Penicillin G acylase is a major industrial biocatalyst that is used in the enzymatic production of 20,000 t a À 1 of 6-aminopenicillanic acid, the industrial b-lactam intermediate, as well as in the enzymatic production of semi-synthetic b-lactam antibiotics. Because efficient recovery and reuse of the biocatalyst is a prerequisite for a viable process, much attention has been focused on the immobilization of penicillin G acylase. Methods that have been studied and will be discussed in this review include covalent attachment to porous organic and inorganic carriers, inclusion in and attachment to biopolymer gels and carrier-free immobilization techniques. Highly active and stable preparations have been developed; mass transfer limitations in the carrier are now a major barrier to further improvement of the biocatalyst performance.

A physical model was derived for the synthesis of the antibiotic cephalexin with an industrial immobilized penicillin G acylase, called Assemblase. In reactions catalyzed by Assemblase, less product and more by-product are formed in comparison with a free-enzyme catalyzed reaction. The model incorporates reaction with a heterogeneous enzyme distribution, electrostatically coupled transport, and pH-dependent dissociation behavior of reactants and is used to obtain insight in the complex interplay between these individual processes leading to the suboptimal conversion. The model was successfully validated with synthesis experiments for conditions ranging from heavily diffusion limited to hardly diffusion limited, including substrate concentrations from 50 to 600 mM, temperatures between 273 and 303 K, and pH values between 6 and 9. During the conversion of the substrates into cephalexin, severe pH gradients inside the biocatalytic particle, which were previously measured by others, were predicted. Physical insight in such intraparticle process dynamics may give important clues for future biocatalyst design. The modular construction of the model may also facilitate its use for other bioconversions with other biocatalysts.