A multicomponent reaction–diffusion model of a heterogeneously distributed immobilized enzyme

Abstract

A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.

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.…”

“…However, the fitted constants are only valid for the reaction conditions the lumped model was derived for. Thus, such a lumped model is not suitable for the rational design of enzyme immobilizates (van Roon et al, 2006). In contrast to that approach, the MEXA approach aims at deriving a mechanistic kinetic model which accounts for all relevant phenomena in the system.…”

“…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). 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).…”