Organophosphorus compounds (OPs) are widely used as pesticides because of their ability to inhibit the activity of acetylcholinesterase (AChE) in the nervous system. Thus., AChE is generally used as a biosensor for pesticide detection. Due to the instability of AChE a more stable enzyme would be desirable for robust applications. We investigated the sensitivity of a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus (AFEST) to seven selected OPs. The IC50 of dichlorvos against AFEST (50.8 +/- 2.6 nM) was 10-fold lower than that of the commercially obtained AChE, indicating that AFEST had higher sensitivity: Its sensitivity for the other OPs was lower than AChE. To enhance the sensitivity of AFEST to OPs, site-directed mutations were introduced in the cap domain of AFEST. The sensitivity of mutant N44S/S48V was enhanced toward all seven OPs compared to the wild-type and was higher than AChE for four OPs, including paraoxon (33 +/- 0.01 nM), dichlorvos (28.0 +/- 0.6 nM), profenofos (43.0 +/- 1.0 nM), and diazinon (3.0 +/- 0.2 nM). The half-lives of AFEST and the mutant N44S/S48V at 37 degrees C were over 15 d. The interactions between the enzymes and select OPs were investigated by molecular docking. The results demonstrated that AFEST and the mutant N44S/S48V have the potential to be biosensor for OP detection. (C) 2012 Elsevier Ltd. All rights reserved.