深海沉积物宏基因组源黄曲霉毒素降解酶的筛选及其降解机制的研究

Aflatoxin (AF) is a group of secondary metabolites mainly produced by Aspergillus flavus and Aspergillus parasiticus. It is highly toxic and carcinogenic. It not only does great harm to human health，but also leads to huge economic losses. Therefore, it is particularly important to detoxify AF safely and efficiently. In this study, a metagenomic library will be constructed from deep-sea sediments of the Pacific, and screening of AFB1-degrading enzymes will be conducted. The gene and amino acid sequences of the AFB1-degrading enzymes will be determined. In addition, heterologous expression, purification, enzymatic characterization and sequence analysis of the AFB1-degrading enzymes will be performed, so as to clarify their effectiveness and novelty. Moreover, the structures of the degradation products will be determined by UPLC-LTQ-Orbitrap, and the degradation mechanisms will be discussed combining with the substrate specificity of the recombinant enzymes. Finally, the toxicity of the degradation products will be evaluated by software simulation and cytotoxicity tests, so as to provide a scientific evidence for application of novel AFB1-degrading enzymes in biological control of AF.

黄曲霉毒素（AF）是黄曲霉、寄生曲霉等真菌产生的次级代谢产物,具有极强的毒性和致癌性。它不仅严重威胁人类健康，而且还造成巨大的经济损失，因此如何安全高效地脱除AF污染是一个亟待解决的重大课题。本申请课题拟从太平洋深海沉积物中获得宏基因组DNA,构建宏基因组文库，筛选深海来源的新型高效AFB1降解酶，确定多个编码AFB1降解酶的基因序列和氨基酸序列。分别在大肠杆菌和毕赤酵母中对筛选获得的新型高效AFB1降解酶进行异源表达，比较其表达效果，对异源表达的重组酶进行分离纯化、活性测定、酶学性质研究和序列分析，明确重组酶的降解活性与新颖性。采用高分辨质谱联用技术 (UPLC-LTQ-Orbitrap)，确定降解产物的结构，并结合酶的底物特异性，分析降解机制。采用软件分析和细胞毒性实验相结合的方法,评价降解产物的毒性,为AFB1降解酶在防治黄曲霉毒素污染方面的应用提供科学依据。

黄曲霉毒素（AF）是黄曲霉、寄生曲霉等真菌产生的次级代谢产物,具有极强的毒性和致癌性。它不仅严重威胁人类健康，而且还造成巨大的经济损失，因此如何安全高效地脱除AF污染是一个亟待解决的重大课题。本研究从太平洋深海沉积物中获取宏基因组DNA，并构建宏基因组文库，筛选到了两种深海来源的新型高效AFB1降解酶（AFB1D1和AFB1D2），确定了其基因序列。AFB1D1与产自地衣芽孢杆菌Bacillus licheniformis的CotA具有90.91%的氨基酸同源性，AFB1D2与产自假蜜环菌Armillariella tabescens的AFO具有87.03%的氨基酸同源性。在大肠杆菌中成功实现了AFB1D1和AFB1D2的克隆与表达。对异源表达的重组酶进行了分离纯化、活性测定、和酶学性质研究，结果表明两个重组酶对AFB1均具有较高的降解活性，且与其同源性较高的CotA和AFO相比，具有嗜冷性和嗜盐性的特点。采用高分辨质谱联用技术 (UPLC-LTQ-Orbitrap)，确定了AFB1D1对AFB1的降解产物为AFQ1和epi-AFQ1。细胞存活率实验表明降解产物（AFQ1和epi-AFQ1）无毒性。AFB1D2对AFB1的降解产物目前仍无法准确鉴定。本研究所发现的新型黄曲霉毒素降解酶，是目前为数不多的成功克隆并表达的黄曲霉毒素降解酶。本研究结果丰富了黄曲霉毒素降解酶的多样性，也为其在食品和饲料中的应用提供了更广阔的应用背景。