title: Boosted large-scale production and purification of a thermostable archaeal phosphotriesterase-like lactonase for organophosphate decontamination.
abstract: Thermostable phosphotriesterase-like lactonases (PLLs) from extremophile archaea, like SsoPox from Sulfolobus solfataricus, are attractive biotechnological tools with industrial applications as organophosphate decontaminants, but their manufacturing still remains an unresolved issue because of the high costs and the low production yields. In this paper, for the first time, an efficient biotechnological process for the production and purification of a recombinant, engineered PLL, SsoPox W263F, expressed in E. coli, has been set up by studying new induction strategies, by designing high cell density cultivations and a new membrane-based downstream process. In fed batches, the enzyme production was boosted of 69-fold up to 4660.0 U L-1using galactose as inducer in the replacement of IPTG; the process was scalable from 2.5 up to 150 L. By coupling a single thermo-precipitation step and an ultrafiltration process, a total enzyme recovery of 77% with a purity grade of almost 80% was reached.
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy. odilefrancesca.restaino@unina2.it.
authors.forename: Odile Francesca
authors.initials: OF
authors.lastname: Restaino
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy.
authors.forename: Maria Giovanna
authors.initials: MG
authors.lastname: Borzacchiello
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Scognamiglio
authors.affiliation: Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Porzio
authors.affiliation: Institute of Protein Biochemistry, National Research Council of Italy, Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Manco
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Fedele
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Donatiello
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: De Rosa
authors.affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Naples, Italy. chiara.schiraldi@unina2.it.
authors.forename: Chiara
authors.initials: C
authors.lastname: Schiraldi
mesh_terms: D061249:Batch Cell Culture Techniques; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis*; D003470:Culture Media / Q000737:chemistry; D003666:Decontamination; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D019766:Genes, Archaeal; D007218:Industrial Microbiology; D000074041:Microorganisms, Genetically-Modified; D010755:Organophosphates / Q000737:chemistry*; D044345:Phosphoric Triester Hydrolases / Q000096:biosynthesis*; D015202:Protein Engineering; D048229:Sulfolobus solfataricus / Q000235:genetics* / Q000378:metabolism
chemical_list: D003470:Culture Media; D010755:Organophosphates; D002265:Carboxylic Ester Hydrolases; C020579:gluconolactonase; D044345:Phosphoric Triester Hydrolases
keywords: High cell density cultivation; Organophosphates; Sulfolobus solfataricus; Thermostable phosphotriesterase-like lactonase; Ultrafiltration