Enzyme cascade for biocatalytic deracemization of D,L-phosphinothricin

•A novel enzyme cascade-based deracemization of phosphinothricin was developed.•A “two stages in one-pot” strategy was developed to avoid by-product inhibition.•High yield and excellent enantiomeric excess of product were achieved.•No extra cofactor was required for the developed enzyme cascade. Deracemization of D,L-phosphinothricin (D,L-PPT) is one of the most promising routes for preparation of optically pure L-PPT. In this work, an efficient multi-enzyme redox cascade was developed for deracemization ofPPT, which includes oxidative reaction and reductive reaction. The oxidative reaction catalyzing oxidative deamination of D-PPT to 2-oxo-4-[(hydroxy)(-methyl)phosphinyl]butyric acid (PPO) was performed by a D-amino acid oxidase and a catalase for removing H2O2. The reductive reaction catalyzing amination of PPO to L-PPT is achieved by a glufosinate dehydrogenase and a glucose dehydrogenase for cofactor regeneration. To avoid the inhibitory effect of glucose on the oxidative reaction, a “two stages in one-pot” strategy was developed to combine these two reactions in deracemization process. By using this strategy, the L-PPT was obtained with a high yield (89 %) and > 99 % enantiomeric excess at substrate loading of 300 mM in absence of addition of extra NADP+. These encouraging results demonstrated that the developed enzyme cascade deracemization process exhibits great potential and economical competitiveness for manufacture of L-PPT from D,L-PPT.