title: Medium-throughput profiling method for screening polysaccharide-degrading enzymes in complex bacterial extracts.
abstract: Polysaccharides are the most abundant and the most diverse renewable materials found on earth. Due to the stereochemical variability of carbohydrates, polysaccharide-degrading enzymes - i.e. glycoside hydrolases and polysaccharide lyases - are essential tools for resolving the structure of these complex macromolecules. The exponential increase of genomic and metagenomic data contrasts sharply with the low number of proteins that have ascribed functions. To help fill this gap, we designed and implemented a medium-throughput profiling method to screen for polysaccharide-degrading enzymes in crude bacterial extracts. Our strategy was based on a series of filtrations, which are absolutely necessary to eliminate any reducing sugars not directly generated by enzyme degradation. In contrast with other protocols already available in the literature, our method can be applied to any panel of polysaccharides having known and unknown structures because no chemical modifications are required. We applied this approach to screen for enzymes that occur in Pseudoalteromonas carrageenovora grown in two culture conditions.
authors.affiliation: Centre National de la Recherche Scientifique, Université Pierre et Marie Curie-Paris 6, UMR 7139 Marine Plants and Biomolecules, Station Biologique, F-29682 Roscoff Cedex, France.
authors.forename: Maude
authors.initials: M
authors.lastname: Fer
authors.forename: Aurélie
authors.initials: A
authors.lastname: Préchoux
authors.forename: Andréa
authors.initials: A
authors.lastname: Leroy
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Sassi
authors.forename: Marc
authors.initials: M
authors.lastname: Lahaye
authors.forename: Claire
authors.initials: C
authors.lastname: Boisset
authors.forename: Pi
authors.initials: P
authors.lastname: Nyvall-Collén
authors.forename: William
authors.initials: W
authors.lastname: Helbert
mesh_terms: D005374:Filtration / Q000379:methods; D006026:Glycoside Hydrolases / Q000032:analysis*; D008403:Mass Screening / Q000379:methods*; D011133:Polysaccharide-Lyases / Q000032:analysis*; D011134:Polysaccharides / Q000378:metabolism*; D031161:Pseudoalteromonas / Q000201:enzymology*
chemical_list: D011134:Polysaccharides; D006026:Glycoside Hydrolases; D011133:Polysaccharide-Lyases