Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues

Xu, Shengbao; Wu, Aibo; Chen, Haode; Xie, Yang; Xu, Yuquan; Zhang, Lei; Li, Jie; Zhang, Dabing

doi:10.1016/j.bioeng.2006.12.002

Cited by 13 publications

(7 citation statements)

References 36 publications

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“…In addition, the sensitivity of our displayed Bm AChE for the representative OP pesticides (dimethoate, isocarbophos and trichlorphon) is much better than the housefly AChE [30]. For dichlorphos, the sensitivity of the displayed Bm AChE was at the same level as with the Drosophila melanogaster AChE [31], but a little less than that of the Bombyx mandarina AChE [32]. Further experimental optimization of the P. pastoris displayed Bm AChE enzyme is expected to meet or exceed the pesticide detection requirements and the displayed Bm AChE enzyme will be used for routine monitoring of CB and OP pesticides.…”

Section: Resultsmentioning

confidence: 79%

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

et al. 2013

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A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGα1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGα1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17×10−8 M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.

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Section: Resultsmentioning

confidence: 79%

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

et al. 2013

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“…Recombinant DmAChE has been previously produced in Pichia pastoris [28], [30]. In this study, the signal for the GPI anchor attachment was removed to induce DmAChE expression and secretion into the culture medium.…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, compared to recombinant enzymes such as Schizaphis graminum AChE and P. papatasi AChE produced in different baculovirus-based insect cells expression systems, our yeast-expressed AChE could be more effectively inhibited by paraoxon and/or carbaryl [33], [34]. Even if the yeast-based recombinant D. melanogaster AChE expression system was also used, our surface display system showed more advantages and got better effects on monocrotophos and omethoate [30], [35], [36]. This might be due to the stability of the enzyme, in our system the yeast suspension was used directly, but not dried powder form in other studies.…”

Section: Discussionmentioning

confidence: 99%

Surface Display of Recombinant Drosophila melanogaster Acetylcholinesterase for Detection of Organic Phosphorus and Carbamate Pesticides

Yin

et al. 2013

PLoS ONE

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Acetylcholinesterase (AChE) is commonly used for the detection of organophosphate (OP) and carbamate (CB) insecticides. However, the cost of this commercially available enzyme is high, making high-throughput insecticide detection improbable. In this study we constructed a new AChE yeast expression system in Saccharomyces cerevisiae for the expression of a highly reactive recombinant AChE originating from Drosophila melanogaster (DmAChE). Specifically, the coding sequence of DmAChE was fused with the 3′-terminal half of an α-agglutinin anchor region, along with an antigen tag for the detection of the recombinant protein. The target sequence was cloned into the yeast expression vector pYes-DEST52, and the signal peptide sequence was replaced with a glucoamylase secretion region for induced expression. The resultant engineered vector was transformed into S. cerevisiae. DmAChE was expressed and displayed on the cell surface after galactose induction. Our results showed that the recombinant protein displayed activity comparable to the commercial enzyme. We also detected different types of OP and CB insecticides through enzyme inhibition assays, with the expressed DmAChE showing high sensitivity. These results show the construction of a new yeast expression system for DmAChE, which can subsequently be used for detecting OP and CB insecticides with reduced economic costs.

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“…The active site of AChE is quite stable and modification(s) in the ester and/or anionic sites should be evidenced by a depletion of enzyme activity [28,29]. Instead, access of organophosphates into a cavity with an active cleft would be a reason for the variation of AChE sensitivity to inhibition by an organophosphate [30,31]. However, this fact should be further clarified in the future.…”

Section: Resultsmentioning

confidence: 99%

Variation of Cholinesterase-Based Biosensor Sensitivity to Inhibition by Organophosphate Due To Ionizing Radiation

Pohanka

Koch

2009

Sensors

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A cholinesterase based biosensor was constructed in order to assess the effects of ionizing radiation on exposed AChE. Although the primary objective of the experiment was to investigate the effect of ionizing radiation on the activity of the biosensor, no changes in cholinesterase activity were observed. Current provided by oxidation of thiocholine previously created from acetylthiocholine by enzyme catalyzed reaction was in a range 395–455 nA. No significant influence of radiation on AChE activity was found, despite the current variation. However, a surprising phenomenon was observed when a model organophosphate paraoxon was assayed. Irradiated biosensors seem to be more susceptible to the inhibitory effects of paraoxon. Control biosensors provided a 94 ± 5 nA current after exposure to 1 ppm paraoxon. The biosensors irradiated by a 5 kGy radiation dose and exposed to paraoxon provided a current of 49 ± 6 nA. Irradiation by doses ranging from 5 mGy to 100 kGy were investigated and the mentioned effect was confirmed at doses above 50 Gy. After the first promising experiments, biosensors irradiated by 5 kGy were used for calibration on paraoxon and compared with the control biosensors. Limits of detection 2.5 and 3.8 ppb were achieved for irradiated and non-irradiated biosensors respectively. The overall impact of this effect is discussed.

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Production of a novel recombinant Drosophila melanogaster acetylcholinesterase for detection of organophosphate and carbamate insecticide residues

Cited by 13 publications

References 36 publications

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

Surface Display of Recombinant Drosophila melanogaster Acetylcholinesterase for Detection of Organic Phosphorus and Carbamate Pesticides

Variation of Cholinesterase-Based Biosensor Sensitivity to Inhibition by Organophosphate Due To Ionizing Radiation

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