title: pHluorin-based in vivo assay for hydrolase screening.
abstract: pHluorin, a pH-sensitive mutant of green fluorescent protein (GFP), acts as a sensor for intracellular pH shifts, triggered by hydrolytic enzymes. This principle was used to develop a pHluorin-based in vivo assay for hydrolase screening. The presented assay was evaluated for Escherichia coli (E. coli) cells, producing heterologous pHluorin and an esterase from Geobacillus stearothermophilus which is considered as a model hydrolase. Subsequently, the utility of this detection system was also demonstrated with recombinantly expressed hydantoinase and amidase in E. coli. This in vivo assay also shows capability for readout with flow cytometric devices. Population shifts of pHluorin-expressing E. coli cells were easily recognized due to pH changes caused by substrate hydrolysis.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany.
authors.forename: Sascha
authors.initials: S
authors.lastname: Schuster
authors.forename: Markus
authors.initials: M
authors.lastname: Enzelberger
authors.forename: Harald
authors.initials: H
authors.lastname: Trauthwein
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Vlada B
authors.initials: VB
authors.lastname: Urlacher
mesh_terms: D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D004950:Esterases / Q000032:analysis* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D049408:Luminescent Agents / Q000032:analysis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: D049408:Luminescent Agents; C485187:PHluorin; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D004950:Esterases