Flow cytometry-based ultra-high-throughput screening assay for cellulase activity
Section snippets
APCC synthesis
We synthesized 3-carboxy-7-(4′-aminophenoxy) coumarin (APCC) as described previously [22], although the reduction from 3-carboethoxy-7-(4′-nitrophenoxy) coumarin to 3-carboethoxy-7-(4′-aminophenoxy) coumarin was achieved using iron powder in ethanol [23] after characterization by nuclear magnetic resonance (NMR) spectroscopy [24]. The substrate for APCC synthesis, (3-carboethoxy-7-(hydroxy) coumarin, was synthesized as described previously [25].
APCC assay in MTPs
Fluorescence-based assays were carried out in
Optimization of reaction in MTPs
The assay design was initially tested in an MTP format using commercial A. niger cellulase. First, we established the optimal concentrations of CMC (0.3%) and HOx (3.4 U/ml), as described in the Supplementary material. No fluorescence was detected in the control reactions, confirming that the observed fluorescence was due to cellulase activity (Fig. 2A). Different amounts of cellulase were then added to the system, and the fluorescence response was measured. The assay showed a linear response to
Acknowledgments
This work was performed as part of the Cluster of Excellence “Tailor-Made Fuels from Biomass”, which is funded by the Excellence Initiative by the German federal and state governments to promote science and research at German universities.
References (27)
- et al.
Cellulases: biosynthesis and applications
Enzyme Microb. Technol.
(1980) Cellulases and related enzymes in biotechnology
Biotechnol. Adv.
(2000)Totally in vitro protein selection using mRNA–protein fusions and ribosome display
Curr. Opin. Chem. Biol.
(1999)- et al.
In vitro enzyme evolution: the screening challenge of isolating the one in a million
Trends Biotechnol.
(2001) - et al.
High-throughput screening of enzyme libraries
Curr. Opin. Biotechnol.
(2000) - et al.
High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments
Chem. Biol.
(2005) - et al.
High-throughput screening of enzyme libraries: in vitro evolution of a β-galactosidase by fluorescence-activated sorting of double emulsions
Chem. Biol.
(2005) - et al.
In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting
Anal. Biochem.
(2004) - et al.
Directed evolution of biocatalysts
Curr. Opin. Chem. Biol.
(1999) - et al.
Hexose oxidase from the red alga Chondrus crispus
J. Biol. Chem.
(1997)