Constitutive cell surface expression of ZZ domain for the easy preparation of yeast-based immunosorbents.

We describe a novel expression cassette that enables efficient and constitutive expression of the ZZ domain derived from Staphylococcus aureus protein A on the yeast cell surface to easily prepare yeast-based immunosorbents. Using this expression cassette containing the PGK1 promoter, a secretion signal derived from α-factor, and a Flo1-derived anchor protein, we successfully created a yeast-based immunosorbent for human serum albumin.

Estimation of enzyme kinetic parameters of cell surface-displayed organophosphorus hydrolase and construction of a biosensing system for organophosphorus compounds.

Yeast cells displaying organophosphorus hydrolase (OPH), which was anchored using a lectin-like cell-wall protein (Flo1p), were used as a biocatalyst for the detection of organophosphorus compounds (OPs). The concentration of p-nitrophenol produced by the hydrolysis of the OPs paraoxon was calculated from the absorbance change at 415 nm during a 30-min reaction. The apparent Michaelis-Menten constant (K'(m)) for surface-displayed OPH was estimated to be approximately 50 µM, which is consistent with the value of purified OPH, and shows that cell-surface expression is a useful strategy to overcome the mass-transport resistance of substrates across the cell membrane. Notably, the long-term storage stability of the enzyme activity exceeded 40 days when cells displaying OPH were preserved at temperatures below -4 °C. A fiber-optic biosensing system was also constructed using a commercial optical-fiber detection device and yeast cells with surface-displayed OPH. A linear relationship was obtained for paraoxon concentrations of up to 50 ppm (182 µM), with a detection sensitivity of 0.0043 A.U. per ppm (R(2) = 0.9574) and a detection limit of 5 ppm (18 µM).

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system.

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 degrees 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 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.

Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae.

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.

Pleading for the use of biodegradable polymers in favor of marine environments and to avoid an asbestos-like problem for the future.

Research results about the movement and accumulation of floating marine debris drifting throughout the world's oceans are reviewed in this paper. A mechanism for this accumulation and movement is strongly associated with surface currents consisting of the Ekman drift and the geostrophic current, because all floating marine debris is passive to surface currents. The basic published mechanism for the North Pacific is common across the world's ocean. After marine debris accumulates in the narrow Ekman convergence zone, it is moved to the east by geostrophic currents. The most important thing is that floating marine debris concentrates in some specific regions, independent of the initial quantity of marine debris. In order to resolve this problem and to avoid an asbestos-like problem, the use of biodegradable polymers is important in our daily life.