Degradation of aflatoxin B1 by recombinant laccase AnLI from Aspergillus niger SF951 expressed in Escherichia coli BL21: a mechanism assessment in silico and in vitro

Abstract

There are approximately 25 % of global cereals contaminated with mycotoxins. As an extremely toxic carcinogen, aflatoxin B₁ (AFB₁) poses a serious threat to public health. Thus, efficient aflatoxin removal remains an urgent issue. Biological detoxification methods have become highly regarded. Because they are non - polluting, highly specific, gentle in operation, and environmentally friendly. In this study, the metagenomic data of the cultured microbiome of Salvia miltiorrhiza Bunge was utilized to excavate the strains carrying laccase genes. Through the mixed culture of coumarin medium and AFB₁, the fungus Aspergillus niger SF951 was successfully screened out. By means of cloning based on the conserved sequence, the full-length gene of a novel laccase AnLI was characterized, along with its deduced amino acid sequences. After heterologously expressing in Escherichia coli BL21 and purification, the recombinant laccase rAnLI was successfully obtained. Under optimized reaction conditions, recombinant laccase rAnLI degraded up to 94.72 % of AFB₁. UHPLC-MS/MS analysis detected four AFB₁ degradation products. Cytotoxicity assays demonstrated that enzymatic degradation of AFB₁ by rAnLI reduced toxicity. Molecular docking analysis further revealed that the binding of AnLI to AFB₁ involved six amino acid residues. Collectively, this research has characterized a novel laccase exhibiting high efficiency in degrading AFB₁. It also offers fresh perspectives on the molecular mechanisms underlying the degradation of AFB₁.