Effect of the microenvironment on the kinetic properties of immobilized enzymes

Abstract

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...

Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain

“…BCCP-FL (71 kDa compared to the exclusion limit of 4 Â 10 6 Da) can be immobilized inside the agarose beads. Bille et al studied the diffusional constraints imposed by the solid matrix on immobilized yeast alcohol dehydrogenase and found that the kinetic properties of the immobilized enzyme are solid matrix-dependent (27). Concentration gradients of substrates are likely to be developed along the radius of the bead (high at the surface, low at the centre) due to diffusion limits imposed by the solid matrix.…”

How enzymes are adsorbed on soil solid phase and factors limiting its activity: A Review

“…Diffusion is also an important step for heterogeneous reactions occurring at the solid surface. Limitation of substrate diffusion towards the enzyme adsorbed on the soil surface could be one of the key factors for the decrease in enzyme activity (Arrio-Dupont and Béchet, 1989;Bille et al, 1989;Douzou and Petsko, 1984;Konecny and Voser, 1977). Enzyme substrate reactions are zero order kinetic.…”

“…The concentration of the substrate remains lowest adjacent to the surface relative to the bulk of the solution. The concentration gradient develops from the bulk of the solution to the soil surface (Bille et al, 1989;Ku and Lentrichia, 1989). There can be three different situations if the diffusion is: slow, equal and fast relative to substrate consumption at the soil surface.…”

d -Amino acid oxidase bio-functionalized platforms: Toward an enhanced enzymatic bio-activity

“…However, covalent linkage requires the modification of one or both parties which may also affect the biological activity. In many cases, these methods give rise to high enzyme loadings maintaining the native structure and biological activity, even increasing the response of the adsorbed enzymes compared to the activity in solution [13][14][15]. However, none of these methods can control the orientation of the enzyme on the solid substrates, the other key factor that determines the enzyme performance in terms of active site accessibility.…”