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

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.

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。

脂肪酶是一类由生物细胞产生的具有特殊三维空间构象的蛋白质，广泛应用于能源、环保、食品、医药、制革和有机合成等领域。脂肪酶的催化特性和稳定性往往与酶的种类、纯度、存在形式（游离或固定化）以及微环境等诸多因素有关。由于游离酶难以回收，限制了其大规模应用。因此，脂肪酶催化反应在很大程度上取决于酶的固定化，它已成为酶工程领域的研究热点。.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。.据此，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，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。以a凝集素Aga2-Aga1为锚定蛋白，(G4S)3为linker，成功地将ROL展示在酿酒细胞表面，获得了具有水解橄榄油活性的全细胞催化剂。该全细胞催化剂水解橄榄油活力为78.9 U·g-1，高于相关文献报道。同时，研究了ROL全细胞催化剂的酶学特性，最适温度为40 °C，最适pH为7.5，具有良好的稳定性和有机溶剂耐受性。海藻酸钠包埋ROL全细胞催化剂催化制备生物柴油得率为81.2%，重复利用7批次，活力仍保持80%以上。项目研究成果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。.项目严格按照任务书内容和要求，有计划地开展研究工作，4年来，完成了任务书规定的工作内容，达到了预期研究目标和考核指标。截止目前，标注该项目已发表SCI论文5篇，中文核心1篇；培养硕博士生4名。.项目经费使用按照《国家自然科学基金财务管理办法》执行，支出合规合理，共支出682,383.61元，结余117,616.39元。结余经费将于2017年用于以生物质为原料，微生物发酵制备油脂，解决全细胞催化制备生物柴油所需油脂原料来源，形成“生物质-微生物油脂-全细胞催化-生物柴油”完整的技术产业链。