Simple‐kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl‐chloride‐activated agarose

Abstract

Yeast alcohol dehydrogenase was successfully immobilized on tresyl-chloride-activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%.Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this Interest immobilized alcohol dehydrogenase.in enzvme technologv has increased in the lastyears because of many potential applications in several domains where the specific properties of the enzymes would be valuable [l-31. Enzyme immobilization is one of the important steps for this achievement.However, several problems still limit a full development of this technology. Among them are the lack of stability of some enzymes [4-71, the lack of a suitable and economical way to regenerate cofactors [8] and the lack of a suitable model which could predict the properties of immobilized enzymes [9, 101.We chose to immobilize yeast alcohol dehydrogenase (EC 1.1.1 .l) which oxidizes several alcohols into their corresponding aldehyde [l 11 whilst reducing the NAD' cofactor. This reaction could therefore be of potential value in a regeneration process, at least as a model [8], as we will show at the end of this work.After optimization of the immobilization procedure in order to obtain the highest activity yield [14], we systematically compared several important properties of the free and the immobilized forms of the enzyme, in order to gather information on the influence of the immobilization using tresylchloride-activated agarose on the modification of these properties. Although pH and temperature-dependence curves were also studied (see section on Measurement of immobilized ADH activity), we mainly focused our attention on the comparison of the activation energies of the reactions and on the kinetic parameters before and after immobilization.This latter comparison was first based on the MichaelisMenten kinetic model, which usually yields a good approximation of the kinetics of two-substrate enzymes provided some experimental precautions are taken. The validity of this model appeared however to be restricted to the soluble Correspondence to

Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase

“…(Kontron Uvikon 930; Kontron, Milano, Italy) at 25°C, using the method described by Bille & Remacle (1986).…”