单宁酶是一种能水解没食子酸单宁的酯键生成没食子酸和其它化合物的酶，而新型单宁酶Tan410不仅可以水解没食子酸单宁，还可以水解阿魏酸酯类、咖啡酸酯类及对硝基苯酚酯类化合物，这打破了单宁酶只能水解没食子酸酯类化合物的论点。为明确Tan410的结构与多底物催化功能间的关系，本研究拟以筛选到的Tan410为研究对象，通过亲和层析、分子筛层析及离子交换层析等技术获取高纯度Tan410；利用悬滴气象扩散和晶体-底物浸泡法分别制备Tan410及酶-底物复合物晶体；通过X-ray衍射晶体学方法解析Tan410及酶-底物复合物的晶体结构，揭示其催化反应机理；结合点饱和突变技术来确定Tan410的催化活性中心和底物结合位点，研究Tan410结构与催化功能间的关系。研究结果不仅有助于阐明单宁酶的分子催化机理，明确单宁酶结构与多底物催化功能间的关系，还为发掘单宁酶的新功能，指导酶分子设计及改造提供重要科学依据。

Tannase is a kind of hydrolytic enzymes which can hydrolyze gallic tannin in plant. In our earlier research, a novel tannase (Tan410) isolated from a soil metagenomic library can not only hydrolyze gallic tannin, but also hydrolyze ferulic acid esters, caffeic acid esters and ρ-nitrobenzoic acid esters. This discovery overturns the argument that tannase only hydrolyze gallic esters compounds. In order to find out its catalysis reaction mechanism, in this project, the crystals of Tan410 and protein-substrate complex which was obtained by using hanging drop vapor diffusion and soaking method, respectively, after Tan410 purified though methods of affinity chromatography, molecular sieve chromatography and anion exchange chromatography, were analyzed by X - ray diffraction crystallography method. The point of saturation mutagenesis technology was used to confirm active-site and substrate binding sites of Tan410 to find out its structural and functional relationship. Consequently, the expected results of this project may not only significantly contribute to the valuable information on tannase catalytic mechanism and its structural and functional relationship, but also provide scientific basis on tannase new function development and modification.