基于表面展示技术脂肪酶全细胞催化剂的酶活影响机制与构象变化规律研究

脂肪酶表面展示技术是将酶蛋白编码基因与锚定蛋白基因融合后，导入宿主菌展示在细胞表面，集脂肪酶表达、纯化和固定化于一体，是制备全细胞催化剂的有效手段。锚定蛋白特性、连接序列种类与长度均影响酶蛋白折叠后翻译，从而改变酶活性构象。因此，展示结构单元的合理匹配是脂肪酶成功展示的关键。截止目前，脂肪酶表面展示制备全细胞催化剂中展示结构单元对酶蛋白活性与构象的影响机理尚不十分清楚。为此，本项目拟以酿酒/毕赤酵母为宿主菌，基于生物信息学和分子生物学交叉，选择4种分子量大小和结构不同的脂肪酶及锚定原理不同的4种锚定蛋白为模型，利用圆二色谱、同步荧光光谱和氘交换检测等技术手段，研究锚定蛋白对酶构象的影响，优化启动子和连接序列，从酶蛋白折叠拓扑构象变化的角度，揭示结构单元对酶活力的影响机制，阐明锚定蛋白/连接序列/酶蛋白/启动子合理匹配规律，为更多脂肪酶或其它外源蛋白质的成功表面展示奠定科学的理论和技术基础。

Lipase surface display techniques is a novel eukaryotic lipase protein displayed technology, which fusions the lipase protein coding genes with anchor protein gene, then the fusion protein is imported into the host micro-organisms. This technique has been successfully used for gene expression, protein purification and immobilization together. It is an effective method for preparation of whole-cell catalyst so far. The displaying structural unit includes the foreign protein, anchor protein and linking sequence. The features of anchor protein and the types of linking sequence and length will both affect the exogenous protein post-translational modification and secretion, resulting in its conformation and activity. Therefore, the reasonable match of structural unit is the key for the success surface displaying foreign proteins. However, it has not been very clear for the matching law of the surface display technique between the displaying structural units and their impact mechanisms on lipase conformation and activities so far. Based on above analyses, the objective of this project is to elucidate the mechanism of effect of displaying structural units on lipse conformation and activity, when lipase is displayed on Saccharomyces cerevisiae / Pichia pastoris cell to make whole-cell catalysts. Firstly, according to the bioinformatics and molecular biology, we select four different types of lipases with different molecular size and structure, and four kinds of anchor proteins with different anchor principle as the experimental models. Then, we investigate the effect of anchor proteins on the enzyme conformation using circular dichroism (CD), synchronous fluorescence spectroscopy (SFS) and deuterium exchange measurement (DEM) techniques. Finally, we optimize the promoters and the linking sequences according to the topology changes of enzyme protein post-translational folding. It is demonstrated the reasonable match rules of the anchor protein / linking sequence / enzyme protein/promoter, and revealed the effect of displaying structural units on the lipase conformation and its catalytic activity. The results in this project should provide the scientific theoretical and technical basis for more other lipases or foreign proteins displayed on S. cerevisiae / P. pastoris cell to make an efficient whole-cell catalyst.