SYA13菌降解玉米赤霉烯酮机制的研究

Zearalenone (ZEN) widely exists in the moldy cereals and dairy products and has the strong reproductive toxicity and carcinogenicity. Because of the traditional method for detoxification is unstable, it can make the nutrient lost. Therefore, for controlling the ZEN pollution, it is necessary to find a new technology with the advantages of the high efficiency, strong specificity as well as no pollution to environment. However, the biodegradation method has the above superiority. This project takes the multi-disciplines including microbiology, molecular biology and modern instrumental analysis science as the theoretical foundation; simulately using the chromatographic analysis technology to separate and purify the ZEN degrading enzyme of the strain SYA13 with high ZEN degradation ability which was separated and identified by our team; using the MALDI-TOF/TOF/MS method to determine the protein sequence of ZEN degrading enzyme; using PCR method to clone the degrading enzyme gene according to the identified protein sequence, using the prokaryotic expression system to express the gene and to identify the degradation function of its production; using the HPLC-MS/MS and MRI technologies to determine the molecular weight and structure of the degradation products, and then confirming the degradation mechanism of the degrading bacterium. This project can lay the foundation for constructing efficient ZEN degradation engineering bacterium, improving the ZEN degrading enzymes production, developing the high-pure and high-active novel detoxification enzyme feed additive , and preventing ZEN toxin poisoning.

玉米赤霉烯酮（ZEN）广泛存在于霉变谷类作物和奶制品中，具有很强的生殖毒害和致癌性。传统的脱毒方法效果不稳定，营养成分损失很大。因此，ZEN污染的控制急切需要一种高效率、特异性强以及对环境没有污染的技术，而生物降解法具有以上优势。本项目以微生物学、分子生物学和现代仪器分析等多学科为理论基础，拟采用层析技术对本课题组分离鉴定的具有高效降解ZEN能力的SYA13菌株的ZEN降解酶进行分离、纯化，MALDI-TOF/TOF/MS方法确定ZEN降解酶的蛋白序列；根据ZEN降解酶的蛋白序列，采用PCR方法克隆ZEN降解酶基因，利用原核表达系统表达该基因并对表达产物的降解功能进行鉴定；采用HPLC-MS/MS和MMR技术确定降解产物的分子量及分子结构，进而揭示ZEN降解菌的降解机制，为今后构建高效ZEN降解工程菌，提高ZEN降解酶产量，开发高纯度高活性新型解毒酶饲料添加剂，预防ZEN毒素中毒奠定基础。

玉米赤霉烯酮（ZEN）广泛存在于霉变谷类作物和奶制品中，具有很强的生殖毒害和致癌性。传统的脱毒方法效果不稳定，营养成分损失很大。因此，ZEN 污染的控制急切需要一种高效率、特异性强以及对环境没有污染的技术，而生物降解法具有以上优势。本项目以微生物学、分子生物学和现代仪器分析等多学科为理论基础，成功分离鉴定了具有高效降解 ZEN 能力的 SYA13 菌株，并对其降解酶进行初步分离，试验结果表明，该菌株的降解酶胞外酶和胞内酶均具有活性，但胞外酶活性大于胞内酶，通过对该菌株的安全性鉴定，发现其无致病性，进行了体内试验，该菌株可以明显缓解ZEN引起的毒性，根据 ZEN 降解酶的蛋白序列，采用 人工合成方法合成了 ZEN降解酶基因， 利用原核表达系统表达该基因并对表达产物的降解功能进行鉴定，发现该降解酶具有很强的降解ZEN能力，该研究结果为今后开发高纯度高活性新型解.毒酶饲料添加剂，预防 ZEN 毒素中毒奠定基础。