D-氨基酸氧化酶特异性固定及其催化研究

D-氨基酸氧化酶（DAAO）在手性氨基酸拆分和药物中间体制备等方面具有广泛应用。酶固定化是提高DAAO催化效率和催化稳定性的一个有效方法。本申请拟研究DAAO定向固定及其催化应用。所提出的酶固定方法分为两个步骤。（1）卟啉铁功能化硅胶球：在硅胶球外包覆巯基硅氧烷；通过卟啉铁的乙烯基与巯基反应，把卟啉铁接枝到硅胶球表面。（2）DAAO的定向固定：通过DAAO表面的氨基与卟啉铁的羧基反应，把D-氨基酸氧化酶固定在功能化硅胶球上。所提出的DAAO固定方法具有如下特点：DAAO与卟啉铁以分子的距离紧邻；DAAO催化后产生的副产物双氧水能够被卟啉铁快速催化而分解为水和氧气；所产生的氧气富集在DAAO周围，可被DAAO的辅因子FAD有效利用；这样的连续循环催化加速了DAAO催化底物的速率。本项目申请将把固定化DAAO应用于手性氨基酸拆分和药物中间体催化合成。

D-Amino acid oxidases are extensively applied to separate racemic mixtures of amino acids and to manufacture the important intermediate compounds. Enzyme immobilization is a method for improvement of enzymatic efficiency and stability. This project is to specifically immobilize D-amino acid oxidase. Spherical silicon will be functionalized with hematin, and D-amino acid oxidase will be immobilized on the spherical silicon by coupling to the hematin. The specific enzyme immobilization enables the DAAO in proximity to the hematin at a molecular distance. The molecular-distance proximity facilitates the immediate decomposition of H2O2 catalyzed by the hematin. The evolved oxygen can be efficiently utilized to oxidize the reduced cofactor FDA of DAAO and H2O2 is produced. The forming of H2O2-O2-H2O2 circle between the DAAO and hematin is a driving force to accelerate the deamination reaction. This project will demonstrate an approach for highly specific enzyme immobilization onto spherical silicon. The approach can be extended to other nanomaterials, possessing potential applications in oxidase-based cascade catalysis processes.