Isolation, Purification, and Characterization of a Laccase-Degrading Aflatoxin B1 from Bacillus amyloliquefaciens B10

Aflatoxins, widely found in feed and foodstuffs, are potentially harmful to human and animal health because of their high toxicity. In this study, a strain of Bacillus amyloliquefaciens B10 with a strong ability to degrade aflatoxin B1 (AFB1) was screened; it could degrade 2.5 mu g/mL of AFB1 within 96 h. The active substances of Bacillus amyloliquefaciens B10 for the degradation of AFB1 mainly existed in the culture supernatant. A new laccase with AFB1-degrading activity was separated by ammonium sulfate precipitation, diethylaminoethyl (DEAE) and gel filtration chromatography. The results of molecular docking showed that B10 laccase and aflatoxin had a high docking score. The coding sequence of the laccase was successfully amplified from cDNA by PCR and cloned into E. coli. The purified laccase could degrade 79.3% of AFB1 within 36 h. The optimum temperature for AFB1 degradation was 40 degrees C, and the optimum pH was 6.0-8.0. Notably, Mg2+ and dimethyl sulfoxide (DMSO) could enhance the AFB1-degrading activity of B10 laccase. Mutation of the three key metal combined sites of B10 laccase resulted in the loss of AFB1-degrading activity, indicating that these three metal combined sites of B10 laccase play an essential role in the catalytic degradation of AFB1.