Liquid Phase Multiplex High-Throughput Screening of Metagenomic Libraries Using p-Nitrophenyl-Linked Substrates for Accessory Lignocellulosic Enzymes

Mariette Smart; Robert J Huddy; Don A Cowan; Marla Trindade

doi:10.1007/978-1-4939-6691-2_13

Liquid Phase Multiplex High-Throughput Screening of Metagenomic Libraries Using p-Nitrophenyl-Linked Substrates for Accessory Lignocellulosic Enzymes

Methods Mol Biol. 2017:1539:219-228. doi: 10.1007/978-1-4939-6691-2_13.

Authors

Mariette Smart^{1

2}, Robert J Huddy^{1

2}, Don A Cowan^{1

2}, Marla Trindade^{3

4}

Affiliations

¹ Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Cape Town, South Africa.
² Centre for Bioprocess Engineering Research, University of Cape Town, Cape Town, South Africa.
³ Institute for Microbial Biotechnology and Metagenomics, University of the Western Cape, Cape Town, South Africa. ituffin@uwc.ac.za.
⁴ Centre for Bioprocess Engineering Research, University of Cape Town, Cape Town, South Africa. ituffin@uwc.ac.za.

PMID: 27900692
DOI: 10.1007/978-1-4939-6691-2_13

Abstract

To access the genetic potential contained in large metagenomic libraries, suitable high-throughput functional screening methods are required. Here we describe a high-throughput screening approach which enables the rapid identification of metagenomic library clones expressing functional accessory lignocellulosic enzymes. The high-throughput nature of this method hinges on the multiplexing of both the E. coli metagenomic library clones and the colorimetric p-nitrophenyl linked substrates which allows for the simultaneous screening for β-glucosidases, β-xylosidases, and α-L-arabinofuranosidases. This method is readily automated and compatible with high-throughput robotic screening systems.

Keywords: Colorimetric substrates; Function-driven screening; High-throughput screening; Lignocellulosic enzymes; Liquid-phase screening; Metagenomics; Multiplex screening; p-Nitrophenyl substrates; α-L-Arabinofuranosidases; β-Glucosidases; β-Xylosidases.

MeSH terms

Cellulases / genetics*
Cellulases / metabolism*
Enzyme Activation
Gene Expression
Gene Library*
High-Throughput Screening Assays*
Liquid-Liquid Extraction / methods*
Metagenome*
Metagenomics* / methods
Substrate Specificity

Substances

Cellulases