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.

β-Glucanase is one of the most extensively used biocatalysts in biofuel, food and animal feed industries. However, the poor thermostability and low catalytic efficiency of most reported β-glucanases limit their applications. Currently, two strategies are used to overcome these bottlenecks, i.e., mining for novel enzymes from extremophiles and engineering existing enzymes. A novel endo-β-1,3-1,4-glucanase of GH16 (Tlglu16A) from the thermophilic fungus Talaromyces leycettanus JCM12802 was produced in Pichia pastoris and characterized. For potential industrial applications, recombinant TlGlu16A exhibits favorable enzymatic properties over most reported glucanases, i.e., remarkable stability over a wide pH range from 1.0 to 10.0 and superior activity on glucan substrates (up to 15,197 U/mg). The only weakness of TlGlu16A is the thermolability at 65 °C and higher. To improve the thermostability, the enzyme thermal stability system was then used to engineer TlGlu16A through optimization of residual charge–charge interactions. Eleven mutants were constructed and compared to the wild-type TlGlu16A. Four mutants, H58D, E134R, D235G and D296K, showed longer half-life time at 80 °C (31, 7, 25, 22 vs. 0.5 min), and two mutants, D235G and D296K, had greater specific activities (158.2 and 122.2 %, respectively) and catalytic efficiencies (k cat/K m, 170 and 114 %, respectively). The engineered TlGlu16A has great application potentials from the perspectives of enzyme yield and properties. Its thermostability and activity were apparently improved in the engineered enzymes through charge optimization. This study spans the genetic, functional and structural fields, and provides a combination of gene mining and protein engineering approaches for the systematic improvement of enzyme performance.