Location: Food and Feed Safety ResearchTitle: Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris Author
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/19/2013
Publication Date: 8/5/2013
Publication URL: http://handle.nal.usda.gov/10113/58265
Citation: Dong, J., Xie, X., He, Y., Beier, R.C., Sun, Y., Xu, Z., Wu, W., Shen, Y., Xiao, Z., Lai, L., Wang, H., Yang, J. 2013. Surface display and bioactivity of Bombyx mori acetylcholinesterase on Pichia pastoris. PLoS One. 8:e70451. doi: 10.1371/journal.pone.0070451. Interpretive Summary: Acetylcholinesterase (AChE) is a key enzyme because it regulates the level of acetylcholine in the blood and terminates nerve impulses. Carbamate (CB) and organophosphorous (OP) pesticides act to paralyze and kill insects by inhibiting their AchE enzyme. Residues of these pesticides may be found in food and the environment. It is feasible to determine the content of CB and OP pesticides according to the degree of inhibition of AChE activity. In this study, we used modern molecular biology tools to facilitate development of a rapid, easy, and sensitive analytical method useful for the detection of anticholinesterase pesticide residues in food and the environment.
Technical Abstract: To construct the Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE), the gene for the anchor protein (AGa1) was obtained from Saccharomyces cerevisiae and was fused with the modified Bombyx mori acetylcholinesterase gene (bmace) and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGa1 was induced in BMMY medium with a final concentration of 1% methanol. Cell surface display of BmAChE was confirmed by flow cytometry and fluorescence microscopy. The enzyme activity of the displayed BmAChE was detected with the Ellman method. In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine and carbamate and organophosphorus pesticides. The assay of flow cytometry and fluorescence microscopy revealed that BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of displayed BmAChE was 787.7 U/g (dry cell weight). The displayed BmAChE was highly sensitive to eserine and ten organophosphorus pesticides, as well as five carbamate pesticides. The displayed BmAChE showed the highest sensitivity to methiocarb at a concentration of 6.58 × 10–10 M required for 50% inhibition of enzyme activity. Results suggest that the BmAChE displayed on the cell surface of P. pastoris GS115 possesses good bioactivity.