Biotechnol Lett (2010) 32:655–659 DOI 10.1007/s10529-010-0204-1 O R I G I N A L R E S E A R C H P A P E R Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system Takeshi Fukuda • Kouta Tsuchiyama • Hirokazu Makishima • Katsumi Takayama • Ashok Mulchandani • Kouichi Kuroda • Mitsuyoshi Ueda • Shin-ichiro Suye Received: 12 November 2009 / Revised: 28 December 2009 / Accepted: 6 January 2010 / Published online: 29 January 2010 The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30 C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity T. Fukuda K. Tsuchiyama H. Makishima S. Suye (&) Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1, Bunkyo, Fukui 910-8507, Japan e-mail: suyeb10@u-fukui.ac.jp K. Takayama Department of Chemistry and Biology Engineering, Fukui National College of Technology, Geshi-cho, Sabae, Fukui 916-8507, Japan A. Mulchandani Department of Chemical and Environmental Engineering, University of California, Riverside, CA 92521, USA K. Kuroda M. Ueda Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems. Keywords Cell surface engineering Flo1p anchor system GPI anchor system Organophosphorus hydrolase Introduction Organophosphorus compounds (OPs) such as pesti- cides, fungicides, and herbicides are highly toxic but are nevertheless used worldwide. The continued use of OPs increases the possibility that delayed cholinergic toxicity and neurotoxicity are induced (Tuovinen et al. 1994). Developed countries inspect imported agricul- tural food products for toxic OPs, and the growing public concern about their safety in modern agricul- ture has stimulated the development of methods for OPs detection. Current analytical techniques, such as chromatography, immunoassay, and the use of enzyme biosensors based on inhibition of cholinester- ase activity, are highly sensitive but have disadvan- tages such as high cost or lengthy procedures (Cortada et al. 2009; Lee et al. 2003). Organophosphorus hydrolase (OPH) effectively hydrolyzes OP esters, including pesticides, such as 123