Identification of a novel D-amino acid oxidase and its application in deracemization of D, L-phosphinothricin

DAAO is applied as a potential catalyst in the biosynthesis of L-PPT. However, its low solubility expression constrains its broader industrial application. Herein, a novel DAAO derived from Cladophialophora carrionii ( Cc DAAO) was identified, which demonstrated superior catalytic performance toward D-Ala (specific activity: 106.38 ± 1.21 U/mg, K m : 1.56 ± 0.06 mM), along with remarkable thermostability and broad substrate spectrum. Under optimal culture conditions, the soluble expression level of Cc DAAO was enhanced through a co-expression strategy with molecular chaperones, and the enzyme activity increased by 36.3% compared with the initial level. Subsequently, Cc DAAO was constructed as a fusion protein (CGD) with catalase from Geobacillus sp. CHB1 ( Gb CAT) and applied in a D-amino acid aminotransferase (DAAT)-mediated cascade system. In a 2 L reaction system, this cascade system achieved complete conversion (> 99%) of 1 M D,L-PPT within 8 h, exhibiting a yield of 11.26 g/L/h for PPO, which represents a significant improvement over existing reports. This study presents a promising practical approach for the industrial production of optically pure L-PPT.