16 hours, transform the ligation product into DH5α competent cells and amplify it, then use a plasmid extraction kit to extract the plasmid, digest it with EcoRI and PstI, and run electrophoresis results showing two b...

Embodiment 3

[0033] Embodiment 3: gene fragment Paox+Pgap+SS1 is connected with cloning vector M+Taox+PgHT+PB

[0034] 1. The gene fragment Paox-+Pgap+SS1 was transferred from the cloning vector Paox+SS1+PB preserved by the inventor, and obtained by double digestion with EcoRI and SpeI endonucleases, purification and recovery;

[0035] 2. The cloning vector M+Taox+PgHT+PB is obtained from Example 2, and the connection method between the gene fragment Paox+Pgap+SS1 and the cloning vector M+Taox+PgHT+PB is the same as in Example 2.

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Abstract

The invention discloses a zearalenone hydrolase with improved trypsin resistance. The zearalenone hydrolase is an enzyme which is produced by single amino acid substitution in a zearalenone hydrolase derived from Clonostachys rosea and having an amino acid sequence of SEQ ID NO. 1 and has stronger trypsin resistance, wherein the amino acid substitution is the substitution at the 262 site. The invention provides a zearalenone hydrolase mutant with improved trypsin resistance by modifying key amino acid residues in a wild zearalenone hydrolase molecule through a protein engineering technology. The resistance half-life period of the enzyme to trypsin is prolonged by 20.8% compared with that of the wild zearalenone hydrolase, and other enzymatic properties are basically consistent with those of the wild zearalenone hydrolase.

Description

technical field [0001] The present invention relates to zearalenone hydrolase, in particular to zearalenone hydrolase with improved resistance to trypsin. Background technique [0002] Zearalenone hydrolase (ZHD101 for short), also known as zearalenone degrading enzyme. The zearalenone degrading enzyme ZHD101 gene is a gene encoding pantolactone hydrolase in Clonosporium rosea, and the protein ZHD101 encoded by the gene can specifically bind and degrade zearalenone. The reaction degradation mechanism is that ZHD101 breaks the ester bond of ZEN into a dihydroxyphenyl derivative with an open side chain, and then loses CO 2 A non-toxic alkyl resorcinol product is obtained which is non-toxic. Therefore, it is often used as a food additive, or as a feed additive to improve feed utilization. [0003] ZHD101 is divided into a hydrolase fold central domain and a cap structure. The junction of the two structures forms a larger groove, which is confirmed by the structure of the su...

Claims

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Application Information

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IPC IPC(8): C12N9/18C12N15/55C12N15/81C12N1/19A23L29/00A23K10/14C12R1/84
CPCC12N9/18C12N15/815A23L29/06A23K10/14
Inventor 姚冬生牛芳园钱丹刘大岭谢春芳刘桂祯黄炯威莫世艺
Owner JINAN UNIVERSITY
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