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The Use of Fungal Laccase for Oxidation of Phosphorothiolates

  • G. Amitai
  • R. Adani
  • G. Sod-Moriah
  • I. Rabinovitz
  • Y. Chai
  • A. Vincze
  • H. Leader
  • B. Chefetz
  • L. Leibovitz-Persky
  • D. Friesem
  • Y. Hadar

Summary

Enzymatic degradation of organophosphorus (OP) insecticides and nerve agents involves the hydrolytic breakdown of the bond between the phosphorus atom and either O-alkyl, 0-aryl (P-OR) or halogen (P-X) moieties. These moieties also serve as leaving groups during irreversible inhibition of acetylcholinesterase (AChE) by these OP’s. However, the P-S bond in phosphorothiolates is more resistant to enzymatic hydrolysis. So far, phosphorothiolates were decomposed rapidly only by chemical oxidation. In the current work we have successfully employed enzymatic oxidation for the rapid degradation of P-S containing OP’s. Degradation kinetics was followed by measuring the residual inhibitory activiy of AChE by the OP compound at specified time intervals. Purified laccase isolated from the white rot fungus Pleurotus ostreatus (Po) together with as 2,2’ azinobis (3-ethylbenzthiazoline - 6-sulfonate) (ABTS) as a mediator caused rapid degradation of the persistent nerve agent 0-ethyl S-[N,N-diisopropylaminoethyl] methylphosphonothiolate (VX) (t½=10 min with 6µg/ml enzyme, ksp = 2200 nmole min-1mg-1) and its structural analog 0,0-diethyl S-[N,N-diisopropylaminoethyl] phosphorothiolate (DiPr-Amiton) (ksp =1833 nmole min-1mg-1). The optimal pH for VX degradation by Pleurotus laccase and ABTS was 7.4 whereas DiPr-Amiton was decomposed at a higher rate at pH 8. The maximal rate of VX and DiPr-Amiton degradation by Po laccase and ABTS was obtained with ABTS:OP molar ratio of 20:1 and 10:1, respectively. The complete degradation of both optical isomers of VX and identification of oxidative biodegradation products were displayed by 31P NMR and GC/MS analysis. A thermostable laccase purified from the fungus Chaetomium thermophylium (Ct) in the presence of the mediator ABTS (ABTS:VX, 20:1) caused a 52 fold slower degradation of VX than by Po laccase (t1,2 =16 min, with 1 mg/ml enzyme in phosphate buffer 0.05M, pH 7.4, 37°C). The specific activity (ksp) of laccase from Ct for the degradation of VX was 42 nmole min-1mg-1. Thus, fungal

Keywords

Nerve Agent Pleurotus Ostreatus Optical Isomer Fungal Laccase Bimolecular Rate Constant 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer Science+Business Media New York 1999

Authors and Affiliations

  • G. Amitai
    • 1
  • R. Adani
    • 1
  • G. Sod-Moriah
    • 1
  • I. Rabinovitz
    • 1
  • Y. Chai
    • 1
  • A. Vincze
    • 1
  • H. Leader
    • 1
  • B. Chefetz
    • 2
  • L. Leibovitz-Persky
    • 2
  • D. Friesem
    • 2
  • Y. Hadar
    • 2
  1. 1.Israel Institute for Biological Research Ness ZionaIsrael
  2. 2.Faculty of AgricultureHebrew University RehovotIsrael

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