Alcohol dehydrogenase has been immobilized to the basic copolymer and its several derivatives using various techniques. Enzyme coupling to the supports with amino groups by means of glutaraldehyde was found the most suitable. Activity of alcohol dehydrogenase coupled to these amino supports was comparable to that of the enzyme bound to Sepharose. Thermal and pH stability of alcohol dehydrogenase increased essentially upon immobilization. Kinetic properties of the immobilized enzyme differed from those of free alcohol dehydrogenase, pH optimum shifted to alkaline range, and apparent Michaelis constants for substrates and coenzymes increased. Curvatures observed in Lineweaver-Burk plots for coenzymes suggest an involvement of diffusion effects in the reaction catalyzed by alcohol dehydrogenase linked to these polymers.

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.