No abstract

The catalytic activities of alcohol dehydrogenases (ADHs) are investigated in the presence of 5% cyclic ethers. Although most cyclic ethers show an inhibitory effect on ADHs, we found that the catalytic rate constant of 2-butanol oxidation by Thermoanaerobacter brockii ADH was increased from 57 to 190 min −1 by the addition of 1,3-dioxolane, which altered the substrate specificity.Alcohol dehydrogenase (ADH) catalyzes the redox interconversion of a variety of alcohols to their corresponding carbonyls coupled with the redox reaction of co-factor NADIJP) + -NAD(P)H. These reactions are catalyzed in the chiral active site of the ADHs; thus, these enzymes are industrially important for the production of optically pure alcohols and kinetic resolution of racemic alcohols. 1-4 For industrial applications of ADHs, the use of enzymes in organic solvents is often beneficial and many combinations of ADHs, organic solvents, and substrates have been investigated. 5-12 Although these studies clearly show the utility of organic solvents in enzymatic reactions, the effect of organic solvents on the function of ADHs is not fully understood for various reasons such as (1) ADHs have different solvent-resistance properties, (2) the enzyme activities depend on the organic solvent properties and (3) organic solvents affect co-factor binding and release. 10,[12][13][14][15] Recently, non-covalent interactions between enzymes and small molecules have attracted attention as a new method for modulating the enzyme function. The catalytic activity of lipases is increased by the addition of fluorinated carboxylic acids, and the substrate specificity and catalytic activity of cytochrome P450s are drastically altered by the addition of various carboxylic acids and organic solvents. 16-21 These methods successfully altered the enzyme functions by the addition of small molecules, avoiding time-consuming mutagenesis techniques. Based on these reports, we expected that the functions of ADHs should also be modulated by organic solvents.Here, we report the effect of organic solvents on the catalytic activities and substrate specificities of three commercially available ADHs, Saccharomyces cerevisiae ADH (ScADH), horse liver ADH (HLADH), and Thermoanaerobacter brockii ADH (TbADH). The substrates were primary and secondary small alcohols (Fig. 1). We selected cyclic ethers as the organic solvents (Fig. 1). Cyclic ethers have relatively high stability and water solubility which make it possible to adjust the concentration of organic solvents in the reaction mixture. In addition, cyclic ethers have a wide variety of structural variations, such as the size of the ether ring, the number and the position of the oxygen atom, and the presence or absence of functional groups, allowing systematic analysis to clarify the effect of organic solvents.We initially investigated the initial velocity of alcohol oxidation reactions normalized by enzyme concentration without organic solvents (Fig. 2). The reaction was monitored at the 340 nm absorption maximum of NAD(P)H...