Novel approach to quantify immobilized‐enzyme distributions

Abstract

The quantitative intraparticle enzyme distribution of Assemblase, an industrially employed polydisperse immobilized penicillin-G acylase, was measured. Because of strong autofluorescence of the carrier, the generally applied technique of confocal scanning microscopy could not be used; light microscopy was our method of choice. To do so, Assemblase particles of various sizes were sectioned, labeled with antibodies specifically against the enzyme, and analyzed light microscopically. Image analysis software was developed and used to determine the intraparticle enzyme distribution, which was found to be heterogeneous, with most enzyme located in the outer regions of the particles. Larger particles showed steeper gradients than smaller ones. A mathematical representation of the intraparticle profiles, based on in-stationary enzyme diffusion into the particles, was validated successfully for a broad range of particle sizes using data for volume-averaged particle size and enzyme loading. The enzyme gradients determined in this work will be used as input for a physical model that quantitatively describes the complex behavior of Assemblase. Such a physical model will lead to identification of the current bottlenecks in Assemblase and can serve as a starting point for the design of improved biocatalysts that also may be based on intelligent use of enzyme gradients.

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

“…A higher enzyme concentration close to the surface could also explain why the measured substrate concentrations close to the interface are lower than those predicted by the kinetic model. Higher enzyme concentrations close to the interface were observed for gelatin-chitosan particles (Assemblase ®) (van Roon et al, 2005) or for alginate hydrogel beads at low protein loadings (Heinemann, 2003). Therefore, the assumption of a homogeneous distribution of the enzymes within the κ-carrageenan hydrogel bead should be investigated in further studies.…”

Immobilization of Penicillin G Acylase: The Key to Optimum Performance

“…[57] This latter conclusion has recently been confirmed by immunolabeling. [58] The performance of Assemblase in the synthesis of cephalexin from (R)-phenylglycine amide and 7-ADCA (see Figure 3) was investigated in detail and compared with dissolved enzyme. [59] Assemblase was a less efficient acyl transfer catalyst, as judged by the S/H ratio [23] , than the dissolved enzyme, which is ascribed to diffusion limitation in the Assemblase carrier.…”

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

“…With the enzyme in Assemblase predominantly situated in the outer regions of the particle (Van Roon et al 2005a), the yield and selectivity are better than that of a particle with a homogeneous enzyme distribution and much better than one with an active core ( Table 2). This results from an improved supply of 7-ADCA to the enzyme and the reduced secondary hydrolysis of the 'slow' CEX for the case of Assemblase.…”

“…The enzyme loading and distribution were previously measured for multiple particle sizes (Van Roon et al 2005a). It was shown that all size-dependent intraparticle enzyme distributions within Assemblase had the mathematical shape of in-stationary enzyme penetration profiles and could be described with a Fourier number as a function of the particle radius.…”

“…Overall, it was shown that diffusion limitations in Assemblase cause a reduced cephalexin production rate, a reduced yield on substrate, and an increased formation rate of the unwanted by-product PG in comparison with the free enzyme (Schroën et al 2002). On top of that, the intraparticle enzyme distribution of Assemblase was found to be heterogeneous (Van Roon et al 2005a), which is expected to have a severe impact on the macroscopic particle behavior as well.…”