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.

Characterization of novel Acidobacteria exopolysaccharides with potential industrial and ecological applications

“…However, the quantities of GlcA and GalA might be underestimated due to the hydrolyses conditions used in this study, which are efficient for hexoses, pentoses and deoxy sugars but not ideal for uronic acids. The monosaccharide compositions we determined for EPSWH15 and EPS5B5 are in basic agreement with previously published observations36 since mannose (Man), glucose (Glc), galactose (Gal) and xylose (Xyl) are among the most common monosaccharides found in EPS3738.…”

Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa

“…To determine the pyruvate content of the polymers, 1 g·l −1 EPS solutions were hydrolyzed and neutralized as described for the sugar monomer analyses ( 58 ). To start the reaction, 10 µl neutralized sample + 90 µl of ddH 2 O or standard were mixed with 100 µl assay mixture [50 µM N-(carboxymethylamino-carbonyl)-4.4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), 50 µM thiamine pyrophosphate, 100 µM MgCl 2 × 6H 2 O, 0.05 U·ml -1 pyruvate oxidase, 0.2 U·ml -1 horseradish peroxidase, 20 mM K 2 PO 4 buffer pH 6] and incubated at 37 °C, 700 rpm for 30 min in a micro-plate shaker.…”

Pyruvate Substitutions on Glycoconjugates

“…A specifically developed colorimetric/fluorometric assay for ketal-pyruvate detection via enzymatic oxidation has been incorporated in a recently introduced high throughput screening platform for the structural analysis of novel EPS structures [179], which underlines the importance of pyruvylated epitopes. The platform is based on ultra-high performance liquid chromatography coupled with ultra-violet and electrospray ionization ion trap detection following EPS isolation.…”