Construction and characterization of a novel glucose dehydrogenase-leucine dehydrogenase fusion enzyme for the biosynthesis of L-tert-leucine.
Microb Cell Fact
; 20(1): 3, 2021 Jan 06.
Article
en En
| MEDLINE
| ID: mdl-33407464
ABSTRACT
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.Palabras clave
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Bacillus cereus
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Bacillus megaterium
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Proteínas Recombinantes de Fusión
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Glucosa 1-Deshidrogenasa
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Leucina-Deshidrogenasa
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Leucina
Idioma:
En
Revista:
Microb Cell Fact
Asunto de la revista:
BIOTECNOLOGIA
/
MICROBIOLOGIA
Año:
2021
Tipo del documento:
Article