Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system. The thermodynamically unfavorable oxidation reaction was driven by the reduction reaction. The efficiency of the biocatalytic reaction was evaluated using 12 different substrate combinations, and a significant variation was observed in substrate conversion, which was subsequently explained by the differences in enzyme kinetics parameters. The reaction with the selected model substrates 2-oxobutanoic acid and L-leucine reached 90.3% conversion with a high total turnover number of 9.0 × 106 under the optimal reaction conditions. Furthermore, complete conversion was achieved by adjusting the ratio of addition of the two substrates. The constructed reaction mode can be applied to other amino acid dehydrogenases in future studies to synthesize a wider range of valuable products.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of l-tert-leucine

Background Biosynthesis of l-tert-leucine (l-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of l-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully. Results In this work, a novel fusion enzyme (GDH–R3–LeuDH) for the efficient biosynthesis of l-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH–R3–LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yield of l-tle by GDH–R3–LeuDH was all enhanced by twofold. Finally, the space–time yield of l-tle catalyzing by GDH–R3–LeuDH whole cells could achieve 2136 g/L/day in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose). Conclusions It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize l-tle and reach the highest space–time yield up to now. These results demonstrated the great potential of the GDH–R3–LeuDH fusion enzyme for the efficient biosynthesis of l-tle.

Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine

Background: Biosynthesis of L-tert-leucine (L-tle), a significant pharmaceutical intermediate, by a cofactor regeneration system friendly and efficiently is a worthful goal all the time. The cofactor regeneration system of leucine dehydrogenase (LeuDH) and glucose dehydrogenase (GDH) has showed great coupling catalytic efficiency in the synthesis of L-tle, however the multi-enzyme complex of GDH and LeuDH has never been constructed successfully.Results: In this work, a novel fusion enzyme (GDH-R3-LeuDH) for the efficient biosynthesis of L-tle was constructed by the fusion of LeuDH and GDH mediated with a rigid peptide linker. Compared with the free enzymes, both the environmental tolerance and thermal stability of GDH-R3-LeuDH had a great improved since the fusion structure. The fusion structure also accelerated the cofactor regeneration rate and maintained the enzyme activity, so the productivity and yeild of L-tle by GDH-R3-LeuDH was all enhanced by 2-fold. Finally, the space-time yield of L-tle catalyzing by GDH-R3-LeuDH whole cells could achieve 2136 g/L/d in a 200 mL scale system under the optimal catalysis conditions (pH 9.0, 30 °C, 0.4 mM of NAD+ and 500 mM of a substrate including trimethylpyruvic acid and glucose).Conclusions: It is the first report about the fusion of GDH and LeuDH as the multi-enzyme complex to synthesize L-tle and reach the highest space-time yield up to now. These results demonstrated the great potential of the GDH-R3-LeuDH fusion enzyme for the efficient biosynthesis of L-tle.