SummaryThis study was carried out to define how the overall rate of reaction would be influenced by different degrees of diffusional resistance to cofactor transport within an oxidoreductase membrane matrix. To accomplish this, 0.7-6.6pM yeast alcohol dehydrogenase was immobilized in an albumin matrix crosslinked with 2.5 or 5.0% glutaraldehyde to give 102-1685 pm thick membranes. The enzyme half-life was at least doubled at pH 7.5 or 8.8 on immobilization. Values of the kinetic constants for the soluble and immobilized enzyme were determined at 25°C and pH 8.8 over the range of 0.01-1.0M bulk solution concentration of ethanol as substrate and 140-1000pM bulk solution concentration of nicotinamide adenine dinucleotide (NAD+) as cofactor, to give essentially single substrate kinetics in NAD+ .Equilibrium partitioning of ethanol and NAD' between the solution and membrane was measured and used in the data analysis. The four kinetic constants for the soluble enzyme agreed with literature values; and all increased with immobilization of the enzyme. The Michaelis constants for NAD+ and for ethanol were greater for the immobilized enzyme. The diffusional resistance to NADi transport, presented in terms of the Thiele modulus, showed that the overall rate of reaction was decreased by about 50% even at values of the modulus as low as 2.0.

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