Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou, Guangdong Province, China.
PLoS One. 2013 Aug 5;8(8):e70451. doi: 10.1371/journal.pone.0070451. Print 2013.
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.
构建了毕赤酵母（P. pastoris）细胞表面展示系统的家蚕乙酰胆碱酯酶（BmAChE），并研究了其生物活性。将修饰后的家蚕乙酰胆碱酯酶基因（bmace）与酿酒酵母的锚蛋白（AGα1）融合，并转化入毕赤酵母菌株 GS115。将携带融合基因 bmace-AGα1 的重组菌诱导表达，使 BmAChE 展示在家蚕细胞表面。荧光显微镜和流式细胞术检测结果表明，BmAChE 成功地展示在家蚕毕赤酵母 GS115 的细胞表面上。采用 Ellman 法检测到展示的 BmAChE 的酶活性为 787.7 U/g（湿细胞重量）。此外，通过与毒扁豆碱、氨基甲酸酯和有机磷农药的抑制试验验证了展示的 BmAChE 的生物活性。展示的 BmAChE 的 IC50 为 4.17×10(-8) M，对毒扁豆碱和五种氨基甲酸酯农药以及七种有机磷农药均高度敏感。结果表明，展示的 BmAChE 具有良好的生物活性。
