A novel method of determination of the internal enzyme distribution within porous solid supports and the deactivation rate constant

D., D.; Hossain, M. M.

doi:10.1002/bit.260280404

Cited by 13 publications

(2 citation statements)

References 23 publications

(4 reference statements)

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“…In several papers the intraparticle biocatalytic distribution was fitted from macroscopic data of systems with relatively straightforward kinetics (21)(22)(23)(24)(25). To the best of our knowledge, up to now little or no research has focused on the modeling of the intraparticle distribution in systems with complex kinetics under non-steady-state conditions.…”

Section: Introductionmentioning

confidence: 99%

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

Roon¹,

Joerink²,

Rijkers³

et al. 2003

Biotechnology Progress

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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.

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Section: Introductionmentioning

confidence: 99%

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

Roon¹,

Joerink²,

Rijkers³

et al. 2003

Biotechnology Progress

View full text Add to dashboard Cite

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“…Van der Wielen (1994) modeled the enzymatic deacylation of penicillin G into phenylacetic acid and 6-aminopenicillanic acid taking coupled electrostatic transport of ionic species into account. The influence of heterogeneous enzyme distributions in systems with nonionic reactants and with relatively simple enzyme kinetics (zeroth order, first order, and MichaelisMenten) was investigated by Do and Hossain (1986) and Hossain and Do (1989), who fitted some extreme intraparticle enzyme distributions to macroscopic data sets.…”

Section: Introductionmentioning

confidence: 99%

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

Roon

Arntz

Kallenberg

et al. 2006

Appl Microbiol Biotechnol

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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.

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Diffusion and chemical reaction rates with nonuniform enzyme distribution: An experimental approach

Ladero¹,

Santos²,

Garcı́a-Ochoa³

2001

Biotechnol. Bioeng.

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The study of the effects of nonuniform distributions of immobilized β‐galactosidase on the overall reaction rate of the hydrolysis of lactose are presented. Diffusion inside the particles has been characterized by measuring the diffusion rates of two β‐galactosidase substrates: lactose and ONPG in a commercial silica‐alumina support. Effective diffusivities have been determined by the chromatographic method under inert conditions. The results obtained for tortuosity can be explained assuming that the transport only takes place in the macropores. The distribution of the immobilized enzyme has been measured by means of confocal microscopy technique. The enzyme has been tagged with FITC and immobilized in particles of different diameters, the internal local concentrations of the enzyme have been determined with the aid of an image computer program. As expected, a more nonuniform internal profile of the enzyme was found when the particle diameter was bigger. Experiments under reaction conditions were carried out in batch reactors using lactose and ONPG as substrates and particles of the immobilized β‐galactosidase of different diameter (1 · 10−4 to 5 · 10−3 m) as catalyst, employing a temperature of 40°C for lactose and 25 and 40°C for ONPG, respectively. The mass balance inside the particle for the substrates has been solved for the internal profiles of the immobilized enzyme inside particles of different size and the enzymatic reactions considered. The calculated and the experimental effectiveness factor values were similar when particles under 2.75 · 10−3 m in diameter were employed. For the same Thiele modulus, a particle with nonuniform distribution of enzyme showed a higher effectiveness as a catalyst than particles with a more uniform distribution. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 458–467, 2001.

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A novel method of determination of the internal enzyme distribution within porous solid supports and the deactivation rate constant

Cited by 13 publications

References 23 publications

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

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

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

Diffusion and chemical reaction rates with nonuniform enzyme distribution: An experimental approach

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