伏马毒素B1降解菌分离鉴定及其酶解机制研究

Fumonisin B1 (FB1), which can widely contaminate feed and its raw materials, has hepatotoxic and nephrotoxic effects. It can cause serious economic losses in animal livestock. Until now, there is a limited number of the FB1-transforming bacteria reported and the relevant study is pitifully inadequate. In particular, it is still not clear for the enzymatic characteristic and the mechanism of enzymatic hydrolysis. We have enriched the FB1-transforming bacteria from the chicken intestines, and found that the crude enzyme excreted by FB1-transforming bacteria play a key role on transforming FB1. On the basis of the results, the FB1-transforming bacteria are further isolated and identified by PCR-DGGE. After fermentation, the key FB1-transforming enzyme was purified by multiple column chromatography and identified by MALDI-TOF/MS. The structure and amino acid sequence of transforming enzyme are analyzed in depth to reveal the enzymatic characteristic and kinetics of enzymatic hydrolysis. In addition, the mass spectrometry is utilized to identify the types and structures of enzymes-hydrolyzed products. Based on this, we reveal the pathway of enzymatic hydrolysis and elucidate the metabolism mechanism. Moreover, the toxicity of enzymes-hydrolyzed products was evaluated by Caco-2 cytotoxicity test to reveal the safety of the transforming enzyme. As expected, the results of the current study will systematically reveal the hydrolysis mechanism of FB1-transforming enzyme, and it will provide scientific basis for the application of FB1-transforming enzyme in animal livestock.

伏马毒素B1（FB1）具有肝毒性和肾毒性，可广泛污染饲料及原料，对畜牧业造成了不可低估的经济损失。当前，已知可降解FB1的菌株数量十分有限，相关研究严重缺乏，特别对于关键降解酶的酶学特性及酶解机理等方面仍不清晰。本课题组已从鸡肠道中富集出可高效降解FB1的菌液，并证实富集菌群分泌的降解酶是FB含量降低的关键因素。本项目将在此基础上，利用PCR-DGGE法进一步分离鉴定可降解FB1的菌株；发酵培养后，通过柱层析和MALDI-TOF/MS技术对关键降解酶进行纯化鉴定，深入分析降解酶结构及氨基酸序列，明确其酶学特性，揭示酶解动力学规律；基于质谱鉴定技术，解析FB1酶解产物的种类和结构，明确其酶解通路，进而阐明降解酶对FB1的酶解机制；开展Caco-2细胞毒性试验，评价酶解产物的安全性。本项目的完成将全面揭示FB1降解酶的酶解机理，并为脱毒酶制剂在畜牧业生产中的实际应用提供科学依据。

伏马毒素B1（FB1）具有肝毒性和肾毒性，可广泛污染饲料及原料，对畜牧业造成了不可低估的经济损失。本项目分离出一种可高效降解伏马毒素B1（FB1）的菌群SAAS79。利用16S rDNA高通量测序和抗生素筛选法确证 Pseudomonas属对FB1的降解起关键作用。研究了SAAS79在不同条件下对FB1降解性能的影响。结果表明SAAS79对伏马毒素类具有高特异性的降解特性，在28℃、pH=5-7条件下对5 μg/mL FB1降解率达100%，而对其它毒素无任何降解力。菌群SAAS79经超声破碎提取后，通过高温处理与蛋白酶处理明确了胞内酶对FB1的降解起关键作用。利用Amicon Ultra-4超滤管对FB1降解酶进行粗分离，证明 >100kD胞内酶在2 h内对5 μg/mL FB1的降解率达90%；并考察了时间、温度及pH对FB1降解酶效率的影响，研究了其酶解动力学参数。采用UPLC-QTOF/MS对FB1酶解产物在正负离子模式下进行全扫描和产物离子扫描，明确FB1的酶解产物3种（均为母体去除丙三酸基团），降解酶2种（脱羧基转移酶和脱氨基转移酶），并绘制了FB1酶解通路；另外，扩增出了潜在中间产物降解酶的基因序列，为后续工作指明方向。采用MARC-145细胞评价FB1酶解产物的细胞毒性。通过MTT法测定细胞活性，同时测定了细胞内MDA及GSH等，明确FB1酶解产物的毒性相较于FB1明显降低。本项目获得的菌群SAAS79及其粗酶制剂极有潜力应用于饲料工业中的FB1脱毒，同时扩增出的基因资源为大规模生产酶制剂提供了可能性，为开发新型饲料FB1脱毒酶制剂奠定了基础。