Study on Mesophilic and Thermophilic Alcohol Dehydrogenases in Gas-Phase Reaction Archana H. Trivedi, † Antje C. Spiess,* ,† Thomas Daussmann, ‡ and Jochen Bu 1 chs † Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52056 Aachen, Germany, and Julich Chiral Solutions GmbH, Prof.-Rehm-Str. 1, D-52428 Ju ¨lich, Germany The initial reaction rate and the thermostability of the mesophilic alcohol dehydrogenase (ADH) from Lactobacillus breVis (LBADH), and the thermophilic ADH from Thermoanaerobacter sp. (ADH T) in gas-phase reaction were compared. The effects of water activity, cofactor-to-protein molar ratio, and reaction temperature on the reduction of acetophenone to 1-phenylethanol were studied. An optimal water activity of 0.55 in terms of productivity was found for both ADHs. The cofactor-to-protein molar ratio was chosen slightly higher than equimolar to increase both activity and thermostability. An excellent optimal productivity of 1000 g‚L -1 ‚d -1 for LBADH and 600 g‚L -1 ‚d -1 for ADH T was found at 60 °C, while the highest total turnover numbers with respect to the enzyme were achieved at 30 °C and amounted to 4.2 million for LBADH and 1.7 million for ADH T, respectively. Interestingly, the ADH from the mesophilic L. breVis showed the higher thermostability in the nonconventional medium gas phase. Introduction Alcohol dehydrogenase (ADH) is widely used as a catalyst for the synthesis of chiral alcohols and hydroxy esters (1). However, as long as the use of ADH is restricted to aqueous reaction media, the scope of industrial bioconversions, especially for the production of fine chemicals, is necessarily limited by a variety of constraints: the operational instability of ADH enzymes and their cofactors (2), the insolubility and lack of stability of many substrates and products in aqueous solutions (3, 4), and the difficulty in recovery of some products and enzymes from aqueous medium (5). In principle, most of these problems might be overcome by switching from conventional (aqueous) to nonconventional media; here the gas-phase catalysis represents a promising alternative (6). Enzymatic gas-phase reactions involve the conversion of a gaseous substrate to a gaseous product using a dry enzyme as catalyst. This method offers many advantages over conventional biocatalysis (7). It can be applied to poorly water-soluble but volatile compounds. The dilute products may be easily recovered using fractionated condensation. Since the substrate and the product are in the gaseous phase and the biocatalyst is in the dry state, the immobilization of the enzyme and its cofactor is much simplified. The recovery and recycling of the immobilized enzyme preparation is possible. Finally, the enzyme and its cofactor in a dry state with controlled water content are more resistant to thermoinactivation; therefore, reactions can be performed at elevated temperature. Most of the previous studies investigated the effects of several parameters on the activity and thermostability of enzymes in gas-phase reaction. These parameters are water activity (6, 8), total gas flow rate (9), amount of added enzyme, enzyme additives (10), temperature (11), and substrate activity (12). Only a few studies were conducted using ADH enzymes such as yeast alcohol dehydrogenase from Saccharomyces cereVisiae (YADH), horse liver alcohol dehydrogenase (HLADH), Sul- folobus solfataricus alcohol dehydrogenase (SSADH), and Lactobacillus breVis alcohol dehydrogenase (LBADH) as either whole cell or isolated enzyme (6, 8, 13). Since gas-phase reactions require elevated temperatures, Pulvin et al. (14) compared the thermostability of mesophilic HLADH with that of the thermophilic SSADH in gas-phase reaction. In this research work, a comparative study of R-specific mesophilic ADH, namely, Lactobacillus breVis alcohol dehy- drogenase (LBADH) and S-specific thermophilic ADH, namely, Thermoanaerobacter sp. alcohol dehydrogenase (ADH T) in gas-phase reaction was carried out. The influence of gas-phase reaction conditions such as water activity, cofactor-to-protein molar ratio, and reaction temperature on the initial reaction rate and the half-life and thus the productivity of the immobilized ADHs was studied. The ADH-catalyzed reduction of prochiral acetophenone to chiral phenylethanol was done using the ADH co-immobilized with NADP + cofactor by physical deposition. The in situ cofactor regeneration was carried out using the same enzyme with 2-propanol as cosubstrate. Materials and Methods Enzymes, Cofactors, Supports, and Other Chemicals. All enzymes and cells were obtained from Julich Chiral Solutions GmbH (Ju ¨lich, Germany). Lyophilized LBADH (EC 1.1.1.2) was a recombinant Lactobacillus breVis alcohol dehydrogenase plain cell extract containing 50 mM PO 4 -buffer (pH 7) and sucrose 5 times the protein (w/w). The protein content was 0.17 mg protein (mg lyophilized powder) -1 with a specific activity of 89 IU (mg protein) -1 measured at 30 °C (detailed method see below). Whole cells of a recombinant Thermoanaerobacter sp. alcohol dehydrogenase (ADH T) had a specific activity of 3.3 IU mg -1 cell. Lyophilized ADH T, the second source of ADH T, was a recombinant Thermoanaerobacter sp. alcohol dehydrogenase heat-treated cell extract (7.5 min at 70 °C) * To whom correspondence should be addressed. Ph: +49-241-80-28111. Fax: +49-241-80-22265. E-mail: spiess@biovt.rwth-aachen.de. † RWTH Aachen University. ‡ Julich Chiral Solutions GmbH. 454 Biotechnol. Prog. 2006, 22, 454-458 10.1021/bp050316g CCC: $33.50 © 2006 American Chemical Society and American Institute of Chemical Engineers Published on Web 02/01/2006