title: Flow cytometry-based ultra-high-throughput screening assay for cellulase activity.
abstract: We have developed a novel, ultra-high-throughput screening assay for the detection of cellulase activity based on fluorescence-activated cell sorting (FACS) and double emulsion technology. Cellulase activity is detected using a series of coupled enzymes, including hexose oxidase (HOx), which generates hydrogen peroxide from the reducing sugars released by cellulases in the presence of any natural or artificial substrate. The assay can be adapted to suit a microtiter plate format, but the highest throughput is achieved by using FACS to screen high-complexity cellulase clone libraries. Using this approach, we achieved a 12-fold enrichment of positive (cellulase-expressing) cells in cellulase reference libraries after just one sorting round.
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, D-52074 Aachen, Germany.
authors.forename: Raluca
authors.initials: R
authors.lastname: Ostafe
authors.forename: Radivoje
authors.initials: R
authors.lastname: Prodanovic
authors.forename: Ulrich
authors.initials: U
authors.lastname: Commandeur
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D001234:Aspergillus niger / Q000201:enzymology; D002480:Cellulase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D010544:Peroxidases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D000429:Alcohol Oxidoreductases; C021354:hexose oxidase; D010544:Peroxidases; C026195:bromide peroxidase; D002480:Cellulase