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.
Takayama
Katsumi
K
Department of Chemistry and Biology Engineering, Fukui National College of Technology, Geshi-cho, Sabae-shi, Japan. takayama@fukui-nct.ac.jp
Suye
Shin-ichiro
S
Kuroda
Kouichi
K
Ueda
Mitsuyoshi
M
Kitaguchi
Tetsuya
T
Tsuchiyama
Kouta
K
Fukuda
Takeshi
T
Chen
Wilfred
W
Mulchandani
Ashok
A
D043303:Aryldialkylphosphatase / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D002462:Cell Membrane / Q000201:enzymology*; D005417:Flavobacterium / Q000201:enzymology*; D008856:Microscopy, Fluorescence / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D013045:Species Specificity; D013499:Surface Properties
D043303:Aryldialkylphosphatase