新型菊糖内切酶在催化菊糖降解生成均一性果寡糖中的分子作用机制研究

Fructooligosaccharides are important prebiotics used in healthy foodstuff, which can be produced by inulin hydrolysis with endoinulinase. However, such fructooligosaccharide 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 high purified fructooligosaccharides and blocking the development of fructooligosaccharides functionally. One novel bacterium Paenibacillus inulolyticus has been isolated that can hydrolyze inulin into homogenous fructooligosaccharides. In this project, the endoinulinase produced by this strain 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 catalyzed by endoinulinase. Success in such studies will help 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形成，但由于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成熟，分辨率较低且成本过高，很难获得聚合度均一的高纯度果寡糖产品，严重制约了果寡糖的应用。本项目基于前期已分离到一株能将菊糖分解为聚合度均一果寡糖的新菌Paenibacillus inulolyticus LXfos16，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、结构分析、定点突变、DNA Sulffing、动力学分析和酶学表征等，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与产物纯度的关系，阐述具有产物特异性菊糖内切酶催化菊糖降解生成聚合度均一果寡糖的分子作用机理，为高纯度果寡糖生产奠定理论基础，为食品加工提供新酶种。

果寡糖可由菊糖内切酶催化菊糖部分水解制成，但由于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成熟，分辨率较低且成本过高，很难获得聚合度均一的高纯度果寡糖产品，严重制约了果寡糖的应用。本项目基于前期已分离到的一株菊糖降解菌Paenibacillus inulolyticus LXfos16，拟以该菌所产内切型菊糖酶为研究对象，通过基因克隆、结构分析、定点突变、DNA Sulffing、动力学分析和酶学表征等，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与产物纯度的关系，阐述具有产物特异性菊糖内切酶催化菊糖降解生成聚合度均一果寡糖的分子作用机理。通过本项目研究，获得一株能将菊糖水解为聚合度单一的果寡糖，克隆并表达了该菌合成的环果寡糖果糖基转移酶，表征了其功能，确定了其催化形成高纯果寡糖的催化机理。本项目研究为高纯度果寡糖生产奠定理论基础，为食品加工提供了新酶种。