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Applied and Environmental Microbiology, August 2004, p. 4681-4685, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4681-4685.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Altering the Substrate Specificity of Organophosphorus Hydrolase for Enhanced Hydrolysis of Chlorpyrifos

Catherine Mee-Hie Cho,1,2 Ashok Mulchandani,1 and Wilfred Chen1*

Department of Chemical and Environmental Engineering,1 Environmental Toxicology Program, University of California, Riverside, California 925212

Received 23 April 2004/ Accepted 27 April 2004

Chlorpyrifos is one of the most popular pesticides used for agriculture crop protection, and widespread contamination is a potential concern. However, chlorpyrifos is hydrolyzed almost 1,000-fold slower than the preferred substrate, paraoxon, by organophosphorus hydrolase (OPH), an enzyme that can degrade a broad range of organophosphate pesticides. We have recently demonstrated that directed evolution can be used to generate OPH variants with up to 25-fold improvement in hydrolysis of methyl parathion. The obvious question and challenge are whether similar success could be achieved with this poorly hydrolyzed substrate, chlorpyrifos. For this study, five improved variants were selected from two rounds of directed evolution based on the formation of clear haloes on Luria-Bertani plates overlaid with chlorpyrifos. One variant, B3561, exhibited a 725-fold increase in the kcat/Km value for chlorpyrifos hydrolysis as well as enhanced hydrolysis rates for several other OP compounds tested. Considering that wild-type OPH hydrolyzes paraoxon at a rate close to the diffusion control limit, the 39-fold improvement in hydrolysis of paraoxon by B3561 suggests that this variant is one of the most efficient enzymes available to attack a wide spectrum of organophosphate nerve agents.

* Corresponding author. Mailing address: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521. Phone: (909) 787-2473. Fax: (909) 787-5696. E-mail: Wilfred{at}engr.ucr.edu.

Applied and Environmental Microbiology, August 2004, p. 4681-4685, Vol. 70, No. 8
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.8.4681-4685.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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