Takayama Katsumi, Suye Shin-ichiro, Kuroda Kouichi, Ueda Mitsuyoshi, Kitaguchi Tetsuya, Tsuchiyama Kouta, Fukuda Takeshi, Chen Wilfred, Mulchandani Ashok
Department of Chemistry and Biology Engineering, Fukui National College of Technology, Geshi-cho, Sabae-shi, Japan.
Biotechnol Prog. 2006 Jul-Aug;22(4):939-43. doi: 10.1021/bp060107b.
The gene encoding organophosphorus hydrolase (OPH) from Flavobacterium species was expressed on the cell surface of Saccharomyces cerevisiae MT8-1 using a glycosylphosphatidylinositol (GPI) anchor linked to the C-terminal region of OPH. Immunofluorescence microscopy confirmed the localization of OPH on the cell surface, and fluorescence intensity measurement of cells revealed that 1.4 x 10(4) molecules of OPH per cell were displayed. Seventy percent of OPH whole-cell activity was detected on the cell surface by protease accessibility assay. The activity of OPH was highly dependent on cell growth conditions. The maximum activity was obtained when cells were grown in a synthetic dextrose medium lacking tryptophan (SD-W) buffered by 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES, 200 mM, pH 7.0) at 20 degrees C, and cobalt chloride was added at 0.1 mM. S. cerevisiae MT8-1 displaying OPH which exhibited a higher activity than Escherichia coli displaying OPH using the ice nucleation protein (INP) anchor. The use of S. cerevisiae MT8-1, which has a "generally regarded as safe (GRAS)" status, as a host for the easy expression of the OPH gene provides a new biocatalyst useful for simultaneous detoxification and detection of organophosphorus pesticides.
使用与有机磷水解酶（OPH）C末端区域相连的糖基磷脂酰肌醇（GPI）锚定，将来自黄杆菌属的编码有机磷水解酶（OPH）的基因表达在酿酒酵母MT8-1的细胞表面。免疫荧光显微镜检查证实了OPH在细胞表面的定位，细胞荧光强度测量显示每个细胞展示了1.4×10⁴个OPH分子。通过蛋白酶可及性分析在细胞表面检测到70%的OPH全细胞活性。OPH的活性高度依赖于细胞生长条件。当细胞在缺乏色氨酸的合成葡萄糖培养基（SD-W）中生长时，用2-[4-(2-羟乙基)-1-哌嗪基]乙烷磺酸（HEPES，200 mM，pH 7.0）缓冲，在20℃下添加0.1 mM氯化钴时，可获得最大活性。展示OPH的酿酒酵母MT8-1比使用冰核蛋白（INP）锚定展示OPH的大肠杆菌表现出更高的活性。使用具有“一般认为安全（GRAS）”地位的酿酒酵母MT8-1作为宿主来轻松表达OPH基因，提供了一种可用于同时解毒和检测有机磷农药的新型生物催化剂。
