2024-03-28T19:34:08Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/27972021-12-27T15:41:08Zcom_10261_69com_10261_2com_10261_34com_10261_5com_10261_101com_10261_86com_10261_1col_10261_322col_10261_287col_10261_354col_10261_339
Montes, Tamara
Grazú, Valeria
López Gallego, Fernando
Hermoso, Juan A.
García, José Luis
Manso, Isabel
Galán, Beatriz
González García, Ramón
Fernández-Lafuente, Roberto
Guisán, José Manuel
2008-01-30T18:01:45Z
2008-01-30T18:01:45Z
2006-11-10
Applied and Environmental Microbiology 73(1): 312–319 (2007 January).
0099-2240
http://hdl.handle.net/10261/2797
10.1128/AEM.02107-06
17098917
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.
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Genetic Modification of the Penicillin G Acylase Surface To Improve Its Reversible Immobilization on Ionic Exchangers
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