Many industrial biocatalysts are used as immobilized enzyme preparations where the protein is tethered on the surface of an insoluble porous carrier. In this way the biocatalyst can be separated easily from the reaction solution and protein-free product is obtained. Recovery of the enzyme for multiple rounds of substrate conversion and improved operational stability of the biocatalyst are advantages often brought about by the immobilization, which may therefore contribute to a reduction of the process costs connected to the catalyst. There are however common disadvantages of rendering an enzyme insoluble through attachment on a carrier. A substantial decrease in specific enzymatic activity is often observed in the immobilized enzyme as compared to the soluble counterpart. A significant portion of the measured loss in catalytic effectiveness of the immobilized enzyme may be apparent and reflect the influence of mass transfer effects on the overall reaction rate. Hindered diffusion in the pores of the carrier particles may promote the formation of gradients in the concentrations of substrates and products along the radial dimension, which may strongly impact on the enzyme activity. Enzymatic reactions that lead to formation or uptake of protons may also produce substantial pH gradients along the characteristic length of the carrier pore, which in turn can alter the activity and stability of the immobilized enzyme. Although theory for the analysis of the performance of immobilized enzymes has been available for decades, there are only few examples published in the literature where actual measurements of intra-particle concentration gradients have been correlated with the kinetic properties of carrier-bound biocatalysts. The evidence gained from a detailed study is expected to have major implications on carrier selection and design of the immobilization process. Cost considerations dictate that the quest for the optimum carrier is a relevant issue of biocatalytic process development in the industry. We report here on the characterization of an industrially used amidase immobilized on epoxy-activated carriers (Sepabeads).

The relative rates of enzymatic reaction and mass transfer of substrate will be determined by the size of the (spherical) particles, the diameter of the pores, and the loading of enzyme activity per unit mass of carrier. Steady state kinetic analysis was used to evaluate the effect of variation in these three parameters on enzyme coefficients, the maximum initial rate (Vmax) and the Michaelis-Menten constant (Km). Using standard grade Sepabeads EC-EP which exhibit a pore diameter of between 30 and 40 nm we observed significant (up to 3-fold) increases in Km in response to an increase in enzyme loading and particle size. When using a carrier that shows a pore diameter about 6 times that of Sepabeads EC-EP, the Km of the immobilized amidase was independent of the enzyme loading in the range 7 - 70 mg protein / g dry carrier. The aggregate data analyzed by using the dimensionless Thiele modulus portray the diffusional effects on the enzymatic conversion rate in carriers featuring a relatively small and high pore diameter. The protein binding capacity increased with increasing specific surface area on the carrier, that is when the particle or pore diameter decreased. However, when using particles with relatively small pores, a substantial fraction of the available surface area appeared to be unavailable for binding of protein.

It was unexpected to observe a substantial effect of particle characteristics and enzyme loading on Vmax, which is recorded under conditions where the enzyme is saturated with substrate. Considering that the observable decrease in Vmax might be traceable to a variation in intra-particle pH resulting from the hydrolysis of an amide substrate and the hindered diffusion of the acidic product, we determined the difference between the pH of the bulk solution and the average pH in the carrier particle. Following impregnation of Sepabeads with a pH-responsive fluorescein label, fluorescence measurements were used to determine the pH in the particles in a time resolved manner while the enzymatic reaction took place. A significant difference (by about 1 pH unit) in the pH values measured inside and outside of the carrier indicates that product concentration gradients may have a large impact on the activity of the immobilized enzyme. The acidification of the carrier relative to the bulk increased with increasing enzyme loading and substrate concentration. Implications of these results for the design of a suitable carrier for amidase-catalyzed conversions are discussed.