Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids.However,compared to the widely studied a-AADHs,l imited knowledge is available about b-AADHs that enable the synthesis of b-amino acids.Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants,the only knownmember of b-AADH family.C rystal structure analysis,s ite-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from a-AADHs.Anumber of rationally engineered variants were then obtained with improved activity (by 110-800 times) towardvarious aliphatic b-amino acids without an enantioselectivity trade-off.T wo b-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (> 99 %e e) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish as olid foundation for further design of b-AADHs for the asymmetric synthesis of b-amino acids.
While chiral fused-ring tetrahydroisoquinoline (THIQ) and tetrahydro-β-carboline (THβC) scaffolds have attracted considerable interest due to their wide spectrum of biological activities, the synthesis of optically pure chiral fused-ring THIQs and THβCs remains a challenging task. Herein, a group of active imine reductases were identified to convert the imine precursors into the corresponding enantiocomplementary fused-ring THIQs and THβCs with high enantioselectivity and conversion, establishing an efficient and green chemoenzymatic approach to fused-ring alkaloids from 2arylethylamines.
An enzymatic method for the synthesis of ethyl (R)‐3‐hydroxyglutarate from ethyl (R)‐4‐cyano‐3‐hydroxybutyate was developed by using free and immobilized recombinant Escherichia coli BL21(DE3)pLysS harboring a nitrilase gene from Arabidopsis thaliana (AtNIT2). The hydrolysis of ethyl (R)‐4‐cyano‐3‐hydroxybutyate proceeded with the freely suspended cells of the biocatalyst under the optimized conditions of 1.5 mol L−1 (235.5 g L−1) substrate concentration and 6.0 wt % loading of wet cells at pH 8.0 and 25 °C, with 100 % conversion obtained in 4.5 h. Furthermore, immobilization of the whole cells enhanced their substrate tolerance, stability, and reusability. Under the optimized conditions (100 mmol L−1 tris(hydroxymethyl)aminomethane hydrochloride buffer, pH 8.0, 25 °C), the immobilized biocatalyst could be reused for up to 16 batches, with a biocatalyst productivity of 55.6 g gwet cells−1 and a space‐time productivity of 625.5 g L−1 d−1. These results demonstrated that the immobilized whole cells might be used as a biocatalyst in the industrial production of ethyl (R)‐3‐hydroxyglutarate, a key intermediate for the synthesis of rosuvastatin.
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