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Enzymatic bioremediation of organophosphorus insecticides by recombinant organophosphorous hydrolase
Authors:Mukesh Kapoor  R Rajagopal
Institution:a Department of Zoology, University of Delhi, Delhi 110007, India
b Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India
Abstract:The gene for organophosphorous hydrolase (OPH) (GenBank accession no. M20392) was chemically synthesised with a codon bias toward E. coli, followed by its cloning and heterologous over-expression in E. coli under induction with 0.1 M isopropyl-d-thiogalactopyranoside (IPTG). The protein was localised in the membrane fraction and no amount of the expressed protein was soluble, thus hindering its purification and further downstream utility. The expressed enzyme was solubilised by mild treatment with ionic detergent sodium dodecyl sulphate (SDS 1.0% (w/v))]. An innovative step of incubation at 4 °C was used to precipitate SDS, resulting in catalytically active OPH in the supernatant and detergent at the bottom. This solubilised, SDS-free, recombinant OPH was able to detoxify parathion and methylparathion ranging between 10-80% and 3.6-45% in enzyme reaction cycles after immobilization on Ca-alginate and agar-agar, respectively.
Keywords:Organophosphorous hydrolase  Enzyme  Cloning  Expression  Immobilization  Enzymatic insecticide detoxification
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