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

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.

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

“…In a previous study by Ohlhoff et al [14], a metagenomic library consisting of more than 150,000 fosmid clones with an average insert size of 31 kb was constructed. A total of 46,000 clones were screened for α-L-arabinofuranosidase (AFase) activity and 13 putative AFase positive clones were identified [15]. Here we performed preliminary thermostability screening by incubating cell-free extracts at a range of temperatures for 60 min.…”

“…AFase positive clones identified during primary screening in [15] were first subjected to thermostability assays to identify those with desirable properties. Briefly, E. coli harbouring recombinant fosmids were inoculated into microtiter plates with LB containing 0.01% ( w / v ) L-arabinose and 15 μg/mL chloramphenicol and incubated for 16 h. A 200 μL aliquot of the overnight cultures was lysed with 10 μL Bugbuster™ protein extraction reagent (Novagen, USA) and the extracts incubated at 25, 40, 50, 60, 70, 80 or 90 °C for 60 min.…”

“…This screening approach thus provides access to previously unknown genes and their novel enzymes with unique structural and kinetic properties [12, 13]. Ohlhoff et al [14] constructed a fosmid metagenomic library from high temperature compost which was subsequently screened for several classes of lignocellulases [15]. In this study, three AFases were chosen from these initial screens and were characterized with the aim of bioprospecting for unique biochemical properties that could be of use in industrial applications.…”