Rhodococcus linzhousis FB002降解玉米赤霉烯酮(ZEN)的作用机理研究

Zearalenone (ZEN) is one of mycotoxins, causing contamination on grain and feed in China and also widely in the world, ZEN is harmful to animals and human. Biodetoxification of mycotocins showed features of secutity and efficiency, related research have been carried out, most of them are limited in screening strains capable of degrading ZEN, little is known about the ZEN degradation mechanism,the structure and the toxicity of the degradation products, which restricts the application of biodetoxification.In the previous work of our research team, a strain showed ZEN degradation activity was isolated from the field severely contaminated by Fusarium sp., this strain was later identified as a novel species of Rhodococcus and named Rhodococcus linhzousis FB002. In this study, we are going to purify ZEN degradation products by Semipreparative Chromatography, then IR(Infrared Spectroscopy), NMR (Nuclear Magnetic Resonance),etc will be used to analyze the structure of degradation products for preliminarily degradation process deducing. The key active enzymes functioning on ZEN or its degradation products will be separated and purified using FPLC(Fast Protein Liquid Chromatography). The proteins will be sequenced using de novo sequencing technique and the genes encoding for these proteins will be cloned in E. coli to perform heterologous expression, purified proteins will be used in confirmation of their roles in ZEN degradation process. Thus, the ZEN degradation mechanism will be elucidated in this study which will provide technical support in innovation of new, green, safe and efficient enzymic preparations in biodetoxification of ZEN.

真菌毒素玉米赤霉烯酮（ZEN）是造成我国乃至世界粮食及饲料污染的重要因素之一。生物脱毒法具有安全、高效的特点，但大多相关研究局限在降解菌株的筛选。对ZEN降解机理、降解产物的结构、毒性等还只知之甚少。本研究组已经从赤霉病高发地区土样中筛选出能够完全降解ZEN的一株红球菌Rhodococcus linzhousis FB002,该项目中拟通过纯化ZEN降解产物，利用红外光谱（IR）、核磁共振（NMR）等方法鉴定降解产物结构，初步推断降解过程。利用快速蛋白液相色谱（FPLC）分离纯化在ZEN降解过程中的关键酶，使用de novo蛋白测序技术分析蛋白氨基酸序列。将降解酶基因进行克隆及表达，验证纯化后的蛋白在ZEN降解途径中的作用，阐明降解机理。为研发绿色、安全、高效的ZEN生物脱毒剂，最终解决饲料中真菌毒素污染问题提供技术支撑。

真菌毒素玉米赤霉烯酮（ZEN）是造成我国乃至世界粮食及饲料污染的重要因素之一。生物脱毒法具有安全、高效的特点，但大多相关研究局限在降解菌株的筛选。对ZEN降解机理、降解产物的结构、毒性等还只知之甚少。本研究组已经从赤霉病高发地区土样中筛选出能够完全降解ZEN的一株红球菌Rhodococcus linzhousis FB002，可高效降解ZEN。在项目实施期内，我们对该菌株进行了更详细的种属鉴定，最终确定为Rhodococcus qingshengii。将ZEN与该菌株共培养，摸索出降解产物分离纯化条件，进而通过LC-MS对其结构进行分析，根据此结果推导出ZEN的降解路径为内酯水解过程。据此查找该菌中内酯水解酶信息，获取该基因序列，并在大肠杆菌中异源表达，但活性较低，需进一步优化反应条件。对ZEN降解产物安全性进行了评价，通过酿酒酵母BLYES雌激素荧光检测，得出R. qingshengii可将ZEN降解为无雌激素活性结构，达到完全脱毒的目的。同时，本项目也对芽孢菌降解ZEN的机理进行了初探，目前获得若干株可高效脱毒的芽孢菌，并对其中效果最优菌株的脱毒机理进行了研究。有望通过进一步的研究，优化降解酶活性，为研发绿色、安全、高效的ZEN生物脱毒剂，最终解决饲料中真菌毒素污染问题提供技术支撑。