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.
['Liao|Langxing|L|', 'Zhang|Yonghui|Y|', 'Wang|Yali|Y|', 'Fu|Yousi|Y|', 'Zhang|Aihui|A|', 'Qiu|Ruodian|R|', 'Yang|Shuhao|S|', 'Fang|Baishan|B|http://orcid.org/0000-0002-8428-5079']
D001409:Bacillus cereus D001410:Bacillus megaterium D042843:Glucose 1-Dehydrogenase D007930:Leucine D050803:Leucine Dehydrogenase D011487:Protein Conformation D011993:Recombinant Fusion Proteins
Fusion enzyme L-tert-leucine Leucine dehydrogenase Glucose dehydrogenase Whole cells