Search

Aflatoxin B 1 (AFB 1 ) is a known toxic human carcinogen and can be detoxified by laccases, which are multicopper oxidases that convert several environmental pollutants and toxins. In this study, a new laccase that could catalyze AFB 1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH 4 ) 2 SO 4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB 1 detoxification. AFB 1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB 1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and [2,2' -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB 1 degradation by Lac 2. The product of AFB 1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q 1 (AFQ 1 ) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB 1 present in food and feeds.

Laccase, a multicopper oxidase, is well known for its industrial potentials to remove environmental pollutants due to its low substrate specificity to oxidize phenols and thus catalytic versatility. Many efforts focused on the metabolic mechanism, yet to decipher the structural determinants responsible for the differentiation between substrates. Aflatoxin B 1 (AFB 1), a new substrate for laccase, is a mycotoxin with a formidable environmental threat to public health and food safety. In the present study, we combined biochemical, in silico mutational and molecular-docking data to gain an insight to the function of key residues in the active cavity close to the T1 copper site in a characterized recombinant laccase from Cerrena unicolor (r Cu L). Kinetic data for computer-assisted virtual mutants established the binding affinity of hydrogen bonds and residues (Asn336, Asp207, Val391, and Thr165) in r Cu L to AFB 1. The augmented binding affinity to AFB 1 may be related to the conformational rearrangements of the laccase and its ability to hydrogen-bond with the substrate. Furthermore, the optimal pH and temperature for r Cu L and variants mediated AFB 1 degradation may depend on their pH stability and thermostability. Our findings reinforce the importance of the structure-function relationship of fungal laccases in degrading AFB 1 , providing mechanistic guidance for future biocatalyst and bioengineering applications. • Consistency of in vitro and in silico study provides a new method in degrading AFB 1. • Verification of critical amino acid residues of C.unicolor laccases on AFB 1. • Mutations of Asn336, Val391 and Asp207 would increase the affinity toward AFB 1. [ABSTRACT FROM AUTHOR]