pH gradients in immobilized amidases and their influence on rates and yields of ?-lactam hydrolysis

Abstract

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. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 267–277, 1999.

Dual-lifetime referencing (DLR): a powerful method for on-line measurement of internal pH in carrier-bound immobilized biocatalysts

“…Apparent inactivation refers to diffusional limitations and their consequences on the observable enzymatic rate, and arises from two factors. First, if the characteristic times for mass transport into and out of the porous carrier are of the same order of magnitude as the characteristic time for the enzymatic reaction, the overall conversion rate for the immobilized preparation will be lower than that for the native enzyme used in homogeneous biocatalysis [4,5,7]. Second, diffusional limitations may induce concentration gradients between bulk liquid and the interior of the carrier, which may steer the microenvironment of the immobilized enzyme away from optimal for activity, stability or both [5].…”

“…Second, diffusional limitations may induce concentration gradients between bulk liquid and the interior of the carrier, which may steer the microenvironment of the immobilized enzyme away from optimal for activity, stability or both [5]. Moreover, the chemical transformation may be itself negatively affected with respect to selectivity or equilibrium position under these conditions [4,7]. Knowledge-based selection of a carrier whose geometrical properties (particle size, pore size) support optimum performance of the enzymatic reaction by attenuating the diffusion effects would facilitate development of efficient heterogeneous catalysts for applied biocatalysis.…”

Direct Measurement of pH Profiles in Immobilized Enzyme Carriers during Kinetically Controlled Synthesis Using CLSM

“…The magnitude of the pH gradients was quantified experimentally using fluorescent pH indicators attached covalently to the immobilized enzyme. The pH difference between bulk fluid and particle amounted to 1.5-3 units for hydrolysis reactions (5,6). The expressiveness of this method was limited because only average pH values in the particles could be observed.…”

Equilibrium position, kinetics, and reactor concepts for the adipyl-7-ADCA-hydrolysis process

“…From the apparent equilibrium constant, the thermodynamic equilibrium constant can be calculated if the dissociation constants of the substrate and products are known (Š vedas et al, 1980;Spieß et al, 1999).…”