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.

Electrochemical study in both classical cell and microreactors of flavin adenine dinucleotide as a redox mediator for NADH regeneration

“…For signals at approximately −0.45 V (reduction) and approximately −0.1 V (oxidation), linear dependences (9) and (10) were observed between the magnitude of the current and the square root of the potential scan rate. This evolution is in agreement with a mass transfer limited phenomenon.…”

“…Because of the high cost of the pyridinic cofactor (NADH), as well as for its very interesting reactivity, continuous regeneration represents an important industrial challenge. The literature contains many works concerning this coenzyme regeneration through chemical [7], photochemical [8], enzymatic [9,10], biological or electrochemical [11,12] means. Fig.…”

Microbial Conversion with Cofactor Regeneration using Genetically Engineered Bacteria

“…[3] Reactions catalyzed by oxido-reductases requiring a nicotinamide coenzyme NAD(P)H have been used in various fields, and to circumvent the high cost and instability of the coenzyme, the recycling of NAD(P)H has been intensively investigated and well developed. Formate dehydrogenase, as the NADH regeneration system, has been used for the production of (R)-2-pentanol, [14] deoxyfructose 6phosphate, [15] alcohols, [16,17] d-amino acids, [18] and l-amino acids. [19,20,21] To retain the cofactor in the reaction mixture, a representative method was developed using polymer-bound NADH, which had two advantages, the increase in molecular size and the improvement of stability.…”

ADH based NAD+ regeneration in a microreactor

“…Up to now, many in situ methods have been studied and developed for regeneration of coenzymes on the macroscale. These methods can be classified into several different categories such as chemical, photochemical, enzymatic, biological or electrochemical . Among them, the enzymatic methods seem to be the most conventional and useful.…”