title: Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis.
abstract: Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.
authors.affiliation: Chair of Chemistry of Biogenic Resources, Technische Universität München.
authors.forename: Broder
authors.initials: B
authors.lastname: Rühmann
authors.affiliation: Chair of Chemistry of Biogenic Resources, Technische Universität München.
authors.forename: Jochen
authors.initials: J
authors.lastname: Schmid
authors.affiliation: Chair of Chemistry of Biogenic Resources, Technische Universität München; sieber@tum.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Sieber
mesh_terms: D002241:Carbohydrates; D002851:Chromatography, High Pressure Liquid; D057166:High-Throughput Screening Assays; D011108:Polymers; D011134:Polysaccharides / Q000032:analysis*
chemical_list: D002241:Carbohydrates; D011108:Polymers; D011134:Polysaccharides