Rohan Karande et al.
FULL PAPERS
phosphate buffer. Elution was performed at the same flow
rate by applying a linear gradient of 2M NaCl min in
phase was separated and dried by adding sodium sulphate
prior to analysis on a Focus gas chromatography (Focus GC,
Thermo Electron Corporation, Dreieich, Germany) using a
chiral RT-ßDex-sm column (30 mꢂ0.25 mmꢂ0.25 mm;
Restek GmbH, Bad Homburg, Germany). The organic
phase samples were diluted 1:10 times in ice-cold diethyl
ether, dried with sodium sulphate, and analyzed as described
above.
À1
10 mM phosphate buffer pH 7.5. Fractions were concentrat-
ed 5-fold by filtration (Centricon, 10 KDa, Millipore Corpo-
ration, Schwalbach, Germany) at 3990ꢂg (48C), aliquoted
in 1-mL Eppendorf tubes and stored at À208C. A final
purity of 60 to 70% was reached (based on PAGE analysis)
and no contaminating activities were found in the control
experiments.
Determination of TADH and FDH Activity, and
Protein Concentration
Acknowledgements
TADH and FDH activities were measured by UV absorp-
tion of NADH at 340 nm using a Cary 300 Bio UV-VIS
spectrophotometer (Varian, Darmstadt, Germany). The
assay mixture for TADH contained 160 mM 3-methylcyclo-
hexanone and 0.2 mM NADH in 100 mM Bis-Tris to a total
volume of 200 mL, pH 6.5 at 458C. This mixture was thermo-
statted at 458C and the reaction was started by adding
TADH.
The assay mixture for FDH contained 200 mM ammoni-
um formate and 0.5 mM NAD in 100 mM phosphate buffer
pH 6.5 at 308C to a total volume of 200 mL. This mixture
was thermostatted at 308C and the reaction was started by
adding FDH.
We thank Prof. Martina Pohl (Forschungszentrum Jꢀlich) for
the kind gift of plasmids containing formate dehydrogenase
genes. Furthermore, we thank Dr. Jonathan Collins for his
expert support during vacuum distillation. This work was fi-
nanced by the ZACG (Zentrum fꢀr Angewandte Chemische
Genomik), the European Union (EFRE) and by the Ministry
of Innovation, Science, Research and Technology of North
Rhine-Westphalia.
References
For TADH activity the decrease of NADH was followed
at 340 nm for 60 sec, while for FDH the increase of the
NADH signal was monitored. Enzyme activities were calcu-
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2520
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2011, 353, 2511 – 2521