Enzymes and microemulsions. Activity and kinetic properties of liver alcohol dehydrogenase in ionic water-in-oil microemulsions

Abstract

The activity and the kinetic properties of horse liver alcohol dehydrogenase have been studied in water-in-oil microemulsions containing sodium dodecyl sulfate (SDS) or hexadecyl trimethylammonium bromide (CTAB), 1-butanol or I-pentanol or 1-hexanol or t-butanol, water and cyclohexane alone o r with octane. In the anionic microemulsions (i.e. containing sodium dodecyl sulfate), the enzyme quickly lost its activity, but was efficiently protected by the coenzyme and some adenine nucleotides. In the cationic microemulsions (i.e. containing hexadecyl trimethylammonium bromide), the enzyme activity was more stable and with higher alcohols was stable for at least 20 min. The Michaelis constant of NAD' calculated with respect to the water content was nearly constant and higher than in water. The maximum velocity in anionic microemulsions depends on the water content whereas in cationic microemulsions, the maximum velocity did not show a clear dependence on the water content and was close to the maximum velocity found in water. The pH dependence of K , and V,,, in these microemulsions was similar to that observed in water. The kinetic data for an hydrophobic substrate, cinnamyl alcohol, showed that this alcohol partitions between the pseudo-phases and thus the apparent Michaelis constant and the concentration at which substrate-excess inhibition appeared were increased. The catalytic properties of the enzyme in microemulsions were illustrated by the preparative reduction of cinnamaldehyde with cofactor recycling. The rate determination of NAD' reduction and of 1-butanol/cinnamaldehyde redox reaction showed that at low water content (2.8%), the NAD' reduction rate was close to zero whereas the redox reaction rate was about half of the rate at higher water content. Probably at low water content the coenzyme binding-dissociation rates are reduced much more than the binding-dissociation rates of the substrates and the rates of the ternary complex interconversion. The cationic microemulsions seemed to be a very favorable medium for enzyme activity, the tetraalkyl ammonium surfactant causing less denaturation than the anionic detergent dodecyl sulfate.The use of water-soluble enzymes for chemical synthesis suffers from several limitations. The most obvious are the low water solubility of many organic substances and the fact that the presence of water makes the reversal of some enzyme reactions for synthetic purposes difficult. In order to overcome these problems, much attention has been paid over the last few years to the solubilization of biological macromolecules in micellar solutions [I -51. Most of the published work deals with three component systems: water, organic solvent and surfactants. Water-soluble enzymes like ribonuclease, peroxidase, a-chymotrypsin, trypsin, lactate dehydrogenase, pyruvate kinase, pyrophosphatase, lysozyme and alcohol dehydrogenase have been solubilized [l -141 in swollen reverse micelles from water, octane or isooctane and di(2-ethylhexy1)sodium sulfosuccinate (AOT). In some instances, other su...

Description of enzyme kinetics in reversed micelles

“…The most extensive kinetic data were reported by Samama et al [12]. They studied the enzymatic conversion of butanol to butanal by horse liver alcohol dehydrogenase (EC 1.1.1.1) and reported a strong deviation from the expected pattern in cetyltrimethylammonium-bromide-reversed micelles when varying the concentration of I-butanol.…”

“…Both Samama and coworkers [12] and Eremin and coworkers [6] noted that substrate inhibition in reversed micelles occurred at a relatively low overall substrate concentration. It is noteworthy that in both cases the inhibition started when the substrate concentration was of the same order of magnitude as the concentration of reversed micelles.…”

“…It was found for most enzymes that the Michaelis constant approached the aqueous value when expressed in overall concentration [5, 81, but this was not the case for Tlwrmoanuerobiunz hrockii alcohol dehydrogenase [l 11 and liver alcohol dehydrogenase [12]. In favour of the second approach was the observation that the Michaelis constant, expressed with respect to the overall volume, did depend on the volume fraction of water (w,,) present [5, 81 and on the micelle concentration [5,8,91.…”

Enzyme and organic solvents: Horse liver alcohol dehydrogenase in non‐ionic microemulsion: Stability and activity

“…The ultraviolet absorption at 280 nm of the surfactant complicates the utilization of cinnamaldehyde (Amax = 320 nm), which has been used previously for this purpose [4]. We therefore carried out the redox activity determination with retinal as oxidant and the cosurfactant butanol as reductant.…”

“…In [4], we described the behavior of horse liver alcohol dehydrogenase in microemulsions made of ionic detergents: SDS and cetyltrimethylammonium bromide. We describe here the reactivity of this enzyme in a microemulsion made with the non-ionic detergent Triton X-100 [5].…”