title: An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination.
abstract: In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces.
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy.
authors.forename: Immacolata
authors.initials: I
authors.lastname: Del Giudice
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy.
authors.forename: Rossella
authors.initials: R
authors.lastname: Coppolecchia
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy.
authors.forename: Luigia
authors.initials: L
authors.lastname: Merone
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy.
authors.forename: Elena
authors.initials: E
authors.lastname: Porzio
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy.
authors.forename: Teresa Maria
authors.initials: TM
authors.lastname: Carusone
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Mandrich
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937, Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Worek
authors.affiliation: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131, Naples, Italy. g.manco@ibp.cnr.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Manco
mesh_terms: D000595:Amino Acid Sequence; D001711:Biotransformation; D003666:Decontamination / Q000379:methods*; D019020:Directed Molecular Evolution; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010575:Pesticides / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000235:genetics
chemical_list: D050505:Mutant Proteins; D010575:Pesticides; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases
keywords: detoxification; lactonase; pesticide; phosphotriesterase; semi-rational engineering approach