Medium-throughput profiling method for screening polysaccharide-degrading enzymes in complex bacterial extracts
Résumé
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. (c) 2012 Elsevier B.V. All rights reserved.