Epimedium mediator and its application to improve the degradation rate of laccase to mycotoxin
A mycotoxin and degradation rate technology, applied in the field of agricultural biology, can solve the problems of high price, poor stability, and potential toxicity, and achieve the effect of efficient degradation, low cost, and wide application range
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0032] The preparation of embodiment 1 recombinant laccase BsCotA
[0033] Take the BL21(DE3) / BsCotA Escherichia coli engineering strain containing the recombinant plasmid, inoculate it in 50mL LB culture medium, and culture it with shaking at 37°C and 220rpm for 12h, then transfer it to 300mL LB medium at a ratio of 2%, at 37°C and 220rpm Shake the culture for about 3 hours (OD600≈0.6), add the inducer IPTG at a final concentration of 1 mM, induce at 16°C for 15 hours, and collect the cells by centrifugation. The bacteria were resuspended in disodium hydrogen phosphate-citric acid buffer (20 mM, pH 7.5). The bacterium was lysed by ultrasonic disruption, the broken bacterium fragments were centrifuged to remove, purified by Ni affinity chromatography column, and the electrophoretic pure eluted fraction was collected and dialyzed to Tris-HCl protein stock solution (50mM Tris-HCl , pH7.4, 150mM NaCl).
Embodiment 2
[0034] Example 2 Preparation of Epimedium water extract mediator
[0035] 2.1 Dry the Epimedium plants in an oven at 38°C for 4 hours. Put the dry plants into the pulverizer for pulverization, 1 min / time, twice in total. Pure water was used as the extractant, the ratio of solid to liquid was 33g / L, the pH was adjusted to about 8.7, the temperature was 45°C, the power was 600W, ultrasonic extraction was performed for 20min, and the precipitate was discarded by centrifugation. The supernatant was transferred to a round-bottomed flask at a temperature of 45° C. and a rotation speed of 50 rpm, and was rotated to evaporate. Add about 20mL of distilled water to the flask, mix well and pour into two 50mL centrifuge tubes. Freeze at -80°C for 2 hours. freeze-dried to obtain an aqueous extract.
[0036] Prepare 50mg / mL Epimedium water extract solution, mix by inversion at room temperature for 1h, centrifuge at 12000rpm for 5min, and discard the precipitate.
[0037] 2.2 Dry the Ep...
Embodiment 3
[0041] Example 3 BsCotA-Epimedium water extract mediator system degrades aflatoxin B1
[0042] Dissolve aflatoxin B1 in dimethyl sulfoxide to prepare a 50mg / L stock solution, and follow the following reaction system: 50mM Tris-HCl (pH 7.0), 20μL aflatoxin B1 solution (50mg / L), 20μL epimedium Aqueous solution of Huohu water extract (50mg / mL), 20μL BsCotA (300U / L). The system without adding laccase BsCotA was used as the control, and the reaction system was set up for 3 repetitions. The reaction was carried out at 30° C., and three times the volume of acetonitrile was added to terminate the reaction after 10 hours. The degradation rate of aflatoxin B1 was analyzed by high performance liquid chromatography (HPLC). The liquid chromatography is a Shimadzu Nexera UHPLC high-performance liquid chromatography analysis system, and the chromatographic separation column is Zorbax SB-C18 (4.6×250mm, 5μm), mobile phase A (water with 0.06% TFA), mobile phase B (water with 0.05% TFA Aceton...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More