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Flow cytometry-based ultra-high-throughput screening assay for cellulase activity

 
: Ostafe, R.; Prodanovic, R.; Commandeur, U.; Fischer, R.

:

Analytical biochemistry 435 (2013), Nr.1, S.93-98
ISSN: 0003-2697
Englisch
Zeitschriftenaufsatz
Fraunhofer IME ()
cellulase; double emulsions; FACS; fluorescence assay; hexose oxidase; vanadium bromoperoxidase

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 sub- strate. 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 sort-
ing round.

: http://publica.fraunhofer.de/dokumente/N-230261.html