纯化酶催化降解微囊藻毒素的途径与分子机理研究

The main harm of cyanobacterial blooms is to produce and release of carcinogenic microcystins (MCs), which is a very serious threat to people drinking water safety. Microbial degradation is one of the most efficient ways to remove the MCs which has stable structure. But the pathway and molecular mechanism of the microbial degradation is still unclear. The strain named Sphingopyxis sp.USTB-05 is used as the research object in this topic. The MC-degrading genes of USTB-05 are to be cloned and the enzymes which are expressed and purified from the MC-degrading genes respectively are to be used to the catalytic degradation of microcystin-LR or microcystin-LR, from which all products are to be separated and purified and used to the determination of the molecular structure by the methods of "Marfey" and infrared absorption spectrum analysis. Then, the site-directed mutagenesis technology is used to change the DNA sequences which encode protein in order to get the amino acids necessary for the enzyme active site, by which degradation enzyme structures and functions are to be analyzed. Finally the MC-degrading pathway and molecular mechanism by the purified enzymes are to be determined, which lay a theoretical foundation for building genetic engineering bacteria to degrade MCs efficiently.

蓝藻水华最主要危害是产生并释放使人致癌的微囊藻毒素(Microsystins, MCs)，因此对人们饮用水安全构成严重的威胁。微生物降解法能最有效去除结构稳定的MCs，，但其降解途径及分子机理仍不清楚。本课题拟以鞘氨醇单胞菌USTB-05为研究对象，对USTB-05菌参与降解MCs的基因进行克隆与表达，对所诱导表达的酶进行分离纯化，并研究其酶学性质。用分离纯化后的酶依次催化降解微囊藻毒素LR（MC-LR）或微囊藻毒素RR（MC-RR），对降解产物分离纯化，并采用"Marfey"法及红外吸收光谱分析法测定分析每一步降解产物的分子结构。利用定点诱变（site-directed mutagenesis）技术，改变编码蛋白质基因中DNA顺序，研究酶活性部位的必要氨基酸，从而研究降解酶的结构和功能的关系。最终研究确定纯化酶催化降解MCs的途径与分子机理，为构建高效降解MCs的基因工程菌奠定理论基础。

蓝藻水华最主要危害是产生并释放使人致癌的微囊藻毒素(Microsystins, MCs)，因此对人们饮用水安全构成严重的威胁。微生物降解法能最有效去除结构稳定的MCs，但其降解途径及分子机理仍不清楚。本课题主要研究结果如下：.（1）通过酶学性质分析，USTB-05-A蛋白酶氨基酸序列与Ras转化酶1（Rce1）具有同源性；USTB-05-B蛋白酶氨基酸序列与D-氨基肽酶（D-AMINOPEPTIDASE）具有同源性；USTB-05-C蛋白酶氨基酸序列与基质金属蛋白酶( metallopeptidase)具有同源性。（2）通过降解基因定点诱变及克隆表达，所表达的蛋白具有降解MC-LR的活性，说明基因诱变的点不是基因的功能区域。（3）通过纯化酶USTB-05-A、USTB-05-B、USTB-05-C依次催化降解MC-LR，最终推测纯化酶催化降解MCs的可能途径是：首先USTB-05-A基因所表达的酶断开环状MC-LR结构中的Adda-L-Arg肽键，把环状的MC-LR水解成线性的MC-LR（H-HN-Adda-D-Glu-Mdha-D-Ala-L-Leu-D-Masp-L-Arg-OH）；接着USTB-05-B基因所表达的酶识别水解线性MC-LR中的D-Ala-L-Leu肽键，生成四肽（Adda-D-Glu-Mdha-D-Ala-OH）及三肽（Leu+MeAsp+Arg），其后产物不能再被酶USTB-05-A降解，但四肽（Adda-D-Glu-Mdha-D-Ala-OH）能被酶USTB-05-C继续降解，生成Adda和三肽（Glu+Mdha+Ala），其后产物不能被酶USTB-05-A和酶USTB-05-B降解；而酶USTB-05-C还能直接催化降解线性的MC-LR（H-HN-Adda-D-Glu-Mdha-D-Ala-L-Leu-D-Masp-L-Arg-OH），生成Adda 和六肽（Glu+Mdha+Ala+Leu+MeAsp+Arg），其后产物不能再被酶USTB-05-A和酶USTB-05-B降解。以上研究表明，一旦环状MC-LR的环被打开，变成线性MC-LR，USTB-05-B和USTB-05-C就能同时催化降解线性MC-LR。.研究结果揭示了USTB-05菌降解MC-LR的降解途径及分子机理，为构建高效降解MCs的基因工程菌奠定了理论基础。