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Acid-resistant zearalenone detoxifying enzyme and encoding gene thereof, and application of enzyme and gene

A technology of zearalenone and detoxification enzymes, applied in the field of enzyme engineering, can solve the problems of inhibition, zearalenone detoxification enzyme activity (low expression level, etc.), achieve good degradation effect and improve detoxification efficiency

Active Publication Date: 2017-08-29
北京中农探味科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the optimum pH of the enzyme is 8.5, and the degradation ability of the enzyme at pH 5.0 and 6.5 is only 15% and 25% of that at pH 8.5, respectively, which shows that the acidic condition seriously inhibits the detoxification enzyme of zearalenone activity; the detoxification enzyme is mainly mixed into pig and chicken feed as an additive to degrade zearalenone in pigs and chickens, and the pH value of the intestinal tract of pigs and chickens is between 5.5 and 6.5, so there is an urgent need for corn The acid-resistant transformation of erythralenone detoxification enzyme makes the optimal pH of its activity change from 8.5 to slightly acidic
Not only that, the above technical scheme also connects the zearalenone detoxification enzyme coding gene obtained by cloning to the plasmid pPIC9K and transforms Pichia pastoris to obtain a recombinant Pichia pastoris strain capable of expressing zearalenone detoxification enzyme, but zearalenone detoxification enzyme The activity (expression level) of mycocetone detoxification enzyme is very low, and 1mL of recombinant yeast fermentation supernatant can only degrade 18.8μg of ZEN in 2 hours

Method used

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  • Acid-resistant zearalenone detoxifying enzyme and encoding gene thereof, and application of enzyme and gene
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  • Acid-resistant zearalenone detoxifying enzyme and encoding gene thereof, and application of enzyme and gene

Examples

Experimental program
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Effect test

Embodiment 1

[0046] Example 1 Acid-resistant modification and codon preference optimization of zearalenone detoxification enzyme

[0047] 1. Acid-resistant transformation of zearalenone detoxification enzyme: on the basis of zlhy-6 detoxification enzyme (ZL201110082679.X, SEQ ID NO.6), arginine R at position 52, lysine K at position 66, Arginine R at position 118, Lysine K at position 148, Arginine R at position 175, Arginine R at position 185, amino acid R at position 189 and Arginine at position 204 were replaced by glutamic acid. Zearalenone detoxification enzyme (ZDE-3) was obtained, and the specific amino acid sequence is shown in SEQ ID NO.1.

[0048] 2. The coding gene of zearalenone detoxification enzyme is optimized according to the yeast codon preference on the basis of amino acid residue transformation, and the zearalenone detoxification enzyme gene (zde-3) is obtained, and the specific nucleoside See SEQ ID NO.2 for the acid sequence.

Embodiment 2

[0049] The construction of embodiment 2 recombinant expression vector

[0050] 1. Add an alpha signal peptide coding sequence, i.e. alpha-zde-3, to the front end of the zearalenone detoxification enzyme gene (zde-3) designed in Example 1, and introduce EcoRI at both ends of the sequence ( GAATTC )Restriction sites. See SEQ ID NO.3 for the full sequence nucleotide sequence. The nucleotide sequence is obtained by chemical synthesis.

[0051] 2. Construction of recombinant expression vector containing zearalenone detoxification enzyme coding gene sequence

[0052] (1) Digest the synthetic α-zde-3 nucleotide sequence and plasmid pAO815 (Invitrogen, USA) with the restriction endonuclease EcoRI, 20 μL of restriction enzyme digestion system: 15 μL of target fragment or plasmid, 2 μL of 10×EcoRI Buffer , EcoRI enzyme 1 μL, ddH 2 O 2 μL, enzyme digestion conditions 37 ℃ reaction 4h.

[0053] (2) Vector pAO815 dephosphorylation reaction system: 15 μL vector after enzyme digestion,...

Embodiment 3

[0059] Example 3 High Expression of Zearalenone Detoxification Enzyme

[0060] The recombinant expression vector pAO815-α-zde-3, pAO815-(α-zde-3) 2 , pAO815-(α-zde-3) 4 and pAO815-(α-zde-3) 6 After linearizing it with Sal I, the linearized vector was introduced into Pichia pastoris GS115 by electroporation, and passed through MD selective medium (1.34% YNB Yeast Nitrogen Base without Amino Acids), 2% Glucose (D-glucose), 4×10 -5 Biotin (Biotin), 2% agar (Agar)) to screen high-expression strains.

[0061] A single colony grown on the selective medium was picked and inoculated into 5mL BMGY (1% glycerol, 1% yeast extract (Yeast Extract), 2% peptone (Peptone), 1.34% YNB, 4×10 -5 Biotin, 100mM potassium phosphate (PH6.0) culture medium, cultured at 30°C, 250rpm for 36h, centrifuged at room temperature at 5000rpm for 5min to collect the bacteria. Cells were treated with 2mL BMMY (0.5% methanol, 1% Yeast Extract, 2% peptone (Peptone), 1.34% YNB, 4×10 -5 Biotin, 100mM potassium...

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Abstract

The invention relates to the field of enzyme engineering, and in particular discloses acid-resistant zearalenone detoxifying enzyme and an encoding gene thereof, and application of the enzyme and the gene. The amino acid sequence of the zearalenone detoxifying enzyme is optimized, 8 amino acid residues (with 8 amino acid difference from zlhy-6 detoxifying enzyme) of the detoxifying enzyme are changed, the optimal reaction pH of recombinant expression zearalenone detoxifying enzyme is reduced from 8.5 to 6.5 and is very identical with the pH of pig and chicken intestinal canals, and the enzyme activity of the detoxifying enzyme still maintains 40 percent or more when the pH is reduced to 5.0, so the detoxifying efficiency of the zearalenone detoxifying enzyme serving as a feed additive to actual application is greatly improved. The zearalenone detoxifying enzyme gene is subjected to codon optimization, and the copy number of the gene in a recombinant yeast genome is increased to be 4, so the expression quantity of the zearalenone detoxifying enzyme is greatly increased.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to an acid-resistant zearalenone detoxification enzyme, its coding gene and its application. Background technique [0002] Zearalenone (Zearalenone, ZEN), also known as F-2 toxin, is a 2,4-dihydroxybenzoic acid lactone compound, a mycotoxin mainly produced by the secondary metabolism of Fusarium graminearum. ZEN has estrogenic activity and is toxic to the reproductive system. Excessive intake of ZEN can cause vulva and mammary gland enlargement and even vaginal and rectal prolapse; affect mammalian breast development, lead to delayed lactation, false pregnancy, and even infertility or miscarriage , fetal malformation and stillbirth. ZEN can induce tumors, not only stimulate the growth of human breast cancer MCF-7 cells by increasing the activity of cytochrome CYP1A1 enzyme, but also one of the causes of the increased incidence of esophageal cancer. ZEN also has obvious toxic effe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12N15/56C12N15/81C12N15/66C12N1/19A23L5/20A23K10/14C12R1/84
CPCA23K10/14A23L5/25C12N9/24C12N15/66C12N15/815C12N2800/22
Inventor 刘阳邢福国
Owner 北京中农探味科技有限公司
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