Collect. Czech. Chem. Commun.
1984, 49, 2415-2423
https://doi.org/10.1135/cccc19842415
Use of immobilized alcohol dehydrogenase for the reduction of NAD to NADH
Jan Kovářa, Marie Navrátilováa, Ladislav Skurskýa, Jaroslav Drobníkb, František Švecb and Jiří Fischerc
a Department of Biochemistry, Purkyně University, 611 37 Brno
b Institute of Macromolecular Chemistry, Czechoslovak Academy of Sciences, 162 06 Prague 6
c Lachema, 621 33 Brno
Abstract
The possibility was examined of preparative NAD to NADH reduction in the presence of an excess of ethanol by alcohol dehydrogenase immobilized on a copolymer of glycidylmethacrylate with ethylenebismethacrylate. The effect of pH, ethanol concentration and of compounds reacting with acetaldehyde on the conversion and on the purity of NADH formed is described. Under optimal conditions of the reduction the NADH if preferentially bound to a DEAE ion exchanger. The best results as regards conversion degree and purity of product were obtained by cyclic flow of the reaction mixture, containing NAD and ethanol in glycine buffer (pH 8.5) of low ionic strength, through a column of immobilized enzyme and a column of DEAE-cellulose. NADH picked up by the ion exchanger can be eluted by concentrated ammonium carbonate buffer and the final product obtained by lyophilization of the effluent. The purity of NADH formed as determined spectrophotometrically, by HPLC, and measurement of the coenzyme activity using lactate dehydrogenase, is comparable to the purity of the commercial product.
