Bacillus oleronius GX01抑制黄曲霉毒素B1（AFB1）产生和降解AFB1机制的研究

Aflatoxin is one of the most serious pollution mycotoxins in the world, which is great harm to human and animal. Our research group has isolated one of Bacillus sp.strain, named Bacillus oleronius GX01, which can degrade aflatoxin B1(AFB1). The degradation rate can reach more than 83% by the secretion of its extracellular enzymes. And the extracellular secretion of active substances can strong inhibit aflatoxin AFB1 production. Therefore, based on our previous work, the project will use chemical analysis technology such as the high performance liquid chromatography, gel chromatography and MALDI/TOF/MS and use genomic library such as the real-time quantitative PCR and Western blot and other molecular biology means to further separate, purify and characterize of this enzyme, clone the gene of degrading enzyme, express of the degrading enzyme gene by using a protein expression system, construct the gene engineering bacteria with independent intellectual property rights, and determine the molecular weight and molecular structure of the degradation products of AFB1 to reveal the degradation mechanism of this degrading bacteria. At the same time, the active components will be isolated and purified to inhibit the production of AFB1 from Bacillus oleronius GX01. The inhibition mechanism of AFB1 production is elucidated from the morphological and molecular biology. Our results can provide the theoretical basis for the biological control of aflatoxin contamination.

黄曲霉毒素是世界上污染最为严重的一种真菌毒素，对人和动物危害巨大。课题组先期分离到一株芽孢杆菌Bacillus oleronius GX01，其分泌的胞外酶对黄曲霉毒素B1（AFB1）的降解率达83%以上，并且分泌的胞外活性物质具有很强抑制黄曲霉产生AFB1的作用。因此，本项目拟在前期工作基础上，采用高效液相色谱、凝胶层析、MALDI/TOF/MS等分析化学技术和基因组文库、实时定量PCR、免疫印迹等分子生物学手段进一步分离、纯化和鉴定该降解酶，克隆该降解酶基因，利用蛋白表达系统表达降解酶基因，构建具有自主知识产权的基因工程菌，并对AFB1降解产物进行分子量和分子结构确定，揭示该降解菌的降解机制。同时分离纯化该菌株抑制AFB1产生的活性成分，从形态学、分子生物学角度初步阐明该菌株抑制AFB1产生机制。该研究成果可为黄曲霉毒素污染的生物防控奠定理论基础和实践基础。

黄曲霉毒素是世界上污染最为严重的一种真菌毒素，对人和动物危害巨大。物理化学方法脱毒不仅需要消耗大量的资源，而且会显著降低食品和饲料的营养效率。采用酶学的方法来去除黄曲霉毒素是目前研究热点。本项目组重新分离获得一株芽孢杆菌 Bacillus amyloliquefaciens B10，其具有抑制黄曲霉生长以及降解黄曲霉毒素B1（AFB1）能力，B10菌株与AFB1共培养96 h后，可将2.5 μg/mL的AFB1几乎完全降解，其分泌胞外酶对AFB1的降解率可达72.9%，成功分离纯化了B10菌株降解AFB1的降解酶，该酶为漆酶。并成功克隆表达了该漆酶，该漆酶基因的开放阅读框为837 bp，编码278个氨基酸，经预测其分子量30.9 kDa，等电点为5.84。B10菌株重组漆酶能够高效地降解AFB1，反应36 h时AFB1降解率可达79.3%。并确定漆酶H87，C132和H149位点对AFB1降解活性起到关键作用。确定了该菌株的最佳抑制黄曲霉生长的最佳培养条件为温度40 ℃，培养时间36 h，接种量3％，转速120 r/min，pH 6，装液量30 mL，最大抑菌率可达46.56 %。明确了该菌株胞外粗提蛋白提取物能有效抑制黄曲霉生长，破坏菌丝正常形态，并从中筛选出几丁质结合蛋白、壳聚糖酶、葡聚糖酶等8种可能的有效抑菌成分，其活性有待后续研究进行验证。该研究成果可为黄曲霉毒素污染和防控黄曲霉毒素B1中毒防控奠定理论基础和实践基础。