Deoxynivalenol toxin degrading enzyme as well as encoding gene and application thereof
A technology of deoxynivalenol and coding gene, applied in the field of enzyme engineering, can solve the problems of non-allowance, loss of nutrients, change of food quality, etc., and achieve the effect of controlling food pollution
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Embodiment 1
[0027] Example 1 Acquisition of deoxynivalenol degrading enzyme gene Dasag and expression of deoxynivalenol degrading enzyme
[0028] Construction of Plasmid Library of Deoxynivalenol Degrading Enolase Gene
[0029] (1) After activating the standard strain Fusarium oxysporum ACCC No.36245 (purchased from China Agricultural Microorganism Culture Collection and Management Center), it was inserted into PDA liquid medium (potato 200g / L, glucose 20g / L, agar 20g / L, The pH is natural), and then the suspension of the strain was added to 100ml of the same medium, and the shaker condition was 220 rpm, 30°C, 72 hours. After the cultivation was completed, the bacteria were collected by centrifugation at 12000 rpm. RNA extraction (Trizol method);
[0030] (2) Using RNA as a template to reverse transcribe and synthesize a cDNA sequence;
[0031] 2. Acquisition of deoxynivalenol degrading enzyme gene
[0032] Using the cDNA sequence obtained in step 1 as a template, a PCR reaction was pe...
Embodiment 2
[0042] Example 2 Activity detection of deoxynivalenol degrading enzyme
[0043] Sample processing: Recombinant Pichia pastoris ASAG2 strain (experimental group) and wild type Pichia pastoris GS115 strain (control group, empty plasmid integration strain) induced expression. After 5 days, 800 μL of the supernatant was taken and placed in a 1.5 mL centrifuge tube, and then 200 μL of 100 ppm DON was added to make the final concentration reach 20 ppm. After the addition, react at 30°C for 0h-5h for post-treatment. Take 10 μL of the processed sample and enter the high performance liquid chromatography to detect the residue of DON. The detection method is carried out according to the GB / T 23504-2009 method. HPLC detection conditions: Agilent Eclipse XDB-C18, 150mm×4.6mm (5μm) chromatographic column, mobile phase: water / methanol=80% / 20%, UV detector detection, column temperature 30°C, flow rate 1.0ml / min, the injection volume is 10 μL. Through the detection of high performance li...
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