Key Laboratory of Feed Biotechnology, Ministry of Agriculture and Rural Affairs, Institute of Feed Research, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, China.
Laboratory of Pet Nutrition and Food, Institute of Feed Research, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, China.
Toxins (Basel). 2024 Oct 16;16(10):445. doi: 10.3390/toxins16100445.
The global prevalence of aflatoxin B1 (AFB1) and zearalenone (ZEN) contamination in food and feed poses a serious health risk to humans and animals. Recently, enzymatic detoxification has received increasing attention, yet most enzymes are limited to degrading only one type of mycotoxin, and free enzymes often exhibit reduced stability and activity, limiting their practicality in real-world applications. In this study, the laccase gene from ZEN/AFB1-degrading ZJ-2019-1 was cloned and successfully expressed in BL21, achieving a protein yield of 7.0 mg/g. The recombinant CotA (rCotA) completely degraded AFB1 and ZEN, with optimal activity at 70 °C and pH 7.0. After rCotA treatment, neither AFB1 nor ZEN showed significantly cytotoxicity to mouse macrophage cell lines. Additionally, the AFB1/ZEN degradation efficiency of rCotA was significantly enhanced by five natural redox mediators: acetosyringone, syringaldehyde, vanillin, matrine, and sophoridin. Among them, the -rCotA was the most effective mediator system, which could completely degrade 10 μg of AFB1 and ZEN within 1 h. Furthermore, the chitosan-immobilized rCotA system exhibited higher degradation activity than free rCotA. The immobilized rCotA degraded 27.95% of ZEN and 41.37% of AFB1 in contaminated maize meal within 12 h, and it still maintained more than 40% activity after 12 reuse cycles. These results suggest that media-assisted or immobilized enzyme systems not only boost degradation efficiency but also demonstrate remarkable reusability, offering promising strategies to enhance the degradation efficiency of rCotA for mycotoxin detoxification.
食品和饲料中黄曲霉毒素 B1（AFB1）和玉米赤霉烯酮（ZEN）污染的全球流行对人类和动物的健康构成了严重威胁。最近，酶法解毒受到了越来越多的关注，但大多数酶仅限于降解一种类型的霉菌毒素，并且游离酶通常表现出稳定性和活性降低，限制了它们在实际应用中的实用性。在这项研究中，从 ZEN/AFB1 降解菌 ZJ-2019-1 中克隆并成功表达了漆酶基因，在 BL21 中实现了 7.0 mg/g 的蛋白产量。重组 CotA（rCotA）可完全降解 AFB1 和 ZEN，最适活性为 70°C 和 pH7.0。rCotA 处理后，AFB1 和 ZEN 对小鼠巨噬细胞系均无明显细胞毒性。此外，五种天然氧化还原介体：乙酰丁香酮、丁香醛、香草醛、苦参碱和槐定碱显著增强了 rCotA 的 AFB1/ZEN 降解效率。其中，-rCotA 是最有效的介体系统，可在 1 h 内完全降解 10 μg 的 AFB1 和 ZEN。此外，壳聚糖固定化 rCotA 系统的降解活性高于游离 rCotA。固定化 rCotA 在 12 h 内降解污染玉米粉中 27.95%的 ZEN 和 41.37%的 AFB1，在 12 次重复使用循环后仍保持超过 40%的活性。这些结果表明，介质辅助或固定化酶系统不仅可以提高降解效率，而且还具有显著的可重复使用性，为提高 CotA 对霉菌毒素解毒的降解效率提供了有前景的策略。
