Abstract: D-amino acid oxidase is a class of flavine adenine dinucleotide containing oxidordeuctase that catalyzes the oxidative dehydrogenation of D-amino acids, which results in corresponding α-keto acids, hydrogen peroxide, and ammonia. D-amino acid oxidases are widely distributed in nature, mainly found in multiple eukaryotic and few prokaryotic organisms. As a classic biocatalyst, D-amino acid oxidases have played important roles in the synthesis of various medicine, pesticides as well as fine chemicals, owing to the mild reaction conditions, broad substrate spectrum and excellent enantioselectivity. Herein, we review the structural characteristics, catalytic mechanism, and protein engineering of D-amino acid oxidase, as well as its application in biocatalysis. Different molecular modification strategies for the engineering of D-amino acid oxidase and the representative achievements are further described. The preparation of 7-aminocephalosporanic acid，chiral amino acids，amines and α-keto acids by using D-amino acid oxidase is also summarized. Finally, the current problems in the biocatalytic application of this enzyme are discussed. The subsequent research could focus on the mining and engineering of novel D-amino acid oxidase. Based on the molecular mechanism of enantioselectivity and substrate recognition, the rational design could be applied to modify the catalytic performance. Furthermore, the newly obtained enzymes could be utilized to construct novel biosynthetic pathways for functional chemicals.

Key words: D-amino acid oxidase, molecular modification, biocatalysis, chiral amino acids, chiral amines

摘要： D-氨基酸氧化酶是一类含有黄素腺嘌呤二核苷酸的氧化还原酶，能够催化D-氨基酸氧化脱氢，生成相应的α-酮酸，过氧化氢和氨。该类酶在自然界中分布广泛，主要来源于真核生物和少数原核生物。作为一种经典的生物催化剂，D-氨基酸氧化酶具有反应条件温和、底物谱广泛、对映体选择性好等特点，在合成医药、农药和精细化学品等方面具有重要的应用价值。本文综述了D-氨基酸氧化酶的基本蛋白结构特征及其催化机制，重点介绍了D-氨基酸氧化酶底物特异性和热稳定性分子改造的策略和代表性成果，以及该类酶在生物催化中的应用，例如制备7-氨基头孢烷酸、手性氨基酸、胺类化合物和α-酮酸。最后，探讨了D-氨基酸氧化酶目前在生物催化应用过程中存在的问题。后续的研究可围绕新酶的挖掘与改造展开工作。基于对映体选择性和底物识别的分子机制，理性设计酶的催化性能，并以挖掘或改造获得的D-氨基酸氧化酶作为新酶元件，用于构建功能化学品生物合成新途径。

关键词: D-氨基酸氧化酶, 分子改造, 生物催化, 手性氨基酸, 手性胺