A kind of zearalenone degrading enzyme and its application
A technology for degrading zearalenone and enzymes, which is applied in the field of enzyme engineering and can solve the problems of few types of zearalenone degrading enzymes and poor stability
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0028] This example specifically illustrates the screening method for zearalenone-degrading bacteria.
[0029] (1) Enrichment of strains
[0030] A portion (2 g) of the sample soil was separately added to the Erlenmeyer flask and incubated with sterile water for 1 hour at 30°C. Take 100 μL of the suspension, add it to 5 mL of enriched medium containing 2 μg / mL zearalenone, and incubate in a constant temperature shaker (200 rpm) at 37° C. for 72 h.
[0031] The enriched medium containing zearalenone is: 1.52g / L KH 2 PO 4 ,2.44g / L Na 2 HPO 4 ,0.2g / L MgSO 4 .7H 2O, 0.5g / L (NH 4 ) 2 SO 4 ,0.05g / L CaCl 2 , 10.0g / L NaCl, 2μg / mL zearalenone.
[0032] (2) Screening of zearalenone-degrading bacteria by plate method
[0033] Aspirate 100 μL of the enrichment solution cultured for 3 days, centrifuge at 3,000 rpm for 5 min, discard the supernatant, suspend the precipitate with 100 μL of screening medium containing 2 μg / mL zearalenone, spread it on the plate, and cultivate at 3...
Embodiment 2
[0039] This embodiment specifically illustrates the method for preparing the concentrated enzyme solution of zearalenone degrading enzyme by utilizing zearalenone degrading bacteria.
[0040] 1. Cultivation: Culture (YPD medium) inoculated with 2% (v / v) bacterial suspension (OD600=0.8) in a constant temperature shaker at 30°C (200rpm) for 36h to the end of logarithmic growth, The liquid culture was centrifuged for 12 min (5000 rpm, 4 °C), the centrifuged supernatant was filtered with a 0.22 μm filter membrane, and the filtrate was concentrated 9 times at 50 °C using a vacuum rotary evaporator to obtain a crude enzyme solution;
[0041] The formulation of the medium used is as follows:
[0042] YPD medium: Preparation method: Weigh 10g of yeast powder and 20g of peptone, dissolve in 900mL of ultrapure water, pressurize at 121°C for 20min, add 100ml of 20g / L glucose (Glucose is sterilized at 115°C for 20min).
[0043] 2. Add the crude enzyme solution in step 1 to an anionic sep...
Embodiment 3
[0048] This example illustrates the method of cloning the zearalenone degrading enzyme LD08 gene.
[0049] The concentrated enzyme solution obtained in Example 2 was separated by SDS-PAGE, the corresponding protein band was cut out, and MALDI-TOF / TOF / MS analysis and identification were carried out, and it was identified as a lipohydrolase. Combined with the genome information of Exophialaspinifera, the amino acid sequence of the enzyme is obtained as SEQ ID NO.2, and the nucleotide sequence is obtained as SEQ ID NO.1.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More