Generation of a mutagenized organophosphorus hydrolase for the biodegradation of the organophosphate pesticides malathion and demeton-S

摘要

The bacterial organophosphorus hydrolase (OPH) enzyme hydrolyses and detoxifies a broad range of toxic organophosphate pesticides and warfare nerve agents by cleaving the various phosphorus-ester bonds (P-O, P-F, P-CN, P-S); however, OPH hydrolyses these bonds with varying efficiencies. The aim of this study was to generate a variant OPH enzyme with improved hydrolytic efficiency against the poorly hydrolysed P-S class of organophosphates. The gene encoding OPH was sequentially mutated at specific codons by saturation mutagenesis and screened for improved activity against the P-S substrates demeton-S methyl and malathion. Escherichia coli lysates harbouring the variants displayed up to 177- and 1800-fold improvement in specific activity against demeton-S methyl and malathion, respectively, compared to the wild-type lysates. The specificity constants of the purified variant proteins were improved up to 25-fold for demeton-S methyl and malathion compared to the wild-type. Activity was associated with organophosphate detoxification as the hydrolysed substrate lost the ability to inhibit acetylcholinesterase. The improved hydrolytic efficiency against demeton-S translated to the improved ability to hydrolyse the warfare agent VX. OPH variant enzymes were generated that displayed significantly improved ability to hydrolyse and detoxify organophosphates harbouring the P-S bond. The long-term goal is to generate an environmentally-friendly enzyme-mediated bioremediation approach for the removal of toxic organophosphate compounds in the environment.