Quantitating intraparticle O₂ gradients in solid supported enzyme immobilizates: Experimental determination of their role in limiting the catalytic effectiveness of immobilized glucose oxidase

Abstract

Enzymatic O2 -dependent oxidations are receiving increased attention for use in fine chemicals synthesis. Solid supported oxidation catalysts often show poor efficiency due to pronounced O2 diffusion restriction. Internal O2 supply therefore constitutes a key parameter for optimizing the enzyme immobilization. We herein describe an optical sensing method for quantitation of space-averaged intraparticle O2 concentrations in porous Sepabeads carriers. The method applies phosphorescence lifetime measurements on Sepabeads labeled with an O2 sensitive indicator dye. Using glucose oxidase immobilized at different loadings (0.005-12 mg/g) on labeled Sepabeads, we analyzed in real time during the enzymatic reaction the formation of O2 concentration differences between bulk liquid and the intraparticle environment. We show that the O2 gradient at apparent steady state increased with increasing enzyme loading, so that O2 eventually became totally depleted from inside the highly loaded carriers. We also show that the residual intraparticle O2 concentration was correlated with the catalytic effectiveness factor (η) of the enzyme immobilizate used, thus providing a direct measure of the magnitude of O2 diffusion limitation. Once corrected for diffusional effect, η was no longer dependent on enzyme loading and its constant value now described the intrinsic activity of immobilized glucose oxidase. Three common procedures of enzyme immobilization, involving adsorption, cross-linking, and covalent attachment, are shown to differ widely concerning the obtained intrinsic activity. Therefore, intraparticle O2 concentration data enable distinction between diffusional restriction and activity loss as the two principal factors limiting the effectiveness of immobilized O2 dependent enzymes, and thus they inform rational design of an optimally active oxidation biocatalyst on solid support.

Advanced characterization of immobilized enzymes as heterogeneous biocatalysts

“…The quantification of spaced averaged intraparticle oxygen concentrations in porous polymethacrylate enzyme carriers was accomplished by labeling the enzyme carrier with an O 2 -sensitive luminophore and application of the phase modulation technique. Formation of a large gradient between O 2 concentrations in bulk solvent and the internal environment of the carrier was detected, clearly indicating limitations in the supply of O 2 co-substrate into the solid support [172,173]. In-operando determination of oxygen gradients between homogeneous liquid phase and internal catalytic environment was performed during determination of catalytic activity of heterogeneous oxidases as biocatalysts.…”

“…Mid-IR spectroscopy [164,165] Raman spectroscopy Wide range of carriers Global reaction rates of stirred suspension of immobilized enzymes Immersed probes were used [157] [172,173] been applied [27, for biocatalyst screening and characterization as well as for study and optimization of the reactor operation. Table 5 presents a summary of the immobilized enzyme system studied, with a focus on applied aspects.…”

Biosilica and bioinspired silica studied by solid-state NMR

“…In operando characterization of the catalyst (i.e. : in realistic conditions of applications of the biocatalyst) have been carried out by applying diverse analytical methods directly within the solid support: examples include optochemical sensing [68][69][70][71] as well as spectroscopic imaging and electrochemical sensing (see table 5 in reference [51]). These methods yield direct handle on reagents' local concentration, gradients and diffusion properties with both temporal and spatial information.…”

Oriented Coimmobilization of Oxidase and Catalase on Tailor-Made Ordered Mesoporous Silica

“…56,60 However, since chemical reaction and diffusion are intrinsically coupled events in transformations catalyzed by solid enzyme immobilizates, η is not independent of E bound and the dependence generally starts to show at high enzyme loadings 7,[14][15][16] . Material properties thus become important in the effort of enhancing the degree of uncoupling of η from E bound .…”