title: Synthesis and evaluation of a series of 6-chloro-4-methylumbelliferyl glycosides as fluorogenic reagents for screening metagenomic libraries for glycosidase activity.
abstract: Screening of large enzyme libraries such as those derived from metagenomic sources requires sensitive substrates. Fluorogenic glycosides typically offer the best sensitivity but typically must be used in a stopped format to generate good signal. Use of fluorescent phenols of pKa < 7, such as halogenated coumarins, allows direct screening at neutral pH. The synthesis and characterisation of a set of nine different glycosides of 6-chloro-4-methylumbelliferone are described. The use of these substrates in a pooled format for screening of expressed metagenomic libraries yielded a "hit rate" of 1 in 60. Hits were then readily deconvoluted with the individual substrates in a single plate to identify specific activities within each clone. The use of such a collection of substrates greatly accelerates the screening process.
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Hong-Ming
authors.initials: HM
authors.lastname: Chen
authors.affiliation: Department of Microbiology & Immunology, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1; Centre for High-Throughput Biology, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Armstrong
authors.affiliation: Department of Microbiology & Immunology, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1.
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Hallam
authors.affiliation: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1; Centre for High-Throughput Biology, University of British Columbia, 2036 Main Mall, Vancouver, BC, Canada V6T 1Z1. Electronic address: withers@chem.ubc.ca.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D003374:Coumarins / Q000737:chemistry*; D015698:Genomic Library; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification*; D006027:Glycosides / Q000138:chemical synthesis* / Q000737:chemistry; D054879:Halogenation; D057166:High-Throughput Screening Assays; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D056186:Metagenomics
chemical_list: C030561:4-methylumbelliferyl glycosides; D001426:Bacterial Proteins; D003374:Coumarins; D006027:Glycosides; D006923:Hymecromone; D006026:Glycoside Hydrolases
keywords: Assay; Glycosidase; High-throughput; Metagenomics; Screening