658 ) l l e c ry2000 d /gm1500 s t iun1000 ( y iiv t 500 t c H a 0 P SigOPH MT8-1/pWMOPH MT8-1/pWIFSSigOPH MT8-1/pWIFLSigOPH MT8-1pWIFSOPH MT8-1/pWIFLOPH MT8-1/pWIFS O MT8-1/pWM Fig. 3 Comparison of OPH activity of yeast cells transformed with various plasmids. The y axis shows the OPH activity, which expressed as formation of 1 nmol of PNP per min per mg dry cells (units/mg dry cell). The x axis shows the strains. Strains MT8-1/pWIFSSigOPH, MT8-1/pWIFLSigOPH, MT8- 1/pWIFSOPH, and MT8-1/pWIFLOPH are constructed using Flo1p anchor system (present study). MT8-1/pWM-SigOPH and MT8-1/pWMOPH that were constructed previously (Takayama et al. 2006), are positive controls using GPI anchor system. MT8-1/pWIFS is a negative control using Flo1p anchor system, which was not inserted OPH gene to the vector. Data are averages based on three independent measurements whereas, MT8-1/pWIFLOPH had 1,600 units/mg dry cell (Fig. 3). Matsumoto et al. (2002) demonstrated that the yeast cells displaying FL-anchor protein had higher ﬂocculation ability than that of the FS-anchor protein and that the ﬂocculation ability of the FL-anchor protein was sufﬁcient to absorb on the cell surface. Therefore, a large amount of the FL-OPH fusion protein would be displayed stably on the cell surface compared with the FS-OPH fusion protein, and MT8-1/pWIFLOPH would exhibit high activity. This result shows that the C-terminal region of the FL-anchor protein (consisting of 3,297–4,341 bp of DNA sequence) was a signiﬁcant domain for ﬂoccu- lation of the Flo1p-OPH fusion protein on the cell surface. Effect of the OPH secretion signal on OPH activity Next, the effect of the OPH secretion signal on OPH activity was investigated. MT8-1/pWIFSOPH and MT8-1/pWIFLOPH contained only the secretion signal sequence of Flo1p in the 50-upstream region of Flo1p-OPH sequence in each transformed plasmid, whereas, MT8-1/pWIFSSigOPH and MT8-1/pWIFL- 123 Biotechnol Lett (2010) 32:655–659 SigOPH contained two secretion signal sequences for OPH in the 50-upstream region of OPH and a secretion signal sequence for Flo1p in the 50-upstream region of Flo1p in each transformed plasmid. MT8-1/pWIFS- SigOPH showed high activity (1,440 units/mg dry cell) compared with MT8-1/pWIFSOPH (960 units/ mg dry cell) (Fig. 3). While the ﬂocculation ability of the FS-anchor protein was insufﬁcient, in contrast to the FL-anchor protein (Matsumoto et al. 2002), the function of the OPH secretion signal might contribute to the efﬁcient expression of OPH, and would comple- ment its activity. Furthermore, MT8-1/pWIFLSig- OPH showed highest activity (1,920 units/mg dry cell). A synergy between the FL/FS anchor and the secretion signal of OPH would be occurred toward activity of OPH on the yeast cell surface. Conclusions We succeeded in displaying OPH on the yeast cell surface using Flo1p anchor system, which resulted in higher OPH activity than GPI anchor system. In particular, MT8-1/pWIFLSigOPH showed 8-fold higher OPH activity than MT8-1/pWMSigOPH or MT8-1/pWMOPH. Microbial biosensors for OPs have been recently developed using recombinant E. coli intracellularly expressing the OPH gene, but the response was slow because of the mass-transport limitation of substrates and products across the cell membrane (Ranina et al. 1996). Therefore, E. coli was engineered to express OPH on the cell surface and the INP anchor was used to improve OPH display on the surface of E. coli (Shimazu et al. 2001). In the present study, we constructed a biocatalyst for OPs detoxiﬁcation and detection based on the yeast- displayed OPH using Flo1p anchor system. The OPH activity of this strain was about 20-fold more efﬁcient than that obtained with E. coli using the INP anchor system. The strains we used may not only be useful for the detoxiﬁcation of OP nerve agents but also for the rapid detection of OPs. For example, the ﬂuorescence intensity of GFP depends on pH; GFP displayed on E. coli showed 65% attenuation in ﬂuorescence for a pH change from 8 to 6 (Shi and Su 2001). To detect OPs by monitoring pH changes in the ﬂuorescence of GFP based on the protons generated by the hydrolysis performed by OPH,