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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. [ABSTRACT FROM AUTHOR]

D‐Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of D‐amino acids, making it one of the most promising routes for synthesizing optically pure L‐amino acids, including L‐phosphinothricin (L‐PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate D‐PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward D‐PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel‐pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M3rd‐SHVG was obtained, exhibiting a >2000‐fold increase in relative activity. The kinetic parameters showed that M3rd‐SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M3rd‐SHVG reshapes the tunnel‐pocket and corrects the direction of enzyme–substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel‐pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M3rd‐SHVG as biocatalyst, 500 mM D, L‐PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production. [ABSTRACT FROM AUTHOR]

BACKGROUND: Sea cucumber Acaudina leucoprocta is a low‐value marine product with high contents of heavy metals in the body wall. However, it could be used as a substitute source of land‐based animals for value‐added products L‐proline (L‐Pro) and L‐hydroxyproline (L‐Hyp). For high recoveries and purities of L‐Pro and L‐Hyp from A. leucoprocta hydrolysate, the preparative separation of these imino acids with cation exchange resins was investigated. RESULTS: Among 11 cation exchange resins tested, Dowex 50WX8 resin offered the best separation capacity for L‐Pro and L‐Hyp from each other and impurities. The static equilibrium adsorption data fitted well with the Langmuir isotherm, and the adsorption kinetic followed the pseudo‐second‐order model. For the most effective ion exchange process, the adsorption and desorption parameters of L‐Pro and L‐Hyp were optimized with the column packed with Dowex 50WX8 resin. Under optimal conditions, the preparative separation of L‐Pro and L‐Hyp was easily and effectively achieved. The L‐Pro purity increased from 55.7% in the extract of A. leucoprocta hydrolysate to 94.3% in the eluent, and the L‐Hyp purity increased from 27.8% to 95.7%; their final recovery yields from the extract of A. leucoprocta hydrolysate were 93.3% and 89.4%, respectively. CONCLUSION: Our study provided an effective approach for large‐scale production of imino acids extracted from low‐value sea products, and offered a scientific reference for the separation of other valuable components. © 2018 Society of Chemical Industry [ABSTRACT FROM AUTHOR]