Cephalosporin C amidase was covalently attached using a protein loading of 7.0-200 mg protein/g dry carrier on four epoxy-activated Sepabeads differing in particle size and pore diameter. Initial-rate kinetic analysis showed that for Sepabeads with small pore diameters (30-40 nm), the apparent K-M of the amidase for hydrolysis of cephalosporin C at 37 degrees C and pH 8.0 increased similar to 3-fold in response to increased particle size (similar to 120-400 mu m) and increased amount of immobilized enzyme (7.0-70 mg protein/g dry carrier) while maximum specific activity (3.2 U/mg protein; 25% of free amidase) was affected only by particle size In contrast, for Sepabeads with wide pores (150-250 nm), the K-M was independent of the enzyme loading. Internal effectiveness factors calculated from observable. Thiele modulus reflected the dependce of K-M on geometrical parametrs of the particles A new method for determination of the overall intraparticle pH was developed based on luminescence lifetime measurements in the frequency domain Sepabeads were doubly labeled using a lipophilic variant of the pH-sensitive dye fluorescein, and Ru(II) tris(4,7-diphenyl-1,10-phenantroline) whose phosphorescence properties are independent of pH. Luminescent lifetime measurements of doubly labeled particle suspensions showed superior signal-to-noise ration compared to fluorescence intensity-based measurements using singly labeled particles. The difference at apparent steady state (Delta pH) between bulk (external pH) and intraparticle pH (internal pH) was as large; as similar to 0 6 units The Delta pH was dependent on substrate concentration, particle size, and pore diameter. Therefore, these results characterize the role of carrier characteristics and reaction parameters in the formation of concentration gradients for substrate and acidic product during hydrolysis of cephalosporin C by immobilized amidase. The strong pH dependence of the immobilized amidase underscores the importance of considering intraparticle pH gradients in the design of an efficient carrier-bound biocatalyst. Biotechnol, Bioeng. 2010, 106 528-540 (C) 2010 Wiley periodicals, Inc.