Investigations of Reaction Kinetics for Immobilized Enzymes—Identification of Parameters in the Presence of Diffusion Limitation

Abstract

A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.

Model-based experimental analysis of enzyme kinetics in aqueous–organic biphasic systems

“…If the enzymes are immobilized in hydrogel beads, diffusion has to be taken into account (Berendsen et al, 2006;van Roon et al, 2006).…”

“…Moreover, until today experimental data have been almost exclusively collected from the bulk phase (Berendsen et al, 2006;Goncalves et al, 2008;Polakovic et al, 2001;Schroen et al, 2002;van Roon et al, 2006). These data can provide only integral information about the reaction system and hardly allow any conclusions about the interdependent phenomena occurring inside the bead (Heinemann, 2003).…”

“…The immobilization of enzymes in hydrogels provides a stabilized aqueous environment for the enzymes while being suspended in an organic solvent. However, the productivity in such gel-stabilized biphasic systems may be limited by diffusion of the reactants (Berendsen et al, 2006;Goncalves et al, 2008;Guisan et al, 1994;van Roon et al, 2006). Hence, it is essential to study the influence of the hydrogel matrix on the transport kinetics.…”

“…Moreover, enzyme immobilizates facilitate downstream processing and reduce the overall process costs since they can easily be recovered and reused (Ansorge-Schumacher, 2007;Cao, 2005;Fernandes and Cabral, 2008;Kurlemann and Liese, 2004;Mateo et al, 2007;Sheldon, 2007). The rational design of enzyme immobilizates is more complex than that of homogeneous systems since mass transfer and diffusion can be rate limiting (Bauer et al, 2002;Berendsen et al, 2006;Buchholz, 1989;Diender et al, 2002;Fernandes and Cabral, 2008;Halling, 1987Halling, , 1994. Usually it is assumed that the kinetic parameters of immobilized enzymes are identical to those of enzymes in solution (Schroen et al, 2002;van Roon et al, 2006).…”

“…Usually it is assumed that the kinetic parameters of immobilized enzymes are identical to those of enzymes in solution (Schroen et al, 2002;van Roon et al, 2006). Nevertheless, immobilization of enzymes can affect their kinetic parameters, observed so far for immobilization techniques based on covalent binding (Berendsen et al, 2006;Buchholz, 1989). Up to date it is still unknown if immobilization of enzymes in hydrogel beads also alters their kinetic parameters.…”