title: Pseudomonas aeruginosa amidase: aggregation in recombinant Escherichia coli.
abstract: The effect of cultivation parameters such as temperature incubation, IPTG induction and ethanol shock on the production of Pseudomonas aeruginosa amidase (E.C.3.5.1.4) in a recombinant Escherichia coli strain in LB ampicillin culture medium was investigated. The highest yield of soluble amidase, relatively to other proteins, was obtained in the condition at 37°C using 0.40 mM IPTG to induce growth, with ethanol. Our results demonstrate the formation of insoluble aggregates containing amidase, which was biologically active, in all the tested growth conditions. Addition of ethanol at 25°C in the culture medium improved amidase yield, which quantitatively aggregated in a biological active form and exhibited in all conditions an increased specific activity relatively to the soluble form of the enzyme. Non-denaturing solubilization of the aggregated amidase was successfully achieved using L-arginine. The aggregates obtained from conditions at 37°C by FTIR analysis demonstrated a lower content of intermolecular interactions which facilitated the solubilization step applying non-denaturing conditions. The higher interactions exhibited in aggregates obtained at suboptimal conditions compromised the solubilization yield. This work provides an approach for the characterization and solubilization of novel reported biologically active aggregates of this amidase.
authors.affiliation: Centro de Investigačão de Engenharia Química e Biotecnologia do Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro 1, Lisbon, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Borges
authors.forename: Rita
authors.initials: R
authors.lastname: Pacheco
authors.forename: Amin
authors.initials: A
authors.lastname: Karmali
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D018929:Cell Culture Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside; D000431:Ethanol; D001120:Arginine; D000581:Amidohydrolases; C019665:amidase