Enzyme Distribution Derived from Macroscopic Particle Behavior of an Industrial Immobilized Penicillin‐G Acylase

Abstract

The macroscopic kinetic behavior of an industrially employed immobilized penicillin-G acylase, called Assemblase, formed the basis for a discussion on some simple intraparticle biocatalytic model distributions. Assemblase catalyzes the synthesis of the widely used semisynthetic antibiotic cephalexin. Despite the obvious advantages of immobilization, less cephalexin and more of the unwanted byproduct d-(-)-phenylglycine are obtained due to diffusional limitations when the immobilized enzyme is employed. To rationally optimize Assemblase, the parameters particle size, enzyme loading, and enzyme distribution, which severely determine the macroscopic particle performance, were studied on the basis of macroscopic observations. Laser diffraction measurements showed that the particle sizes in Assemblase vary as much as 100-fold. The relative and total enzyme loadings in Assemblase and fractions thereof of different sizes were determined by initial-rate d-(-)-phenylglycine amide hydrolysis, cephalexin synthesis experiments, and active-site titration. These experiments revealed that the loading of penicillin-G acylase in Assemblase was inversely correlated with the particle diameter. Apart from enzyme loadings, estimates on the intraparticle enzyme distribution came from cephalexin synthesis experiments, where mass-transport limitations were present. Although this method cannot provide the level of detail of specific labeling experiments, it is simple, fast, and cheap. Within the set of simple model predictions, a heterogeneous enzyme distribution with most biocatalyst present in the outer region of the particle (within the outer 100 microm) gave the best description of the observed behavior, although no exact correlation was established. Highly detailed determination of intraparticle enzyme distributions must come from immunolabeling.

Novel approach to quantify immobilized‐enzyme distributions

“…Thus, enzyme distribution (which is often overlooked) can strongly influence macroscopic particle dynamics. In fact, for Assemblase, previous experiments on its external macroscopic kinetic behavior have indicated a heterogeneous enzyme distribution, but the actual distribution could not be derived from these measurements (van Roon et al, 2003).…”

“…7). This was done for particle sizes ranging from 340 to 570 Am in diameter, which is the main volume-contributing size range in native Assemblase (van Roon et al, 2003).…”

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

“…Typically these samples are analyzed by chromatographic methods like high performance liquid chromatography (HPLC) (Berendsen et al, 2006;Goncalves et al, 2002;Guisan et al, 1994;Magario et al, 2008;Schroen et al, 2002;van Roon et al, 2003;2006). Moreover, capillary electrophoresis (Schroen et al, 2002), pH-titrimetry (Guisan et al, 1994), or enzyme-coupled methods like the glucose oxidase-peroxidase test are applied (Handrikova et al, 1996;Polakovic et al, 2001).…”

Development of immobilized systems with penicillin acylase from Esherichia coli for production of semisyntetic penicillins

“…Zaključeno je, na osnovu ovih rezultata, da je enzim uglavnom prisutan u plitkom omotaču na površini zrna [121].…”

“…Molarna koncentracija ireverzibilnog inhibitora koja dovodi do potpune inhibicije PAC ekvivalentna je molarnoj koncentraciji aktivnih mesta u molekulu enzima. Titracija aktivnog položaja slobodne i imobilisane PAC se izvodi inkubiranjem 0,1 cm 3 slobodne PAC ili 0,2 g imobilisane PAC sa različitim sadržajem enzima, preko noći na pH 8,0 i 4 o C sa različitim koncentracijama rastvora fenilmetilsulfonilflurida (PMSF) da bi se postigla postepena inhibicija aktivnog enzima [210]. Posle 12 h, ispituje se preostala aktivnost enzima u reakciji hidrolize penicilina G standardnim postupkom.…”