“…The quality of the reconstructed model met our requirements for subsequent analysis because 99.45 % of the residues exhibited an average 3D‐1D score of more than 0.2 (Figure S1), and 89.4 % of the residues were in the most favored regions [27] (Figure S2). Since residue D218 stays far away from the active center of the enzyme, its presence in the unfavorable region in the Ramachandran plots shows no influence on the accuracy of the model.…”
Due to its stringent stereospecificity, D‐amino acid oxidase (DAAO) has made it very easy to synthesize L‐amino acids. However, the low activity of the wild‐type enzyme toward unnatural substrates, such as D‐glufosinate (D‐PPT), restricts its application. In this study, DAAO from Rhodotorula gracilis (RgDAAO) was directly evolved using a hydrophilicity‐substitution saturation mutagenesis strategy, yielding a mutant with significantly increased catalytic activity against D‐PPT. The mutant displays distinct catalytic properties toward hydrophilic substrates as compared to numerous WT‐DAAOs. The analysis of homology modeling and molecular dynamic simulation suggest that the extended reaction pocket with greater hydrophilicity was the reason for the enhanced activity. The current study established an enzymatic synthetic route to L‐PPT, an excellent herbicide, with high efficiency, and the proposed strategy provides a new viewpoint on enzyme engineering for the biosynthesis of unnatural amino acids.
“…The quality of the reconstructed model met our requirements for subsequent analysis because 99.45 % of the residues exhibited an average 3D‐1D score of more than 0.2 (Figure S1), and 89.4 % of the residues were in the most favored regions [27] (Figure S2). Since residue D218 stays far away from the active center of the enzyme, its presence in the unfavorable region in the Ramachandran plots shows no influence on the accuracy of the model.…”
Due to its stringent stereospecificity, D‐amino acid oxidase (DAAO) has made it very easy to synthesize L‐amino acids. However, the low activity of the wild‐type enzyme toward unnatural substrates, such as D‐glufosinate (D‐PPT), restricts its application. In this study, DAAO from Rhodotorula gracilis (RgDAAO) was directly evolved using a hydrophilicity‐substitution saturation mutagenesis strategy, yielding a mutant with significantly increased catalytic activity against D‐PPT. The mutant displays distinct catalytic properties toward hydrophilic substrates as compared to numerous WT‐DAAOs. The analysis of homology modeling and molecular dynamic simulation suggest that the extended reaction pocket with greater hydrophilicity was the reason for the enhanced activity. The current study established an enzymatic synthetic route to L‐PPT, an excellent herbicide, with high efficiency, and the proposed strategy provides a new viewpoint on enzyme engineering for the biosynthesis of unnatural amino acids.
“…Overexpression of the otsBA operon inE. coli, which codes for trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, was used to study the production of Trehalose [ 27 ]. Fig.…”
Due to its stringent stereospecificity, D-amino acid oxidase (DAAO) has made it very easy to synthesize L-amino acids. However, the low activity of the wild-type enzyme toward unnatural substrates, such as D-glufosinate (D-PPT), restricts its application. In this study, DAAO from Rhodotorula gracilis (RgDAAO) was directly evolved using a hydrophilicitysubstitution saturation mutagenesis strategy, yielding a mutant with significantly increased catalytic activity against D-PPT. The mutant displays distinct catalytic properties toward hydrophilic substrates as compared to numerous WT-DAAOs. The analysis of homology modeling and molecular dynamic simulation suggest that the extended reaction pocket with greater hydrophilicity was the reason for the enhanced activity. The current study established an enzymatic synthetic route to L-PPT, an excellent herbicide, with high efficiency, and the proposed strategy provides a new viewpoint on enzyme engineering for the biosynthesis of unnatural amino acids.
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