鞘氨醇单胞菌USTB-05生物降解节球藻毒素的途径与分子机理研究

With the global warming and discharge of wastewater containing a huge amount of nitrogen and phosphorus, harmful cyanobacterial bloom is increasing all over the world, and its main harm is to produce and release the carcinogenic nodularins (NODs) and microsystins (MCs), which may threaten the safety of human drinking water. NODs belong to the stable monocyclic pentapeptides. Although many people study on the biodegradation of MCs in the world, almost no information is provided on the biodegradation of NODs in China. Base on the successful findings of NODs can be biodegraded by Sphingopyxis sp. USTB-05 and its crude enzymes, and the metabolite products of NODs are observed, combined the modern molecular biological technique with the computer molecular simulation, we will study on the cloning of biodegradation genes, and the expression and purification of recombinant biodegradation enzymes, and the identification of biodegradation products as well as the mechanism of molecular simulation in this applied project. The pathway and molecular mechanism for the biodegradation of NODs by Sphingopyxis sp. USTB-05 are firstly studied, which will provide our contribution on the enrichment and development of theory for the biodegradation of NODs and the application of genetic engineering bacteria in the removal of NODs from water body.

随着全球气候变暖和氮磷的排放，蓝藻水华在我国和世界仍然处于增加趋势，其最主要危害是产生并释放使人致癌的节球藻毒素(Nodularins,NODs)和微囊藻毒素(Microsystins,MCs)，因此对人们饮用水安全构成了严重威胁。NODs属于单环五肽结构稳定化合物，虽然世界上对MCs生物降解研究较多，但国内对NODs生物降解则基本处于研究空白。我们在发现鞘氨醇单胞菌USTB-05及粗酶具备高效生物降解NODs能力并观察到降解产物基础上，本申报项目拟将现代分子生物学技术和计算机分子模拟相结合，开展NODs降解基因的克隆，具备催化活性重组降解酶的表达与纯化、NODs降解产物结构识别和重组酶催化降解NODs 机理的分子模拟等一系列系统深入研究，全面揭示鞘氨醇单胞菌USTB-05生物降解NODs的途径与分子机理，不仅为丰富和发展NODs的生物降解理论做出贡献，而且为快速高效去除NODs奠定基础。

随着水体富营养化和全球气候变暖，蓝藻水华在世界各地的暴发呈增长趋势，其最主要的危害是产生并释放多种藻毒素，其中产生量最大、分布范围最广泛和造成危害最严重的是肝毒素(Hepatotoxin)，主要包括节球藻毒素(NOD)和微囊藻毒素(Microcystins, MCs)，分属难生物降解的单环五肽和七肽化合物，对人类和动物的饮用水安全构成了严重威胁。.尽管人们普遍开展了MCs的生物降解研究，但对毒性效应更强NOD的生物降解在国内却几乎没有研究报道。本研究在国内外首次采用具有自主知识产权的鞘氨醇单胞菌USTB-05，在细胞和酶水平降解NOD的途径与分子机理进行了系统深入的研究，取得如下成果：1）USTB-05是目前发现的最高效生物降解NOD的菌株，在24 h内可以将初始浓度为25.3 mg/L的NOD全部降解。2）与降解MCs途径类似，USTB-05的第1个重组酶首先断裂连接Adda与Arg的肽键，将环状NOD转化为线型NOD。3）USTB-05的第3个重组酶断裂线型NOD中连接Adda和Glu的肽键，生成Adda作为产物。4）采用计算机分子模拟与分子生物学技术相结合，利用同源建模和分子模拟构建酶的三维结构，预测了降解酶的活性位点。.本项目从细胞和酶水平揭示了USTB-05生物降解不同肝毒素的作用机理，为丰富和发展鞘氨醇单胞菌USTB-05降解肝毒素的基础研究做出贡献。