Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Abstract

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier⁻¹ in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t_1/2=625 h vs. t_1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.

Keywords::

• penicillin acylase
• Streptomyces lavendulae
• immobilization
• epoxy-activated supports
• Sepabeads
• confocal scanning microscopy

Acknowledgements

This work was supported by the Universidad Complutense de Madrid and Grupo Santander (project PR27/05-13880-BSCH), and the Spanish Ministry of Education (project ref. BIO2003-04832). The assistance of the Electronic Microscopy Center ‘Luis Bru’ of the Universidad Complutense in the confocal scanning microscopy measurements is gratefully acknowledged. We would also like to thank Rhöm GmbH and Resindion for supplying us the epoxy-activated supports. Finally, our sincere thanks go to Dr José Luis García for fruitful discussions in the context of this work.