L-amino acid oxidases (LAAOs) can be applied to convert racemic amino acids to D-isomers, which are potential precursors of pharmaceuticals. However, this application is hampered by the lack of available stable and structure-determined LAAOs. In this study, we attempt to address this limitation by utilizing two ancestral LAAOs: AncLAAO-N4 and AncLAAO-N5. AncLAAO-N4 has the highest thermal and temporal stabilities among the designed LAAOs that can be used for deracemization and stereoinversion. AncLAAO-N5 can provide X-ray crystal structures, which are helpful to reveal substrate recognition and reaction mechanisms of LAAOs at the molecular level. Next, we attempted to improve activity of AncLAAO-N4 toward L-Val through a semi-rational protein engineering method. Three variants with enhanced activity toward L-Val were obtained. Taken together, we believe that the activity and substrate selectivity of AncLAAOs give them the potential to be key enzymes in various chemoenzymatic reactions.

L-氨基酸氧化酶 (LAAO) 可用于将外消旋氨基酸转化为 D-异构体，后者是药物的潜在前体。然而，由于缺乏可用的稳定和结构确定的 LAAO，该应用受到阻碍。在这项研究中，我们试图通过利用两个祖先的 LAAO：AncLAAO-N4 和 AncLAAO-N5 来解决这一限制。AncLAAO-N4 在设计的 LAAO 中具有最高的热稳定性和时间稳定性，可用于去外消旋和立体转化。AncLAAO-N5 可以提供 X 射线晶体结构，有助于在分子水平上揭示 LAAO 的底物识别和反应机制。接下来，我们尝试通过半理性蛋白质工程方法提高 AncLAAO-N4 对 L-Val 的活性。获得了对 L-Val 具有增强活性的三种变体。综合起来，