糖基化对烟曲霉壳聚糖酶性质影响的结构生物学研究

Due to the safe and non-toxic concerns, producing oligosaccharids through hydrolysis of chitosan with chitosanse is a popluar method.A powerful endo-chitosanase (CSN) was cloned from Aspergillus fumigatus Y2K and further identified as a member of glycosyl hydrolase family 75. It has already been used in large scale preparation of chito-oligosaccharides. However，the catalytic stereochemistry and structure of this GH-75 enzyme remain unknown. In our previous work, the chitosanase from Y2K was successful expressed in P.pastrios, with the yield of 3 mg/ml and activity of 25 000 U/ml. The analysis showed some properties of the recombination protein, such as activity，thermostability, denaturtant resistance, were altered and this may due to glycosaltion in P.pastrios. In order to elucidate the role of carbohydrates on structure and function in glycoproteins, this chitosanase has been investigated. The MALDI-TOF MS and glycoprotein coloration analysis showed that this chitosanase was O-glycosylated at one single site. On the primary basis, MALDI-TOF MS will be used to further search the glycosaltion site, and fluorescence, circular dichroism, X-ray crystallography will be used to elucidate the role of glycosylation on structure and conformation of chitosanase. We aim to provide theory for designing the industrial chitosanases and screening enzymes with high activity and thermostability.

壳聚糖酶酶法降解脱乙酰几丁质（壳聚糖）生产壳寡糖因其无污染、产物分子量低且功效强等优点成为壳聚糖降解的首选途径。G75家族的烟曲霉壳聚糖酶虽然能够有效降解壳聚糖，具有工业生产潜力。但该酶目前无论催化机理还是结构研究相对甚少。本项目在前期工作中用毕赤酵母高效表达了该酶。酶学性质分析发现该重组壳聚糖酶的活力、热稳定性及某些性质有显著地提高（已发表），通过糖蛋白染色试验和质谱鉴定初步发现该壳聚糖酶仅有一个位点被O-糖基化单糖修饰。为研究该壳聚糖酶的催化机制及糖基化对其性质的影响机理。本项目拟进一步利用质谱鉴定该壳聚糖酶的糖基化位点；解析壳聚糖酶的活性结构域；利用荧光光谱、圆二色谱和蛋白质晶体研究糖基化对该酶构象及三维结构的影响，从而揭示该壳聚糖酶的催化机制及糖基化修饰对壳聚糖酶性质的影响机理，为工业用酶的性质改造提供理论依据，为筛选高活性、耐高温壳聚糖酶提供理论基础。

壳寡糖生产是甲壳素工业高值化研究的重要方向，其生物、生理功效要求寡糖链具有特定的长度。壳聚糖酶是一种专一性水解壳聚糖的水解酶，能有效地作用于壳聚糖的糖苷键，产生聚合度2-10的壳寡糖。G75家族的烟曲霉壳聚糖酶能够有效降解壳聚糖，生成聚合度3-6的壳寡糖。本项目在初期工作中用毕赤酵母高效表达了该酶。酶学性质分析发现该重组壳聚糖酶的活力、热稳定性及某些性质有显著地提高。进一步研究表明，该酶性质的改变与糖基化修饰有关。因此，近一年来本课题组在国家基金主任基金资助的基础上，利用荧光光谱、圆二色谱从构象及二级结构研究了糖基化修饰对壳聚糖酶性质的影响，发现糖基化修饰对酶的二级结构的影响较小；然后，通过糖蛋白染色实验及质谱分析了糖基化类型和位点，确定了该蛋白是O-糖基化修饰，并且确定了糖基化位点，为后期蛋白的性质改造提供了理论基础。