Modelling of the enzymatic kinetically controlled synthesis of cephalexin: Influence of diffusion limitation

Abstract

In this study the influence of diffusion limitation on enzymatic kinetically controlled cephalexin synthesis from phenylglycine amide and 7-aminodeacetoxycephalosporinic acid (7-ADCA) was investigated systematically. It was found that if diffusion limitation occurred, both the synthesis/hydrolysis ratio (S/H ratio) and the yield decreased, resulting in lower product and higher by-product concentrations. The effect of pH, enzyme loading, and temperature was investigated, their influence on the course of the reaction was evaluated, and eventually diffusion limitation was minimised. It was found that at pH >or=7 the effect of diffusion limitation was eminent; the difference in S/H ratio and yield between free and immobilised enzyme was considerable. At lower pH, the influence of diffusion limitation was minimal. At low temperature, high yields and S/H ratios were found for all enzymes tested because the hydrolysis reactions were suppressed and the synthesis reaction was hardly influenced by temperature. The enzyme loading influenced the S/H ratio and yield, as expected for diffusion-limited particles. For Assemblase 3750 (the number refers to the degree of enzyme loading), it was proven that both cephalexin synthesis and hydrolysis were diffusion limited. For Assemblase 7500, which carries double the enzyme load of Assemblase 3750, these reactions were also proven to be diffusion limited, together with the binding-step of the substrate phenylglycine amide to the enzyme. For an actual process, the effects of diffusion limitation should preferably be minimised. This can be achieved at low temperature, low pH, and high substrate concentrations. An optimum in S/H ratio and yield was found at pH 7.5 and low temperature, where a relatively low reaction pH can be combined with a relatively high solubility of 7-ADCA. When comparing the different enzymes at these conditions, the free enzyme gave slightly better results than both immobilised biocatalysts, but the effect of diffusion limitation was minimal.

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

“…Intrinsic enzyme kinetics Schroën et al 2002 derived kinetic equations for product and by-product formation using the King-Altman approach. Duggleby et al (1995) and Svedas et al (1980) found that the enzyme only binds PGA and 7-ADCA if the amino group is neutral (the carboxylic acid group can be Fig .…”

“…In the second, CEX is hydrolyzed to yield 7-ADCA and PG. Schroën et al (2002) showed that transport of multiple reactants limited each reaction differently. 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).…”

“…Schroën et al (2002) showed that transport of multiple reactants limited each reaction differently. 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.…”

“…Schroën et al (2002) adapted their own model for free penicillin G acylase kinetics to one for Assemblase by lumping all relevant phenomena of the biocatalytic particle into a new set of fitted pseudokinetic constants. Although their description of macroscopically observed kinetics was quite good, the fitted constants are only valid for the reaction conditions and biocatalyst preparations they were derived for.…”

“…In the literature, several published papers focus on some of these phenomena. Intrinsic kinetic parameters have been published by Schroën et al (2002). In addition, papers on all sorts of reaction-diffusion systems can be found.…”