Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris
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| Authors: | Jie-Xian Dong Xi Xie Yong-Sheng He Ross C Beier Yuan-Ming Sun Zhen-Lin Xu Wei-Jian Wu Yu-Dong Shen Zhi-Li Xiao Li-Na Lai Hong Wang Jin-Yi Yang |
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| Institution: | 1. Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou, Guangdong Province, China.; 2. Shenzhen Academy of Metrology and Quality Inspection, Shenzhen, Guangdong Province, China.; 3. United States Department of Agriculture, Agricultural Research Service, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit, College Station, Texas, United States of America.; Weizmann Institute of Science, Israel, |
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| Abstract: | 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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