Enzyme Cascade with Four Enzymes in One Pot for the Synthesis of L‐Phosphinothricin

Abstract The biocatalytic oxidative deamination‐ reductive amination process for the production of L ‐phosphinothricin ( L ‐PPT) from D,L‐phosphinothricin ( D,L ‐PPT) is a green and environmentally friendly approach with significant development potential. In this study, the adopted technological route involves the oxidative deamination of the D ‐PPT component in D , L ‐PPT to 2‐oxo‐4‐(hydroxymethylphosphinyl)butanoic acid (PPO) by utilizing D‐amino acid oxidase (DAAO), followed by the catalytic reductive amination of PPO to L ‐PPT using phosphinothricin dehydrogenase (PpDH). In order to enhance the catalytic rate and reduce the inhibitory effect of the intermediate product PPO on the catalytic process, optimization of both oxidative deamination and reductive amination phases was conducted through separate strategies of constant‐pressure oxygen supply and substrate feeding, respectively. A “two‐step one‐pot” method was employed to link the optimized reactions, significantly shortening the production time. Furthermore, the “two‐step one‐pot” catalytic strategy was optimized, ultimately achieving a high 97.7% yield of L ‐PPT with ee of L ‐PPT>99.9% after 5.5 h of reaction under 800 mM D , L ‐PPT catalytic conditions, D‐PPT and PPO could not bedetected in the reaction system, further enchanced the catalytic efficiency, production efficiency and product quality. The aforementioned results demonstrate that this proposed approach has great potential for industrial‐scale production of L ‐PPT as well as significant competitive advantages in terms of economic feasibility.