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Applied and Environmental Microbiology, January 2007, p. 312-319, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.02107-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers{triangledown}

Tamara Montes,1 Valeria Grazú,1 Fernando López-Gallego,1 Juan A. Hermoso,2 Jose L. García,3 Isabel Manso,3 Beatriz Galán,3 Ramón González,4 Roberto Fernández-Lafuente,1* and José M. Guisán1*

Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain,1 Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto de Química-Física Rocasolano, CSIC, Serrano 119, 28006 Madrid, Spain,2 Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain,3 Departamento de Microbiología de los Alimentos, Instituto de Fermentaciones Industriales, CSIC, C/Juan de la Cierva 3, 28006 Madrid, Spain4

Received 6 September 2006/ Accepted 29 October 2006

A new mutant of the industrial enzyme penicillin G acylase (PGA) from Escherichia coli has been designed to improve its reversible immobilization on anionic exchangers (DEAE- or polyethyleneimine [PEI]-coated agarose) by assembling eight new glutamic residues distributed homogeneously through the enzyme surface via site-directed mutagenesis. The mutant PGA is produced and processed in vivo as is the native enzyme. Moreover, it has a similar specific activity to and shows the same pH activity profile as native PGA; however, its isoelectric point decreased from 6.4 to 4.3. Although the new enzyme is adsorbed on both supports, the adsorption was even stronger when supports were coated with PEI, allowing us to improve the enzyme stability in organic cosolvents. The use of restrictive conditions during the enzyme adsorption on anionic exchangers (pH 5 and high ionic strength) permitted us to still further increase the strength of adsorption and the enzyme stability in the presence of organic solvents, suggesting that these conditions allow the penetration of the enzyme inside the polymeric beds, thus becoming fully covered with the polymer. After the enzyme inactivation, it can be desorbed to reuse the support. The possibility to improve the immobilization properties on an enzyme by site-directed mutagenesis of its surface opens a promising new scenario for enzyme engineering.

* Corresponding author. Mailing address: Instituto de Catálisis, CSIC, Campus Universidad Autónoma, 28049 Madrid, Spain. Phone: 34 91 585 48 09. Fax: 34 91 585 47 60. E-mail for Roberto Fernández-Lafuente: rfl{at}icp.csic.es. E-mail for José M. Guisán: jmguisan{at}icp.csic.es.

{triangledown} Published ahead of print on 10 November 2006.

Applied and Environmental Microbiology, January 2007, p. 312-319, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.02107-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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