2008 Annual Meeting
(64a) On the Role of Concentration Gradients In the Performance of Carrier-Bound Immobilized Biocatalysts: The Case of An Industrial Amidase
Authors
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.