Zearalenone toxin degradation enzyme and coding gene and application thereof
A technology of zearalenone and coding genes is applied in the field of enzyme engineering to achieve the effect of controlling food pollution
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Embodiment 1
[0029] Example 1 Acquisition of zearalenone-degrading enzyme gene zlhy-6 and expression of zearalenone-degrading enzyme
[0030] 1. Construction of zearalenone degrading enzyme gene plasmid library
[0031] Break the mouth of the ampoule tube of the standard strain M. pink ACCC No.31535 (purchased from the Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences) and add 300-500 μL of liquid PDA medium (potato 200g / L, glucose 20g / L) , agar 20g / L, PH natural), and then the suspension of the strain was added to 100ml of the same medium, and the shaking conditions were 220 rpm, 30 ° C, and 72 hours. After the cultivation was completed, the cells were collected by centrifugation at 12,000 rpm in a centrifuge. RNA extraction (Trizol method);
[0032] (2) synthesizing a cDNA sequence by reverse transcription using RNA as a template;
[0033] 2. Obtaining the zearalenone degrading enzyme gene
[0034] Using the cDNA sequence obtained in step 1 as a templa...
Embodiment 2
[0045] Embodiment 2 Activity detection of zearalenone degrading enzyme
[0046] Sample processing: Recombinant Pichia strains (experimental group) and wild-type Pichia strains (control group, Pichia strains integrated with empty plasmids) were inserted into BMGY medium and continued to culture to the OD of the bacterial solution 600 = 2-3, the cells were collected by centrifugation at room temperature, resuspended in BMMY medium for 5 days, and 1 mL of culture medium was taken out every day, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. Take 980 μL of the supernatant into a 1.5 mL centrifuge tube, and then add 20 μL of 1000 ppm ZEN to make the final concentration 20 ppm. After the addition, the reaction was performed at 30°C for 2h-10h, and the post-treatment was placed at 4°C for detection. Take 20 μL of the treated sample and put it into high performance liquid chromatography to detect the ZEN residue. The detection method is carried out according ...
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