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The application of wild‐type ω‐transaminase was limited by steric hindrance towards bulky substrates, therefore improvement of the catalytic efficiency and stereoselectivity toward substrates with two bulky substituent adjacent to the carbonyl is of general interest. In this study, according to the double substrate binding pocket theory, a (S)‐selective ω‐transaminase from the Burkholderia vietnamiensis G4, which showed puny catalytic activity toward the β‐keto esters with small steric hindrance, was engineered to accept bulky β‐keto esters, which were not accessible by any wild‐type enzyme. A few desired variants were obtained that exhibited activity toward bulky β‐keto esters. Furthermore, a substrate‐dependent shift in enantio‐preference of HBV variant towards β‐keto esters with linear or branched aliphatic substituents was observed. The best variant was applied to the asymmetric synthesis of aliphatic β‐amino acids at semi‐preparative scale with high yield and enantioselectivity. This study will improve the general understanding and inspire further engineering work for reversing enantio‐specificity of ω‐transaminases. [ABSTRACT FROM AUTHOR]

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α‐AADHs, limited knowledge is available about β‐AADHs that enable the synthesis of β‐amino acids. Herein, we report the crystal structures of a l‐erythro‐3,5‐diaminohexanoate dehydrogenase and its variants, the only known member of β‐AADH family. Crystal structure analysis, site‐directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α‐AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110–800 times) toward various aliphatic β‐amino acids without an enantioselectivity trade‐off. Two β‐amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86–87 %). These results provide important insights into the molecular mechanism of 3,5‐DAHDH, and establish a solid foundation for further design of β‐AADHs for the asymmetric synthesis of β‐amino acids. [ABSTRACT FROM AUTHOR]

Terminal vicinal diols are important chiral building blocks and intermediates in organic synthesis. Reduction of α-hydroxy ketones provides a straightforward approach to access these important compounds. In this study, it has been found that asymmetric reduction of a series of α-hydroxy aromatic ketones and 1-hydroxy-2-pentanone, catalyzed by Candida magnolia carbonyl reductase (CMCR) with glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, afforded 1-aryl-1,2-ethanediols and pentane-1,2-diol, respectively, in up to 99 % ee. In order to evaluate the efficiency of the bioreduction, lyophilized recombinant Escherichia coli whole cells coexpressing CMCR and GDH genes were used as the biocatalyst and α-hydroxy acetophenone as the model substrate, and the reaction conditions, such as pH, cosolvent, the amount of biocatalyst and the presences of a cofactor (i.e., NADP+), were optimized. Under the optimized conditions (pH 6, 16 h), the bioreduction proceeded smoothly at 1.0 m substrate concentration without the external addition of cofactor, and the product ( S)-1-phenyl-1,2-ethanediol was isolated with 90 % yield and 99 % ee. This offers a practical biocatalytic method for the preparation of these important vicinal diols. [ABSTRACT FROM AUTHOR]

Highlights: [•] A new (S)-enantioselective ω-transaminase from Burkholderia vietnamiensis G4 was biochemically characterized. [•] The transaminase exhibited broad substrate specificity and excellent enantioselectivity (>99%). [•] It is the first example that ω-transaminase catalyzed the amino transfer to β-keto ester to give optically pure β-amino acid ester. [•] Asymmetric synthesis of unnatural amino acids, such as (S)-4-aminovaleric acid, (S)-3-aminopentanoic acid ethyl ester and (S)-3-aminobutyric acid ethyl ester, was achieved. [Copyright &y& Elsevier]

A new carboxylic acid reductase (CAR) gene from Segniliparus rotundus DSM 44985 was overexpressed in Escherichia coli . The recombinant enzyme exhibited high activity toward a variety of aromatic and aliphatic carboxylic acids. Especially, it effectively reduced 4-hydroxybenzoic acid ( 8a ) and 4-nitrobenzoic acid ( 19a ), toward which the known Nocardia CAR exhibited no or little activity. The recombinant E. coli cells co-expressing the Segniliparus CAR and Nocardia PPTase genes catalyzed the reductions of vanillic acid ( 20a ) and 3,4-dihydroxyphenylacetic acid ( 25a ) to give vanillyl alcohol ( 20c ) and 3-hydroxytyrosol ( 25c ) with high yield, respectively. The endogenous aldehyde reductases of E. coli should be responsible for the further reduction of the produced aldehydes. These results demonstrated that Segniliparus CAR was a useful addition to the biocatalyst tool-box for the reduction of carboxylic acids and might find applications in the synthesis of valuable bio-based chemicals from renewable resources. [ABSTRACT FROM AUTHOR]