期刊
PROCESS BIOCHEMISTRY
卷 47, 期 12, 页码 2219-2226出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2012.08.021
关键词
Acetylcholinesterase; Archaeoglobus fulgidus; Thermophilic esterase; Organophosphorus compound; Mutagenesis; Inhibition
资金
- National Natural Science Foundation of China
- National Basic Research Program of China (973 Program)
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.
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