水稻α-半乳糖苷酶的分子改造及全细胞催化体系的构建

α-Galactosidase (α-Gal) is exo-glycosidase that cleave with the optical congiguration retained, the terminal nonreducing α-D-galactose residues. It is particular interest in view of their biotechnological applications in food, feed and pharmaceutical industry, and is rated as one of the enzymes with highest application value and most demand. In this project, acid rice α-Gal I with obvious advantage in degradation of galactomanna and alkaline rice α-Gal IV with specific transferase activity will be investigated. The whole-cell biocatalyst of rice α-Gal I and α-Gal IV will be constructed via yeast cell-surface display system. The characteristic structure and key amino acid residues which are relevant to activity and thermal stability of the rice α-Gal will be discussed. For improving the activity and thermal stability, moleculars of α-Gal I and α-Gal IV will be modified via the semi-rational design and the directed evolution, respectively. The properties, kinetic characterization and substrates specificities of rice α-Gal I and α-Gal IV displayed on yeast cell surface will be analyzed further. The catalyst action，mechanism and model on degradation of galactomanna and synthesis of galacto-oligosaccharides will be characterized. This research will provide the theoretical basis for widening application of α-Gal, solve the problems in concern with low level of catalysis activity and thermal stability, complexity of extraction. And it is also important for exploring the structure and function of rice complete genome.

α-半乳糖苷酶（α-Gal）属外切糖苷酶类，专一性催化非还原末端α-半乳糖苷键的水解，在食品、饲料、医药工业等领域应用广泛，被列为我国最具应用价值和市场急需的酶制剂之一。本项目拟选用水稻中对半乳甘露聚糖有显著降解优势的酸性α-GalⅠ和具有独特转糖基活性的碱性α-Gal Ⅳ为研究对象，构建两个酶的酵母表面展示全细胞催化体系；分析影响活性、热稳定性的特征结构和重要氨基酸残基；分别采用半理性设计和定性进化策略对两种酶基因进行分子改造以提高酶的活力和稳定性；进一步研究酵母表面展示α-Gal的理化性质、动力学性质及底物特异性；解析酶在催化半乳低聚糖水解与合成及半乳甘露聚糖降解作用中的功能特性、活性表达机理与作用模式的表征；本研究的提出将为拓宽α-Gal应用领域奠定理论基础；解决普遍存在的酶活力低，热稳定性差，提取工艺复杂等实际应用中的瓶颈问题；并对水稻全基因组构造与机能的解析具有重要意义。

α-半乳糖苷酶（α-Gal）属外切糖苷酶类，专一性催化非还原末端α-半乳糖苷键的水解，在食品、饲料、医药工业等领域应用广泛，被列为我国最具应用价值和市场急需的酶制剂之一。本项目以水稻中对半乳甘露聚糖有显著降解优势的酸性α-GalⅠ和具有独特转糖基活性的碱性α-Gal Ⅳ为主要研究对象，利用同源建模结构预测法分析了源自水稻α-Gal I 和α-Gal VI的二、三级结构。在结构预测的基础上，推测α-Gal I与模板α-Gal在多糖底物特异性上出现显著差异可能是由C-末端的三个差别区域造成的，为理性设计改造α-Gal酶分子底物适应性奠定了基础。通过分子动力学模拟的方法分析了影响α-Gal I 的热稳定性的关键氨基酸和突变位点，为下一步针对热稳定性的酶分子改造打下良好基础。本项目按计划完成了α-Gal酵母表面展示质粒载体的初步构建，其活性表达也正在探讨中。此外，在半乳甘露聚糖酶解改性的调控及产物的DSC热分析、质构性质分析方面均获得颇具价值的进展。值得一提的是，我们首次用纯化的棉籽糖合成酶（α-Gal VI）对棉籽糖生物合成反应动力学性质及催化机制进行了系统研究，对该类酶的基础研究是一个重要贡献。以上述成果为基础，现已申报国家发明专利1项，发表中文核心期刊论文2篇，待发食品科学领域SCI、EI论文各1篇（under review）。