Optimization of alcohol dehydrogenase activity and NAD(H) regeneration in organic solvents

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Abstract
Alcohol dehydrogenase (EC 1.1.1.1.) and NAD were co‐immobilized on different supports and used as biocatalysts in organic solvents. Both horse liver alcohol dehydrogenase and yeast alcohol dehydrogenase were tested as biocatalysts for the oxidation of primary alcohols or the reduction of aldehydes. The influence of the kind of support, solvent, water content of the reaction mixture and kind and concentration of the substrates on the redox reaction rate was investigated. NAD(H) was regenerated with the aid of a second substrate. It was shown that both alcohols and aldehydes can serve as reaction media for biocatalysis at molar concentrations, provided the solvents are saturated with water. Highest enzyme activity (2 IU · mg−1 for horse liver alcohol dehydrogenase) was obtained when a hydrophobic long‐chain aldehyde or pentanol was used as the second substrate. Using high concentrations of the second substrate, an equilibrium shift towards over 95 % conversion of substrate was obtained. High total turnover numbers for NAD and NADH of about one million were realized. Thus, coenzyme costs were reduced to about 0.01 US$ per kilogramme of product. This means that the enzyme cos (bout 1000 US$ per kilogramme of product) have become the limiting factor for the application of both alcohol dehydrogenases in production processes for fine chemicals.