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A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co-polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages.The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions.The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.Immobilized enzyme technology underwent a wide development with the advent of numerous immobilization methods. This allowed fundamental studies of new aspects of enzyme-catalyzed reactions [I], but many applications in the industrial domain were also found [2, 31, mostly in bioconversion processes [4, 51 and in the analytical field [6, 71.Despite this large amount of work, the relationship between the immobilization method used and its effects on the properties of the immobilized enzyme is still difficult to predict [8], mainly because of changes due to the activation procedure and the effect on the microenvironment induced by the support [9]. One of the few approaches to this problem involves the study of the influence of a charged microenvironment on the activity of a-chymotrypsin [lo, 111. This work reports the effect of the charge of the microenvironment of the pK, of two groups present in the active center of the enzyme, through local modification of the pH.Because of this lack of information, we decided to perform a systematic study of the effect of the microenvironment on the properties of the enzyme. For this purpose, a new immobilization method had to be developed in which the influence of the chemical modification of the enzyme and of its microenvironment could be controlled. We developed a three-step strategy, in which the enzyme could be first activated, then inserted into a well-defined microenvironment and finally immobilized by multipoint covalent linkages. The model illustrated in this paper is for yeast alcohol dehydrogenase (ADH), Correspondence to J. Remade, Unite de Biochimie Cellulaire, FacultCs Universitaires de Namur, rue de Bruxelles 61, B-5000 Namur, Belgium Abbreviations. ADH, yeast alcohol dehydrogenase; SPDP, N-succinimidyl 3-(2-pyridyldithi...

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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

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