Zeranol decomposing enzyme and coding gene and application thereof
A technology of erythralenone and coding genes, which is applied in hydrolytic enzymes, genetic engineering, plant genetic improvement, etc., can solve the problems of nutrient loss, chemical reagent residue, toxin degradation effect is not obvious, etc., to achieve the prevention and treatment of mycotoxin poisoning Effect
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Embodiment 1
[0020] Example 1: Acquisition of zearalenone decomposing enzyme gene (ZEN-jlm)
[0021] The inventors used TaKaRa Smart TM The cDNA library construction kit was used to construct the cDNA library of Gliocladium roseum. Randomly picked clones were sequenced, and the measured sequence was compared with the sequence in the international gene bank (GenBank) by BLAST to obtain the red cDNA of enonelolytic enzyme (ZEN-jlm).
[0022] 1. Extraction of total RNA from Gliocladium roseum
[0023] Clonospora rosea was cultured in liquid PDA medium (200 rpm, 30° C., 48 h). After the culture was completed, the precipitate was collected by centrifugation at 12000 r / min for 10 min. Wash with 75% ethanol solution prepared with DEPC treated water. Dry at room temperature, dissolve and precipitate RNA in RNase-free water, and two clear bands of 28S and l8S can be seen by 1% agarose gel electrophoresis; 260 / OD 280 The ratio identifies the purity of the total RNA, and the ratio is 1.95; stor...
Embodiment 2
[0061] Example 2: Recombinant expression of zearalenone decomposing enzyme in Bacillus subtilis
[0062] 1. According to SEQ ID NO: 1, design primers as follows:
[0063] P1: 5'-GTGGGGTTCAAAGAAAGGCCTG-3' (SEQ ID NO: 3);
[0064] P2: 5'-CCCAAGCTT CTAGAACTTGCCAAGGAACTTGGC-3' (introducing a HindIII restriction site) (SEQ ID NO: 4).
[0065] Using the pET30a-ZENjlm plasmid as a template to amplify the ZEN-jlm gene, the PCR system is as follows:
[0066]
[0067]
[0068] Reaction program: pre-denaturation at 94°C for 3min; denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 1min, 35 cycles; extension at 72°C for 10min. 1% agarose gel electrophoresis to detect PCR products.
[0069] 2. Using the Bacillus subtilis genomic DNA as a template, set primers to amplify P 43 Promoter sequence, EcoRI restriction site was introduced into primer P3, and the designed primers were as follows:
[0070] P3: 5'-CGGAATTCCTTGTAGAGCTCAGCATTATTG-3' (SEQ ID NO: 5);...
Embodiment 3
[0106] Example 3: Separation and purification of recombinant erythralenone decomposing enzyme
[0107] Preparation of seed solution: Inoculate the recombinant strain WB800-ZEN-jlm into LB medium, fill a 250mL Erlenmeyer flask with a liquid volume of 50mL, then place it in a constant temperature shaking shaker at 37°C, 200r / min, and cultivate for 24h.
[0108] Preparation and separation and purification of fermentation supernatant: 14L LB medium was placed in a 20L fermentation hall, and after autoclaving, 5% (v / v) inoculum was used to inoculate the shake flask seed solution. During the fermentation, the dissolved oxygen content was 60%, the magnetic stirring speed was 200r / min, and the fermentation was carried out at 37°C for 24 hours. After the fermentation was completed, the fermentation broth was centrifuged at 5000r / min for 10min in a refrigerated centrifuge at 4°C. The supernatant was used for protein precipitation. With 40% saturation (NH 4 ) 2 SO 4 Solution, 4°C, 5h...
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