多种重要农作物的宿主曲霉属真菌可以产生致癌的次生代谢产物，例如黄曲霉毒素。因此，需要新的解毒策略并将其从食物和饲料链中去除。在此，研究了枯草芽孢杆菌多铜氧化酶 CotA (BsCotA) 对黄曲霉毒素 B1 (AFB1) 的解毒作用。这种漆酶是在大肠杆菌中重组产生的，而密码子优化导致获得的活性蛋白量重复。将 CuCl2 添加到培养基中，导致 V max 增加 25 倍，这对应于 Cu2 与酶蛋白的结合得到改善，这对于催化反应至关重要。为了避免 Cu2 的潜在细胞毒性，在微需氧条件下进行培养，与标准需氧条件相比，确实产生了 100 倍多的功能性蛋白质。 V max 从 0.30 ± 0.02 U/mg 增加到 33.56 ± 2.02 U/mg 表明了这一点。使用带有荧光检测的 HPLC (HPLC-FLD) 分析 AFB1 的降解动力学表明理论底物饱和度高于水中的溶解度。在相对较高的浓度（500 μg/L）下，AFB1 在 0.2 μM BsCotA 剂量下以 10.75 μg/Lh（0.17 nmol*min-1*mg-1）分解。根据质谱法（即 HPLC-MS、HPLC-QTOF），AFQ1 和 epi-AFQ1 被鉴定为初始氧化产物。这些分子都不是漆酶的底物，但都在缓冲液中分解。然而，分解似乎不是由于末端呋喃环中的乙烯基醚的水合造成的。根据细菌 SOS 对 DNA 损伤反应的去抑制，在多种稀释度中评估了形成的 AFB1 的遗传毒性，表明与 AFQ1 相比，毒性降低了约 80 倍。这项研究的结果表明，BsCotA 对黄曲霉毒素 B1 的生物解毒具有很高的潜力。版权所有 © 2024 Subagia、Schweiger、Kunz-Vekiru、Wolfsberger、Schatzmayr、Ribitsch 和 Guebitz。

A variety of important agricultural crops host fungi from the Aspergillus genus can produce cancerogenic secondary metabolites such as aflatoxins. Consequently, novel strategies for detoxification and their removal from food and feed chains are required. Here, detoxification of Aflatoxin B1 (AFB1) by the Bacillus subtilis multi-copper oxidase CotA (BsCotA) was investigated. This laccase was recombinantly produced in E. coli while codon optimization led to duplication of the amount of active protein obtained. CuCl2 was added to the cultivation medium leading to a 25-fold increase of V max corresponding to improved incorporation of Cu2+ into the enzyme protein which is essential for the catalytic reaction. To avoid potential cytotoxicity of Cu2+, cultivation was performed at microaerobic conditions indeed leading to 100x more functional protein when compared to standard aerobic conditions. This was indicated by an increase of V max from 0.30 ± 0.02 to 33.56 ± 2.02 U/mg. Degradation kinetics of AFB1 using HPLC with fluorescence detection (HPLC-FLD) analysis indicated a theoretical substrate saturation above solubility in water. At a relatively high concentration of 500 μg/L, AFB1 was decomposed at 10.75 μg/Lh (0.17 nmol*min-1*mg-1) at a dosage of 0.2 μM BsCotA. AFQ1 and epi-AFQ1 were identified as the initial oxidation products according to mass spectrometry (i.e., HPLC-MS, HPLC-QTOF). None of these molecules were substrates for laccase but both decomposed in buffer. However, decomposition does not seem to be due to hydration of the vinyl ether in the terminal furan ring. Genotoxicity of the formed AFB1 was assessed in several dilutions based on the de-repression of the bacterial SOS response to DNA damage indicating about 80-times reduction in toxicity when compared to AFQ1. The results of this study indicate that BsCotA has high potential for the biological detoxification of aflatoxin B1.Copyright © 2024 Subagia, Schweiger, Kunz-Vekiru, Wolfsberger, Schatzmayr, Ribitsch and Guebitz.