An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination

Biotechnol Bioeng. 2016 Apr;113(4):724-34. doi: 10.1002/bit.25843. Epub 2015 Oct 20.


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.

Keywords: detoxification; lactonase; pesticide; phosphotriesterase; semi-rational engineering approach.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biotransformation
  • Decontamination / methods*
  • Directed Molecular Evolution
  • Hydrolysis
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins / genetics
  • Mutant Proteins / isolation & purification
  • Mutant Proteins / metabolism
  • Pesticides / metabolism*
  • Phosphoric Monoester Hydrolases / genetics
  • Phosphoric Monoester Hydrolases / isolation & purification
  • Phosphoric Monoester Hydrolases / metabolism*
  • Protein Conformation
  • Sulfolobus solfataricus / enzymology*
  • Sulfolobus solfataricus / genetics


  • Mutant Proteins
  • Pesticides
  • phosphorylphosphatase
  • Phosphoric Monoester Hydrolases