Stability and activity of alcohol dehydrogenases in W/O‐microemulsions: Enantioselective reduction including cofactor regeneration

Abstract

Microemulsions provide an interesting alternative to classical methods for the conversion of less water‐soluble substrates by alcohol dehydrogenase, but until now stability and activity were too low for economically useful processes. The activity and stability of the enzymes are dependent on the microemulsion composition, mostly the water and the surfactant concentration. Therefore, it is necessary to know the exact phase behavior of a given microemulsion reaction system and the corresponding enzyme behavior therein. Because of their economic and ecologic suitability polyethoxylated fatty alcohols were investigated concerning their phase behavior and their compatibility with enzymes in ternary mixtures. The phase behavior of Marlipal O13‐60 (C13EO6 in industrial quality)/cyclohexane/water and its effect on the activity and stability of alcohol dehydrogenase from Yeast (YADH) and horse liver (HLADH) and the carbonyl reductase from Candida Parapsilosis (CPCR) is presented in this study. Beside the macroscopic phase behavior of the reaction system, the viscosity of the system indicates structural changes of aggregates in the microemulsion. The changes of the enzyme activities with the composition are discussed on the basis of transitions from reverse micelles to swollen reverse micelles and finally, the transition to the phase separation. The formate dehydrogenase from Candida boidinii was used for the NADH‐regeneration during reduction reactions. While the formate dehydrogenase did not show any kinetic effect on the microemulsion composition, the other enzymes show significant changes of activity and stability varying the water or surfactant concentration of the microemulsion. Under certain conditions, stability could be maintained with HLADH for several weeks. Successful experiments with semi‐batch processes including cofactor regeneration and product separation were performed. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 638–646, 2000.

Optimization of adsorptive immobilization of alcohol dehydrogenases

“…Unfortunately the operational instability of ADHs in aqueous solution limits their widespread industrial application (3,4). Application of ADHs in non-conventional media such as organic solvents and in a gas/solid system (enzymatic gas phase system) could overcome some of the problems associated with the ADH instability in aqueous media (5,6).…”

“…The immobilization of alcohol dehydrogenase on various supports has been investigated by several researchers (4,5,(7)(8)(9). Commonly used procedures are adsorption or covalent linkage to a carrier.…”

“…Yeast alcohol dehydrogenase (YADH) was chosen as model enzyme, because of its known high sensitivity and poor stability (3,4), which make it a suitable candidate for the present study. In the present work, non-porous solid glass beads (0.25-0.30 mm) were selected as a support because of their mechanical, chemical and thermal stability (18).…”

Study on Mesophilic and Thermophilic Alcohol Dehydrogenases in Gas-Phase Reaction

“…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).…”

Oxidation of a mixture of 2-(R) and 2-(S)-heptanol to 2-heptanone bySaccharomyces cerevisiaein a biphasic system

“…These reactions mainly correspond to reduction processes. The synthesis of (S )-secondary alcohols from the corresponding methylketones is an example of the use of baker's yeast in stereoselective reduction (Gervais et al , 2000;Griffin et al , 2001;Molinari et al , 1998;Orlich et al , 2000).…”