Analysis of the inactivation of liver alcohol dehydrogenase during storage in Aerosol‐OT/isooctane microemulsions

Abstract

Changes in the enzymatic properties of horse liver alcohol dehydrogenase (HLADH; EC 1.1.1.1) were studied as a function of incubation time in Aerosol‐OT/isooctane microemulsions. The enzyme was characterized by fluorimetric binding studies of the inhibitor isobutyramide to the binary complex, HLADH‐NADH and by determination of Km,app and Vmax,app values for cyclohexanone. The Km,app values for cyclohexanone and the Kd,app for isobutyramide stay constant throughout a 48‐h incubation, whereas the Vmax,app and the total number of inhibitor binding sites decrease. Thus the inactivation process previously described corresponds to progressive loss of functional sites, while the properties of the remaining functional sites are unchanged. If no co‐enzyme is added to the system, the enzyme loses catalytic activity within less than an hour, but if co‐enzyme is added, a fraction of the HLADH enzyme population retains enzyme activity over a long period of time. Hence the presence of bound co‐enzyme significantly inhibits the process(es) leading to inactivation of the enzyme in the microemulsions.

Comparison of Enzymatic Activity and Nanostructures in Water/Ethanol/Brij 35 and Water/1-Pentanol/Brij 35 Systems

“…In general, the enzyme substrate is dissolved in the organic phase and it is supposed that it does not markedly alter the nanostructure of the reaction medium. For example, the bio-oxidation of alcohols to aldehydes has often been studied without considering the possible cosurfactant role of alcohols. − The advantages of such systems are evident: the enzymatic activity can be optimized by choosing a convenient composition of the different constituents. In this way the enzymatic activity can be considerably enhanced compared to the activity in pure aqueous solutions.…”

“…As other authors did in several previous studies, − ,− we have chosen alcohol dehydrogenase (HLADH) as the enzyme. This enzyme catalyzes the oxidation of different primary aliphatic alcohols with comparable activities.…”

“…For example, the bio-oxidation of alcohols to aldehydes has often been studied without considering the possible cosurfactant role of alcohols. [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] The advantages of such systems are evident: the enzymatic activity can be optimized by choosing a convenient composition of the different constituents. In this way the enzymatic activity can be considerably enhanced compared to the activity in pure aqueous solutions.…”