Biotechnol Lett (2010) 32:655–659 primers were used. For the gene encoding OPH, the forward primer was 50-GGTAGATCTTCGATCGG CACAGG CCATCGGATCAAT-30 (BglII site, italic). To amplify the signal sequence domain of OPH, we used the forward primer SigOPH-50: 50-GGTAGAT- CTATGCAAACGAGAAGGGTTGTGCTCAAG-30 (BglII site, italic). In this sequence, the upstream region of OPH signal sequence is underlined. The OPH-30 reverse primer was 50-ACTGCTCGAGCCTCCAG CCTTGTCATCGTCATCCTTGTAATCAGATCCA CCTGACGCCCGCAAGGTC GGTGACAA-30 (XhoI site, italic). pWIFS and pWIFL were the plasmid vectors used for expressing OPH on the yeast cell surface (Fig. 2). The FL gene comprised almost all the sequence of Flo1p and the FS gene consisted of part of Flo1p without 1,044 bp of 30 downstream of the FL gene (Fig. 1c) (Matsumoto et al. 2002). The ampliﬁed fragment was digested and introduced in each plasmid vector (Fig. 2). The resulting plasmids were named pWIFSSigOPH, pWIFLSigOPH, pWIFSOPH, and pWIFLOPH (Fig. 2). OPH DNA was analyzed using a DNA sequencer (Applied Biosystems, Japan, Tokyo, Japan). The plasmids were transformed into S. cere- visiae MT8-1 by the lithium acetate method. Measurement of OPH activity The hydrolytic activities of OPH-displaying cells were assayed by measuring p-nitrophenol (PNP) produced during hydrolysis of Paraoxon (Sigma) as the substrate. Cells were precultured in 5 ml SD-W medium [0.1 mM CoCl2 (Nacalai tesque, Kyoto, Japan)] for 24 h at 20 C and were further cultured for 75 h. Cells were harvested by centrifugation at 3,0009g for 5 min at 25 C and washed three times with 50 mM HEPES buffer (pH 8.0) containing 100 lM CoCl2. Washed cells were resuspended in 50 mM HEPES buffer (pH 8.0) containing 100 lM CoCl2 and adjusted to OD600 = 1. An OD600 value of 1 = 0.313 mg dry cells/ml-1 (Horii et al. 2009). Next, 50 ll of 20 mM Paraoxon in methanol was added and reactions were carried out for 24 h at 30 C. After incubation, cells were removed by ﬁltration using a 0.22 lm Ultrafree-MC ﬁlter unit (Millipore). PNP in the ﬁltrated solution was detected by HPLC using Hitachi L-7100 and TSKgel for ODS- 80Ts (75 9 4.6 mm i.d.) (Tosoh, Tokyo, Japan) at 30 C. The eluent was 90% (v/v) acetonitrile in 50 mM HEPES buffer (pH 8.5) at 0.5 ml min-1 at 2 µ m Ampr ori ICL terminator pWIFS or pWIFL MCS TRP1 UPR-ICL promoter FS or FL gene 2 µ m Ampr ori ICL terminator TRP1 Constructed vector SigOPH or OPH Digestion using restriction enzyme Ligation UPR-ICL promoter FS/FL gene 657 ori Ampr pWM513 OPH PCR amplification SigOPH or OPH Constructed vectors pWIFSSigOPH pWIFLSigOPH pWIFSOPH pWIFLOPH Fig. 2 Scheme for the construction of plasmids for display of OPH on the yeast cell surface using Flo1p anchor system. The gene encoding OPH is ampliﬁed by PCR with pWM513, as the template. pWIFS and pWIFL are the plasmid vectors for expressing the OPH on the yeast cell surface. The ampliﬁed fragment is digested and introduced in each plasmid vector. The resulting plasmids are named pWIFSSigOPH, pWIFLSig- OPH, pWIFSOPH and pWIFLOPH 30 C and the eluate was monitored at 415 nm. The retention time of PNP was 12 min. One enzyme unit is deﬁned as the formation of 1 nmol PNP per min per mg dry cells (Takayama et al. 2006). Results and discussion Effect of the anchor system on OPH activity As shown in Fig. 3, MT8-1/pWIFLOPH using Flo1p anchor system showed 6-fold greater OPH activity compared with MT8-1/pWMSigOPH or MT8-1/ pWMOPH using GPI anchor system, indicating the effective display of OPH with an active site located near the C-terminus. We investigated the effect of Flo1p length on OPH activity. The optimal whole cell activity of MT8-1/ pWIFSOPH was only 960 units/mg cell dry wt 123