Yersinia spp. 来源植酸酶的构效关系解析及酶学性质的分子改造

当前商业化植酸酶的酶学性质难以完全满足饲料工业的要求，改良植酸酶的酶学性质有利于提高其在饲料中的有效性。YiAPPA是目前已知的活性最高的植酸酶，YrAPPA是与YiAPPA结构相似的植酸酶中蛋白酶抗性、酸性条件下酶活和热稳定性最优良的植酸酶。鉴于YiAPPA、YrAPPA在结构、催化机制上的相似性，以及酶学性质的差异性，本课题采用基于结构的基因重组技术和定点突变技术构建两者的杂合酶和点突变体，通过比较杂合酶、点突变体与YiAPPA、YrAPPA的酶学性质，确定与酶学性质相关的关键结构区域和氨基酸残基。进一步结合突变体的性质和模拟的蛋白质结构，分别阐明YiAPPA、YrAPPA具有优良酶学性质的分子机制。并在此基础上，分别对这两种植酸酶的酶学性质进行分子改造，以获得更适用于饲料工业的植酸酶。本研究有助于进一步了解植酸酶的催化机理，为植酸酶的酶学性质改良提供理论依据和设计思路。

The enzymatic properties of current commercial phytases are difficult to fully meet the requirements of feed industry. Improvement of phytase performance can enhance the effectiveness of phytases as feed additive. YiAPPA is characterized as the phytase with the highest activity ever reported. YrAPPA is one of the phytases which show structural similarity to YiAPPA, and exhibits the highest protease resistance, acidic activity and stability among the phytases. Based on the similarities in structural characteristics and catalytic mechanism, and the differences in enzymatic properties, structure-based fragment shuffling is used to generate hybrids between YiAPPA and YrAPPA. The key regions for different enzymatic properties of YiAPPA and YrAPPA are identified by comparing the enzymatic properties of the hybrids, YiAPPA and YrAPPA. Further, site-directed mutagenesis is used to construct point mutants to identify important residues associated with the enzymatic properties of YiAPPA and YrAPPA. Molecular basis of the maintance of fine enzymatic properties and structure-function relationship of YiAPPA and YrAPPA are revealed by analyzing the enzymatic properties and tertiary structure of mutants. And on this basis, the enzymatic properties of this two phytases were modified to obtain phytases which were more suitable for feed industry. This study helps to further understand the catalytic mechanism of phytase. Moreover, this study can also provide effective strategies for commercial production of phytases of improved enzymatic properties.