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Robust NADH-regenerator: improved alpha-haloketone-resistant formate dehydrogenase
H Yamamoto1, K Mitsuhashi, N Kimoto
1Life Science Development Center, CPI Company, Tsukuba Research Center, Daicel Chemical Industries, 27 Miyukigaoka, Tsukuba 305-0841, Japan. h-yamamoto@daicel.co.jp
Applied Microbiology and Biotechnology
|September 1, 2004
Summary
Engineered formate dehydrogenases (FDHs) show enhanced stability and activity for industrial applications. A novel mutant enzyme enabled efficient synthesis of optically active alpha-haloalcohols, crucial chiral building blocks.
Area of Science:
- Biocatalysis
- Enzyme Engineering
- Organic Synthesis
Background:
- Formate dehydrogenases (FDHs) are vital for NADH regeneration in asymmetric reductions.
- FDHs exhibit low activity and instability, particularly against alpha-haloketones, limiting their industrial use.
- Optically active alpha-haloalcohols are valuable synthetic intermediates.
Purpose of the Study:
- To enhance the stability and activity of Mycobacterium vaccae formate dehydrogenase (McFDH) against alpha-haloketones.
- To develop a robust biocatalytic process for synthesizing optically active alpha-haloalcohols.
Main Methods:
- Site-directed mutagenesis of McFDH to create cysteine mutants.
- Enzyme characterization of mutant FDHs for stability and activity.
- Coexpression of engineered McFDH and a carbonyl reductase (KaCR1) in E. coli for synthesis.
Main Results:
- Mutant C256S displayed significant tolerance to ethyl 4-chloroacetoacetate (ECAA).
- Mutant C146S showed activation by organic compounds.
- An optimized mutant, McFDH-26 (C6A/C146S/C256V), was constructed.
- High yield (49.9 g/l) and enantiopurity (>99% e.e.) of ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-ECHB] were achieved.
Conclusions:
- Engineered McFDH variants demonstrate improved stability and performance.
- The optimized biocatalytic system using McFDH-26 and KaCR1 is effective for industrial production of chiral alpha-haloalcohols.
- This study provides a promising strategy for enzymatic synthesis of valuable chiral compounds.