title: Strategy for screening metagenomic resources for exocellulase activity using a robotic, high-throughput screening system.
abstract: Exocellulases play a key role in cleaving the accessible ends of cellulose molecules to release soluble glucose and cellobiose. To date, there have been no screens for exocellulase owing to assay protocol limitations, the high cost of substrates, and low activity of exocellulases compared with endocellulases. This study is the first to demonstrate direct screening for exocellulase activity using a robotic, high-throughput screening (HTS) system. Cell growth in 96-well plates was measured by monitoring optical density over 11-14h at 37°C with agitation. Fluorescence of methylumbelliferyl groups released from 4-methylumbelliferyl-β-D-cellobioside was determined using a VICTOR3 microplate reader. This new HTS system enabled activity verification of more than 10(4) clones per day. As a result, we obtained four exocellulases clones (CelEx-SF301, CelEx-SF309, CelEx-BR12 and CelEx-BR15) from 29,006 metagenomic fosmid clones that had previously been prepared from sweet potato field soil microbes and rumen fluid. This powerful approach could be effectively applied to screen various metagenomic resources for new enzymes.
authors.affiliation: Applied Microbiology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 181 Ipsin-gil, Jeongeup-si, Jeonbuk 580-185 Republic of Korea.
authors.forename: Kyong-Cheol
authors.initials: KC
authors.lastname: Ko
authors.forename: Yunjon
authors.initials: Y
authors.lastname: Han
authors.forename: Dae-Eun
authors.initials: DE
authors.lastname: Cheong
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Choi
authors.forename: Jae Jun
authors.initials: JJ
authors.lastname: Song
mesh_terms: D018533:Biomass; D044602:Cellulases / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D015698:Genomic Library; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D056186:Metagenomics / Q000379:methods*; D012371:Robotics / Q000295:instrumentation; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods; D012988:Soil Microbiology
chemical_list: D004269:DNA, Bacterial; D044602:Cellulases
keywords: 4-Methylumbelliferyl-β-d-cellobioside; BGL; CBH; Cellobiose; Cellopentaose; Cellotetraose; Cellotriose; EG; Endo-β-1,4-glucanase; Enzyme assay; Exo-β-1,4-cellobiohydrolases; Exocellulase; G1; G2; G3; G4; G5; GHs; Glucose; Glycoside hydrolases; HTS; High-throughput screening; High-throughput screening (HTS); LB; Luria-Bertani; MeUmb; MeUmbG(2); Metagenome; Methylumbelliferyl; ORF; Open reading frame; PFGE; Pulsed-field gel electrophoresis; TLC; Thin-layer chromatography; β-Glucosidase