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High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Farrow, Mary F. and Arnold, Frances H. (2011) High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli. Journal of Visualized Experiments , 54 . Art. No. e2942. ISSN 1940-087X . PMCID PMC3217630. https://resolver.caltech.edu/CaltechAUTHORS:20180821-075520096

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Abstract

Cellulase enzymes (endoglucanases, cellobiohydrolases, and β-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production. Of particular interest are fungal cellulases, which are already being used industrially for foods and textiles processing. Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium. We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (“halos”) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine, relative protein fitness. Traditional bacterial whole cell CMC/CR activity assays involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.3791/2942DOIArticle
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217630/PubMed CentralArticle
ORCID:
AuthorORCID
Arnold, Frances H.0000-0002-4027-364X
Additional Information:© 2011 JoVE. Date Published: 8/13/2011. This work was funded by the Gordon and Betty Moore Foundation, and by the UNCF/Merck Science Initiative. No conflicts of interest declared.
Funders:
Funding AgencyGrant Number
Gordon and Betty Moore FoundationUNSPECIFIED
UNCF/Merck Science InitiativeUNSPECIFIED
Subject Keywords:Molecular Biology, Issue 54, cellulase, endoglucanase, CMC, Congo Red
PubMed Central ID:PMC3217630
Record Number:CaltechAUTHORS:20180821-075520096
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180821-075520096
Official Citation:Farrow, M. F., Arnold, F. H. High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli. J. Vis. Exp. (54), e2942, doi:10.3791/2942 (2011).
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:88965
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:21 Aug 2018 20:23
Last Modified:15 Oct 2019 22:55

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