DON 毒素在鸡体内关键代谢酶的筛选鉴定及作用机制

Deoxynivalenol (DON) is one of the most toxic mycotoxin, which contaminate animal feeds and food stuffs broadly all over the world. The contamination of DON brings enormous threat to the development of agriculture and human health. Recent study shows that the microbes in some animal intestines can tansform DON to the nontoxic metabolites, DOM-1. However, the applicaiton of these microbes in the detoxification of animal feeds is limited, because of the strict requirement of growth environment. Thus, the identification of the enzymes which are responsible for the deepoxidation is indispensable. Furthermore, all of the identified microbes which can transform DON to DOM-1 are protected by patents in abroad, which limits the research and application of them in china. In our previous study, 6 individul microbes which can transform DON to DOM-1 were separated from chicken intestines. In this project, we plan to identify the separated microbes further, so that we can obtain the microbes with intellectual property, which can transform DON to DOM-1. Based on the identification of the microbes, we plan to screen and identify the key enzymes which are responsible for the DON transformation in theses identified chicken microbes by constructing a yeast YZAS107 cDNA expression library and analyzing the enzyme activity of recombinant protein. The results of this study are of important theoretical significance to the mechanism of DON deepoxidation, and the development of DON detoxification enzyme preparations.

DON毒素是一种毒性极强的霉菌次生代谢产物，其污染普遍，已给畜牧业发展及人类健康造成了很大的危害。研究发现某些畜禽肠道微生物能将DON代谢成几乎无毒的脱环氧产物DOM-1。由于DON脱环氧代谢肠道微生物对生长环境要求苛刻，在饲料脱毒等方面的应用受到一定限制，迫切需要进一步分离其脱环氧代谢的关键酶。然而，已被分离的DON脱环氧代谢微生物均受国外专利保护，对我国的科研和生产应用造成了障碍。在前期研究中，通过对鸡肠道微生物的筛选，已初步获得DON脱环氧代谢菌株6株。本研究拟对这6株菌株做进一步鉴定，以期获得具有自主知识产权的DON 脱环氧代谢微生物。并在此基础上，通过构建酵母YZAS107 表达文库与重组蛋白的酶活性分析等方法来筛选和鉴定DON脱环氧代谢关键酶，并初步探讨其作用机制。本研究对深入认识DON 脱环氧代谢的生化机制具有重要的理论意义，可为DON毒素脱毒酶制剂的开发提供理论依据。

脱氧雪腐镰刀菌烯醇（Deoxynivalenol），简称DON毒素，是由禾谷镰刀菌等真菌产生的次级代谢产物。DON毒素是霉菌毒素中污染粮食等作物最普遍的一种单端孢霉烯族B族毒素。生物脱毒是目前解决DON污染粮食作物的一种最有前景的方法。.本项目从鸡肠道中分离筛选可以将DON毒素代谢转化为低毒的脱环氧产物deepoxy-DON（简称DOM-1）的微生物。通过联合使用四环素、氨苄青霉素、泰乐霉素、林可霉素四种抗生素，梯度稀释，96孔板筛选等方法富集具有DON脱环氧代谢能力的菌群。对有活性及无活性的菌群进行比较宏基因组学分析，发现有活性菌群中独有的Eggerthella和Gordonibacter都属于Coriobacteriaceae。参考文献筛选Coriobacteriaceae的方法，改良培养基，连续培养5天，先后长出二批克隆，第二批克隆可以代谢DON毒素为DOM-1。经鉴定，该菌株为革兰氏阳性菌，其大小约为0.2～0.3 μm × 0.7～1.0 μm，呈细杆状，无鞭毛。对上述单克隆菌株进行16S rRNA基因测序，并构建系统发育树，通过序列比对发现，该菌株属于伊格尔兹氏菌属（Eggerthella），将该菌株命名为Eggerthella sp. D II-9，该菌株具有DON降解活性pH值范围广（5-10）和温度范围广（30-45℃）的特点，且其活性的维持不依赖于培养体系中DON的持续存在。.综上，本项目筛选出一种新的未经报导的微生物，既可为DON毒素污染的粮食作物脱毒提供候选菌株，也为后续研究脱环氧代谢的机理奠定了基础。