The major enzymes involved in lignin degradation are laccase, class II peroxidases (lignin peroxidase, manganese peroxidase, and versatile peroxidase) and dye peroxidase, which use an oxidative or peroxidative mechanism to deconstruct the complex and recalcitrant lignin. Laccase and manganese peroxidase directly oxidize phenolic lignin components, while lignin peroxidase and versatile peroxidase can act on the more recalcitrant non-phenolic lignin compounds. Mediators or co-oxidants not only increase the catalytic ability of these enzymes, but also largely expand their substrate scope to those with higher redox potential or more complicated structures. Neither laccase nor the peroxidases are stringently selective of substrates. The promiscuous nature in substrate preference can be employed in detoxification of a range of organics.

Enzymatic treatment is an attractive method for mycotoxin detoxification, which ideally prefers the use of one or a few enzymes. However, this is challenged by the diverse structures and co-contamination of multiple mycotoxins in food and feed. Lignin-degrading fungi have been discovered to detoxify organics including mycotoxins. Manganese peroxidase (MnP) is a major enzyme responsible for lignin oxidative depolymerization in such fungi. Here, we demonstrate that eight MnPs from different lignocellulose-degrading fungi (five from Irpex lacteus, one from Phanerochaete chrysosporium, one from Ceriporiopsis subvermispora, and another from Nematoloma frowardii) could all degrade four major mycotoxins (aflatoxin B₁, AFB₁; zearalenone, ZEN; deoxynivalenol, DON; fumonisin B₁, FB₁) only in the presence of a dicarboxylic acid malonate, in which free radicals play an important role. The I. lacteus and C. subvermispora MnPs behaved similarly in mycotoxins transformation, outperforming the P. chrysosporium and N. frowardii MnPs. The large evolutionary diversity of these MnPs suggests that mycotoxin degradation tends to be a common feature shared by MnPs. MnP can, therefore, serve as a candidate enzyme for the degradation of multiple mycotoxins in food and feed if careful surveillance of the residual toxicity of degradation products is properly carried out.

Aflatoxin B₁ (AFB₁) and zearalenone (ZEN) exert deleterious effects to human and animal health. In this study, the ability of a CotA laccase from Bacillus subtilis (BsCotA) to degrade these two mycotoxins was first investigated. Among the nine structurally defined chemical compounds, methyl syringate was the most efficient mediator assisting BsCotA to degrade AFB₁ (98.0%) and ZEN (100.0%). BsCotA could also use plant extracts, including the Epimedium brevicornu, Cucumis sativus L., Lavandula angustifolia, and Schizonepeta tenuifolia extracts to degrade AFB₁ and ZEN. Using hydra and BLYES as indicators, it was demonstrated that the degraded products of AFB₁ and ZEN using the laccase/mediator systems were detoxified. Finally, a laccase of fungal origin was also able to degrade AFB₁ and ZEN in the presence of the discovered mediators. The findings shed light on the possibility of using laccases and a mediator, particularly a natural plant-derived complex mediator, to simultaneously degrade AFB₁ and ZEN contaminants in food and feed.

The co-occurrence of multiple mycotoxins, including aflatoxin B₁ (AFB₁), zearalenone (ZEN) and deoxynivalenol (DON), widely exists in cereal-based animal feed and food. At present, most reported mycotoxins degrading enzymes target only a certain type of mycotoxins. Therefore, it is of great significance for mining enzymes involved in the simultaneous degradation of different types of mycotoxins. In this study, a dye-decolorizing peroxidase-encoding gene BsDyP from Bacillus subtilis SCK6 was cloned and expressed in Escherichia coli BL21/pG-Tf2. The purified recombinant BsDyP was capable of oxidizing various substrates, including lignin phenolic model compounds 2,6-dimethylphenol and guaiacol, the substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), anthraquinone dye reactive blue 19 and azo dye reactive black 5, as well as Mn²⁺. In addition, BsDyP could efficiently degrade different types of mycotoxins, including AFB₁, ZEN and DON, in presence of Mn²⁺. More important, the toxicities of their corresponding enzymatic degradation products AFB₁-diol, 15-OH-ZEN and C₁₅H₁₈O₈ were significantly lower than AFB₁, ZEN and DON. In summary, these results proved that BsDyP was a promising candidate for the simultaneous degradation of multiple mycotoxins in animal feed and food.

Cellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored. In the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley β-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (− 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (kcat/Km; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency. This study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase–substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.

Manganese peroxidase is one of the Class II fungal peroxidases that are able to oxidize the low redox potential phenolic lignin compounds. For high redox potential non-phenolic lignin degradation, mediators such as GSH and unsaturated fatty acids are required in the reaction. However, it is not known whether carboxylic acids are a mediator for non-phenolic lignin degradation. The white rot fungus Irpex lacteus is one of the most potent fungi in degradation of lignocellulose and xenobiotics. Two manganese peroxidases (IlMnP1 and IlMnP2) from I. lacteus CD2 were over-expressed in Escherichia coli and successfully refolded from inclusion bodies. Both IlMnP1 and IlMnP2 oxidized the phenolic compounds efficiently. Surprisingly, they could degrade veratryl alcohol, a non-phenolic lignin compound, in a Mn2+-dependent fashion. Malonate or oxalate was found to be also essential in this degradation. The oxidation of non-phenolic lignin was further confirmed by analysis of the reaction products using LC–MS/MS. We proved that Mn2+ and a certain carboxylate are indispensable in oxidation and that the radicals generated under this condition, specifically superoxide radical, are at least partially involved in lignin oxidative degradation. IlMnP1 and IlMnP2 can also efficiently decolorize dyes with different structures. We provide evidence that a carboxylic acid may mediate oxidation of non-phenolic lignin through the action of radicals. MnPs, but not LiP, VP, or DyP, are predominant peroxidases secreted by some white rot fungi such as I. lacteus and the selective lignocellulose degrader Ceriporiopsis subvermispora. Our finding will help understand how these fungi can utilize MnPs and an excreted organic acid, which is usually a normal metabolite, to efficiently degrade the non-phenolic lignin. The unique properties of IlMnP1 and IlMnP2 make them good candidates for exploring molecular mechanisms underlying non-phenolic lignin compounds oxidation by MnPs and for applications in lignocellulose degradation and environmental remediation.