玉米赤霉烯酮降解酶的基因克隆、外源表达及解毒特性研究

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.

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。

玉米赤霉烯酮（ZEN）是由污染谷物和植物的镰刀菌产生的一类具有类雌激素作用的霉菌毒素，可与子宫内雌激素受体不可逆结合而影响动物的生殖生理，降低动物繁殖性能和产品品质，对畜牧养殖业造成巨大经济损失。传统的物理化学去毒方法因其存在效果不稳定、营养成分损失大、影响饲料适口性等缺点而不能被广泛应用到实际生产中。微生物及生物酶解毒因具有解毒效率高、特异性强、可在温和条件下将毒素转化为无毒代谢产物等优点备受研究者关注。鉴于枯草芽孢杆菌作为益生菌的潜在应用价值，本研究首先通过全基因组测序技术，挖掘到芽孢杆菌降解ZEN的潜在酶基因过氧化物酶（Prx）和α/β水解酶；再通过计算机技术对来源于枯草芽孢杆菌的染料脱色过氧化物酶与ZEN进行分子对接和动力学模拟，预测枯草芽孢杆菌168染料脱色过氧化物酶BsDyP对ZEN具有很高的催化降解潜力； 对染料脱色过氧化物酶BsDyP进行基因克隆，构建重组质粒pET-31b-BsDyP，在大肠杆菌Rosseta（DE3）中表达获得分子量为44kDa的重组蛋白；酶学特性研究表明BsDyP降解ZEN的最适反应pH为8.0，最适温度为42℃，最适酶量为2.0 U /mL，最适初始H2O2浓度为100 μM，Ca2+, Cu2+, Zn2+, Co2+, Mn2+和Fe3+对BsDyP降解ZEN有明显抑制作用，BsDyP介导的ZEN降解动力学参数Km、Kcat和Vmax值分别为40.36μg/mL、0.04s-1和17.74μg/min/mg；采用UPLC-MS/MS方法首次鉴定出染料脱色过氧化物酶BsDyP降解ZEN的产物为ZEN-11,12-氧化物，通过MCF-7细胞实验评估产物ZEN-11,12-氧化物的雌激素活性显著降低，表明BsDyP降解ZEN是一种解毒作用；同时为推进BsDyP的实际应用，首次提出葡萄糖氧化酶可与染料脱色过氧化物酶（GOD-BsDyP）联合应用用于饲料ZEN的降解；通过小鼠和猪等动物试验评估了枯草芽孢杆菌霉菌毒素生物降解剂（MBA）可有效缓解ZEN对动物的毒性作用，改善生产性能、抗氧化机能和提高动物源性食品安全。本研究结果为开发新型、高效、安全的饲料玉米赤霉烯酮降解酶制剂，用于动物的实际生产奠定基础。