- Coupling of Site-Directed mutagenesis and immobilization for the rational design of more efficient biocatalysts: The case of immobilized 3G3K PGA from E. coli
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We have investigated the synthetic performance of the immobilized 3G3K mutant of the Penicillin G acylase (PGA) from E. coli obtained by site-directed mutagenesis. The 3G3K mutant, characterized by a tag consisting of three lysines alternating with three
- Serra, Immacolata,Ceechini, Davide A.,UbiaIi, Daniela,Manazza, Elena M.,Albertini, Alessandra M.,Terreni, Marco
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experimental part
p. 1384 - 1389
(2009/08/07)
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- Influence of substrate structure on PGA-catalyzed acylations. Evaluation of different approaches for the enzymatic synthesis of cefonicid
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The influence of the substrate structure on the catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled acylations has been studied. In particular, the affinity of different β-lactam nuclei towards the active site has been evaluated considering the ratio between the rate of synthesis (vs) and the rate of hydrolysis of the acylating ester (vhl). 7-Aminocephalosporanic acid (7-ACA) and 7-amino-3-(1-sulfomethyl-1,2,3,4-tetrazol-5-yl)thiomethyl-3-cephem-4-carboxylic acid (7-SACA) showed a good affinity for the active centre of PGA. The enzymatic acylation of these nuclei with R-methyl mandelate has been studied in order to evaluate different approaches for the enzymatic synthesis of cefonicid. The best results have been obtained in the acylation of 7-SACA. Cefonicid (8) was recovered from the reaction mixture as the disodium salt in 65% yield and about 95% of purity. Furthermore, through acylation of 7-ACA, a "one-pot" chemo-enzymatic synthesis was carried out starting from cephalosporin C using three enzymes in sequence: D-amino acid oxidase (DAO), glutaryl acylase (GA) and PGA. Cefonicid disodium salt was obtained in three steps, avoiding any intermediate purification, in 35% overall yield and about 94% purity. This approach presents several advantages compared with the classical chemical processes.
- Terreni, Marco,Tchamkam, Joseph Gapesie,Sarnataro, Umberto,Rocchietti, Silvia,Fernandez-Lafuente, Roberto,Guisan, Jose M.
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p. 121 - 128
(2007/10/03)
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