玉米赤霉烯酮(ZEA)具有类雌激素作用，可与子宫内雌激素受体结合，影响动物的生殖生理。课题组筛选到一株高效降解ZEA的枯草芽孢杆菌，其分泌的胞外酶对ZEA的降解率大于80%。在前一自然基金资助下已分离纯化出降解酶蛋白单一组分，分子量为55kDa。本申请拟对纯化后的降解酶进行基因克隆、外源表达和功能特性的研究。首先采用edman降解法和DeNovo测序法测定3-5段特征肽段氨基酸序列；根据特征肽段氨基酸序列设计简并引物，以菌cDNA为模板，通过简并PCR结合cDNA末端快速扩增技术，获得降解酶基因全长及完整的开放阅读框；将该基因转化到毕赤酵母中，构建异源表达重组菌；对重组菌进行高密度发酵培养、产酶条件优化及遗传稳定性研究；进一步研究酶学特性，分析代谢产物分子结构，揭示解毒作用机理，并进行产物毒理试验；最后从细胞水平和动物饲养水平对重组酶解毒功能进行验证。为开发霉菌毒素降解酶制剂奠定理论基础。

Zearalenone (ZEA), a non-steroidal estrogen, can irreversibly combine with the estrogens receptor in uterus so that reproduction physiology of animal are influenced, which lead to enormous hazards on livestock breeding. We have isolated a strain of Bacillus subtilis ANSB01G from intestinal tract chymus of chicks, and over 80% ZEA can be degraded by its extracellular enzyme. The zearalenone degrading enzyme, whose molecular weight is 55 kDa, has been isolated and purified with the supporting by the previous National Natural Science Foundation of China（31301981）. We will study on gene cloning, heterogenous expression and functional characteristics of zearalenone degradation enzyme from this Bacillus subtilis in this applying program . Firstly, amino acid sequences of 3-5 characteristic peptides should be determined using edman degrading method and DeNovo sequencing method, meanwhile each peptide include at least ten amino acid residues. Secondly, degenerate primers will be designed using amino acid sequence of characteristic peptide, gene amplification will be carried out using rapid amplification of cDNA ends with the template of genomic DNA of Bacillus subtilis ANSB01G, then we can get the whole gene of this degrading enzyme with sequence analysis of the PCR product. Thirdly, the gene of ZEA degrading enzyme should be transformed into Pichia Pastoris and the heterologous expression recombinant strain should be constructed. After that we should do the high-dentisy fermentation on this recombinant strain, optimize enzyme-producing condition and study the genetic stability of recombinant strain. We should study the enzymatic characteristics of this enzyme and analyze the molecular structure of metabolism product of ZEA by this enzyme and prove the function mechanism of degrading ZEA. Finally, the function of the recombinant degrading enzyme will be verified by experiments on artificial simulated duodenal and gastric juice and cell assay in vitro and experiment on effects of degrading enzyme on gilts fed ZEA in vivo. In conclusion, this research will lay the theoretical foundation for exploiting the efficient and safe feed additive of mycotoxin degradation enzyme.