定向进化提高磺酰脲除草剂脱毒酯酶SulE催化效率及其机制

磺酰脲除草剂是一类广泛应用的超高效除草剂，但部分长残留期品种如甲磺隆和胺苯磺隆对作物药害严重；而残留期相对较短的品种如噻磺隆和豆磺隆可作为抗除草剂转基因工程的靶标除草剂。因此，研究磺酰脲除草剂微生物降解脱毒具有重要价值。酯酶SulE催化多种磺酰脲除草剂去酯化脱毒，对噻磺隆活性极高，但对其它品种活性很低，这限制了其应用价值。前期建立了高效的SulE定向进化方法，并筛选到几个对多种磺酰脲除草剂去酯化活性显著提高的突变体。本项目拟继续筛选活性进一步提高的SulE突变体，通过研究突变体的酶学特性和动力学，解析突变体及其与底物复合物结构，分析突变体和野生型酶动力学、结构及与底物结合差异，从而揭示突变体催化效率提高的分子机制。预期结果为通过理性设计进一步提高SulE催化效率提供理论依据，为磺酰脲除草剂残留污染修复和抗除草剂转基因工程提供优良基因资源，对推动我国农业及环境生物技术创新具有重要意义。

Sulfonylurea herbicide is a kind of ultra-efficient herbicide widely used in control of agricultural weeds. However, some sulfonylurea herbicides, such as metsulfuron-methyl and ethametsulfuron-methyl, are persist for a long time in soil and severely damage the subsequent rotation crops, while those with relatively short residue period, such as thifensulfuron-methyl and chlorimuron-methyl, are ideal target herbicides for herbicide-resistant transgenic engineering. Therefore, it is of great significance to study the microbial degradation and detoxification of sulfonylurea herbicides. In our previous work, a novel esterase gene sulE was cloned from Hansschlegelia zhihuaiae S113. SulE catalyzes the de-esterification of a variety of sulfonylurea herbicides with a methyl or ethyl ester (such as metsulfuron-methyl, sulfometuron-methyl, chlorimuron-ethyl, tribenuron-methyl, bensulfuron-methyl, thifensulfuron-methyl and ethametsulfuron-methyl) to the corresponding parent acids, which are herbicidal-inactive. SulE exhibits high de-esterification activity to thifensulfuron-methyl but very low activities to other sulfonylurea herbicides, which greatly limited its application. Therefore, it is necessary to improve the activities of SulE to various sulfonylurea herbicides. Previously, we established an efficient directed evolution method of SulE, and screened several mutants of SulE with significant improvement activities to sulfonylurea herbicides. In this research, we plan to screen SulE mutants with higher de-esterification activities, study the properties, kinetics and the structures of the mutants. The expected results provide a theoretical basis for further improving the activity of SulE through rational design, valuable genes for elimination of sulfonylurea residue and herbicide-resistant transgenic engineering, and promote the innovation of agricultural and environmental biotechnology in China.