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Applied and Environmental Microbiology, April 2002, p. 2026-2030, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.2026-2030.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Bacterial Cell Surface Display of Organophosphorus Hydrolase for Selective Screening of Improved Hydrolysis of Organophosphate Nerve Agents

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 19 October 2001/ Accepted 25 January 2002

Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. However, the effectiveness of degradation varies dramatically, ranging from highly efficient with paraoxon to relatively slow with methyl parathion. Sequential cycles of DNA shuffling and screening were used to fine-tune and enhance the activity of OPH towards poorly degraded substrates. Because of the inaccessibility of these pesticides across the cell membrane, OPH variants were displayed on the surface of Escherichia coli using the truncated ice nucleation protein in order to isolate novel enzymes with truly improved substrate specificities. A solid-phase top agar method based on the detection of the yellow product p-nitrophenol was developed for the rapid prescreening of potential variants with improved hydrolysis of methyl parathion. Two rounds of DNA shuffling and screening were carried out, and several improved variants were isolated. One variant in particular, 22A11, hydrolyzes methyl parathion 25-fold faster than does the wild type. Because of the success that we achieved with directed evolution of OPH for improved hydrolysis of methyl parathion, we believe that we can easily extend this method in creating other OPH variants with improved activity against poorly degraded pesticides such as diazinon and chlorpyrifos and nerve agents such as sarin and soman.

* Corresponding author. Mailing address for Ashok Mulchandani: Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521. Phone: (909) 787-6419. Fax: (909) 787-5696.

* Corresponding author. Mailing address for Wilfred Chen: 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, April 2002, p. 2026-2030, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.2026-2030.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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