新型菊糖内切酶高效表达及高纯度果寡糖形成的分子机制

The fructooligosacchrides are important prebiotics used in healthy foodstuff, which can be produced by inulin hydrolysis with endoinulinase. However, such fructooligosacchride products always have a wide range of polymerization degree due to randomly broken glycosidic bond inner molecule inulin. Moreover it is difficult to purify oligosaccharide with different polymerization degree from the hydrolytic products because of the low efficiency and high cost in sugar separation, leading to the difficulty in preparing the high purified fructooligosaccaharides and blocking the development of fructooligosaccharides functionally. Therefore, it is key factor for high purity of fructooligosacchrides to modify endoinulinase increasing product speciality. One novel bacterium Bacillus sp. LX8 has been isolated that can hydrolyze inulin into high homogenous fructooligosaccharides. In this project, the endoinulinase produced by strain LX8 will be studied by gene cloning, enzyme characterizing, structure predicting, site-direct mutating and chemical modifying, mutant bank constructing, and dynamics analyzing. The key amino acids and domains in endoinulinase influencing product specifility and enzyme activity will be discussed, as well as the hydrolytic mechanism catalyzing by endoinulinase. The success in such studies will help to understand the relationship between structure and function and the molecular modification of enzyme.

果寡糖是公认的益生元代表，可由菊糖内切酶催化菊糖部分降解产生，但因内切酶催化糖苷键断裂的随机性，所形成产物是不同聚合度的果寡糖混合物，而目前糖分离技术还不十分成熟，分辨率较低且成本过高，很难获得高纯度果寡糖产品，严重影响果寡糖的功能发挥和应用开发。因此，如何改造生物酶的产物专一性以提高产物形成过程中的聚合度均一性，是获得高纯度果寡糖的关键。本课题组前期工作已分离到一株新菌Bacillus sp. LX8，能够将菊糖分解为聚合度非常均一的果寡糖，本项目拟以该菌所产高产物专一性的内切型菊糖酶为研究对象，通过基因克隆、酶学表征、结构预测、定点突变与化学修饰、突变体库构建和动力学分析等，探讨菊糖酶分子中影响产物专一性和酶活性的关键氨基酸和结构域以及与产物纯度的关系，阐述具有产物特异性菊糖内切酶的催化反应机理，为深入研究酶分子结构与功能关系以及酶的分子改造提供理论依据，为食品加工提供新酶种。

果寡糖是公认的益生元代表，可由菊糖内切酶催化菊糖部分降解产生，但因内切酶催化糖苷键断裂的随机性，所形成产物是不同聚合度的果寡糖混合物，而目前糖分离技术还不十分成熟，分辨率较低且成本过高，很难获得高纯度果寡糖产品，严重影响果寡糖的功能发挥和应用开发。因此，如何改造生物酶的产物专一性以提高产物形成过程中的聚合度均一性，是获得高纯度果寡糖的关键。本课题组前期工作已分离到一株新菌Bacillus sp. LX8，能够将菊糖分解为聚合度非常均一的果寡糖，本项目拟以该菌所产高产物专一性的内切型菊糖酶为研究对象，通过基因克隆、酶学表征、结构预测、定点突变与化学修饰、突变体库构建和动力学分析等，探讨菊糖酶分子中影响产物专一性和酶活性的关键氨基酸和结构域以及与产物纯度的关系，阐述具有产物特异性菊糖内切酶的催化反应机理，为深入研究酶分子结构与功能关系以及酶的分子改造提供理论依据，为食品加工提供新酶种。