Application of caffeic acid as a mediator for laccase degradation of mycotoxins
A technology of mycotoxins and caffeic acid, applied in the field of agricultural biology, can solve the problem of low degradation rate of mycotoxins
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Embodiment 1
[0023] The preparation of embodiment 1 recombinant laccase BsCotA
[0024] Take the BL21(DE3) / BsCotA Escherichia coli engineering strain containing the recombinant plasmid, inoculate it in 50mL LB culture medium, shake at 220rpm at 37°C for 12h, transfer it to 300mL LB medium at a ratio of 2%, shake at 220rpm at 37°C Cultivate for about 3 hours (OD600≈0.6), add inducer IPTG at a final concentration of 1 mM, induce at 16°C for 15 hours, and collect the bacteria by centrifugation. The bacteria were resuspended in disodium hydrogen phosphate-citric acid buffer (20 mM, pH 7.5). Cells were lysed by ultrasonication. The broken bacterial fragments were centrifuged to remove them, purified by Ni affinity chromatography, and the pure eluted fractions were collected and dialyzed to Tris-HCl protein stock solution (50mM Tris-HCl, pH7.4, 150mM NaCl) middle.
Embodiment 2
[0025] Example 2 BsCotA-mediator system degrades aflatoxin B 1
[0026] aflatoxin B 1 Dissolved in dimethyl sulfoxide to prepare a 50mg / L mother solution, according to the following reaction system: 50mM Tris-HCl (pH 7.0), 20μL aflatoxin B 1 Solution (50mg / L), 20μL caffeic acid solution (10mM), 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, after 10 hours, three times the volume of acetonitrile was added to terminate the reaction, and aflatoxin B was analyzed by high performance liquid chromatography (HPLC) 1 degradation rate. The liquid chromatography is a Shimadzu Nexera UHPLC high-performance liquid chromatography analysis system, and the chromatographic separation column is ZorbaxSB-C18 (4.6×250mm, 5 μm), mobile phase A (water of 0.06% TFA), mobile phase B (acetonitrile of 0.05% TFA ); Gradient elution conditions: 0% B elution for 4 minu...
Embodiment 3
[0028] Example 3 Degradation of Zearalenone by BsCotA-Mediator System
[0029] Dissolve zearalenone in dimethyl sulfoxide to prepare a 50mg / L mother solution, according to the following reaction system: 50mM Tris-HCl (pH 7.0), 20μL aflatoxin B 1 Solution (50mg / L), 20μL caffeic acid solution (10mM), 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 h. The degradation rate of zearalenone 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 ZorbaxSB-C18 (4.6×250mm, 5 μm), mobile phase A (water of 0.06% TFA), mobile phase B (acetonitrile of 0.05% TFA ); Gradient elution conditions: 0% B elution for 4 m...
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