Modulation of Penicillin Acylase Properties via Immobilization Techniques: One-Pot Chemoenzymatic Synthesis of Cephamandole from Cephalosporin C Marco Terreni, a, * Giuseppe Pagani, a Daniela Ubiali, a Roberto Ferna´ndez-Lafuente, b Cesar Mateo b and Jose´ M. Guisa´ n b a Pharmaceutical Biocatalysis Laboratories, Dipartimento di Chimica Farmaceutica, Universita ` degli Studi di Pavia, via Taramelli 12, 27100 Pavia, Italy b Laboratorio de Tecnologia Enzimatica, Instituto de Catalisis, C.S.I.C. Cantoblanco, 28049 Madrid, Spain Received 29 January 2001; revised 16 May 2001; accepted 22 June 2001 Abstract—The modulation of penicillin G acylase (PGA) properties via immobilization techniques has been performed studying the acylation of 7-aminocephalosporanic acid with R-mandelic acid methyl ester. PGA from Escherichia coli, immobilized onto agarose activated with glycidol (glyoxyl–agarose), has been used for the design of a novel one-pot synthesis of Cephamandole in aqueous medium and without isolation of intermediates, through three consecutive biotransformations catalyzed by d-amino acid oxidase, glutaryl acylase and PGA. # 2001 Elsevier Science Ltd. All rights reserved. The use of enzymes is a powerful tool for the organic chemist. However, in order to take full advantage of the possibilities of these biocatalysts, mechanistic aspects must be considered, such as the conformational changes some enzymes can undergo during catalysis. For exam- ple, it has been hypothesized that penicillin G acylase (PGA) from Escherichia coli undergoes conformational modifications upon binding of the acyl donor substrate on the active center 1 3 and this has also been supported by crystallography analyses. 4 Consequently, it may be expected that any alteration of the conformation may induce significant changes of the enzyme properties. The use of immobilization protocols yielding enzyme deri- vatives with different rigidity or involving different areas of the protein may enable the modulation of the enzyme catalytic properties. PGA from E. coli can be immobilized both on Eupergit C and on agarose gel activated with glycidol (glyoxyl– agarose). 5 Using Eupergit C, the immobilization of PGA proceeds via a two-step mechanism: 6 9 a pre- liminary hydrophobic absorption to the support is fol- lowed by a covalent attachment involving the nucleophilic groups of the enzyme (Lys, Cys, Tyr) and the epoxy groups of the support. The enzyme is mostly immobilized, indeed, through its hydrophobic areas. On the contrary, using glyoxyl–agarose, 10 the enzyme is immobilized through the areas bearing the highest den- sity of Lys residues. 11 Consequently, the immobilization on Eupergit C and glyoxyl–agarose can induce a differ- ent orientation of the protein. Furthermore, the immo- bilization on glyoxyl–agarose may yield enzyme derivatives with different rigidities and stabilities depending on the activation degree of the support: a limited (not stabilized) or a multipoint (stabilized) link- age can be obtained using a low or a high activated support, respectively. 5 Using PGA from E. coli a new chemoenzymatic synth- esis of Cefazolin from Cephalosporin C has been pro- posed. 12 This approach, performed in fully aqueous medium and without intermediates purification, involves three consecutive biotransformations catalyzed by d-amino acid oxidase (DAAO), glutaryl acylase (GA) and PGA, respectively. In this process, the acylation of 7-ACA with tetrazol-1- yl acetic acid methyl ester (TZAM) catalyzed by immo- bilized PGA was the key step to be optimized in order to obtain the final product in high yield and concentration. 0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0960-894X(01)00463-2 Bioorganic & Medicinal Chemistry Letters 11 (2001) 2429–2432 *Corresponding author. Tel.: +39-0382-507625; fax: +39-0382- 507589; e-mail: markt@pbl.unipv.it