The gene of the NAD‐dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol‐coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site‐directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the T_m values revealed that the stability at temperatures above 50 °C is optimal for the native and the recombinant wt enzyme (T_m 57 °C), whereas the T_m values of the mutant enzymes vary in the range 44–52 °C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.

Shake-flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10-fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.