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The pH gradients developing within immobilized biocatalysts during hydrolysis of penicillin G and glutaryl-7-aminocephalosporanic acid have been estimated both theoretically and experimentally. For the latter a fluorimetric method for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence intensity of an enzyme-bound fluorophore determined with a fiber bundle. The theoretical calculations were based on a model for the hydrolysis with immobilized enzymes using a kinetic expression with five pH-dependent, measurable kinetic and equilibrium constants. The transport reaction differential equation which considers the laminar boundary layer has been solved numerically for the key component. The calculated values agreed well with the experimental data. Under the typical reaction conditions of penicillin G hydrolysis the average pH value in the carrier was 1 and 2.5 pH units below the bulk pH (=8) with and without buffer, respectively. The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5. This demonstrates the existence of considerable pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance. The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzyme stability. These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis. The prediction quality of the model has been tested applying it to fixed bed reactor design. The reduction in rate and yield due to concentration and pH gradients can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors. Thus, enzymatic conversions with high yield and high operational effectiveness are achieved., (Copyright 1999 John Wiley & Sons, Inc.)

Cultures with immobilized hybridoma cells were performed in fixed bed systems. "Steady state" values for volume-specific substrate uptake and metabolite production rates were determined at various perfusion rates and superficial flow velocities of the medium within the carrier matrix. Data from fixed bed volumes between 50 and 600 ml did not show any difference. The volume-specific glutamine and glucose uptake rate turned out to be independent of the superficial flow velocity, but decreased with decreasing glutamine and glucose concentration. The volume-specific oxygen uptake rate increased with increasing superficial flow velocity and substrate concentration, respectively. A similar behavior was observed for the ratio between oxygen and glucose uptake rate. The production rate for monoclonal antibodies was neither affected by the substrate concentration nor by the superficial flow velocity. The metabolic parameters of the immobilized cells were put into kinetic equations and compared to those of suspended cells. It could be concluded that the metabolism of the immobilized cells is determined by the oxygen supply within the macroporous carriers.

Biotransformations catalyzed by free and immobilized enzymes have been carried out in aqueous suspensions with up to 25% (w/w) precipitated substrate or product. For the kinetically controlled synthesis of N-Acetyl-Tyr-Arg-NH(2) with up to 0.8 M insoluble activated substrate N-Acetyl-TyrOEt catalyzed by alpha-chymotrypsin (EC3.4.21.1) the dipeptide yield was found to be >90%. This and the space-time yields were higher than those observed for one-phase aqueous systems and much higher than in systems where the insoluble substrate had been solubilized by addition of organic solvents. In the equilibrium controlled hydrolysis of 0.4 M D-phenylglycine-amide catalyzed by immobilized penicillin amidase (EC 3.5.1.11) the product precipitates. The enzyme immobilized in the support with the smallest pores could be reused without reduction in the rate due to precipitation in the pores. This decreases the number of immobilized enzyme molecules that can be used as biocatalysts. The latter was observed for supports with larger pores as the solubility decreases with increasing particle size. These results demonstrate that biotransformations with insoluble substrates or products using free or immobilized enzymes can be easily carried out in aqueous two-phase systems, without organic solvents, provided that the pore sizes of the supports are sufficiently small and that the rate of mass transfer from the precipitated substrate is large. The latter increases with decreasing particle size. (c) 1995 John Wiley & Sons, Inc.