Fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format

2016

JoVE

Self Cite

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.

Section: '-Bis(dimethylamino)-diphenylaminementioning

confidence: 99%

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Rühmann

2016

JoVE

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“…Detailed parameters are given in (Rühmann et al, 2014). The calibration standards were diluted with neutralized TFA-matrix to compensate the influence on the derivatization process.…”

Section: -Well Derivatization Methods For Monomer Analysis (Step 5)mentioning

confidence: 99%

“…For this purpose, we combined our recently developed high throughput carbohydrate analysis method (Rühmann, Schmid, & Sieber, 2014) with micro-plate based cultivation and sample preparation to create a powerful and fast tool for screening the highly diverse field of microbial EPS (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

High throughput exopolysaccharide screening platform: From strain cultivation to monosaccharide composition and carbohydrate fingerprinting in one day

Rühmann

2015

Carbohydrate Polymers

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Microbial exopolysaccharides (EPS) are multifunctional biogenic polymers, which exist in highly diverse chemical structures. To facilitate a fast determination of the carbohydrate composition of novel isolated strains or modified EPS variants a fast screening and analytical method is required. The platform as realized and described in this article is based on the fast carbohydrate analysis via liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection in 96-well format to detect different sugars, sugar derivatives and substituents such as pyruvate. Monosaccharide analysis from hydrolyzed polysaccharides was validated successfully by 16 commercially available polymers with known structure. The method is sensitive enough to distinguish various types of sphingans which solely differ in small alterations in the monomer composition. Even a quantitative detection of single monomers as present in complex plant polysaccharides like karaya gum, with the lowest recovery, was in accordance with literature. Furthermore, 94 bacterial strains for the validation of the screening platform were completely analyzed and 41 EPS producing strains were efficiently identified. Using the method a carbohydrate-fingerprint of the strains was obtained even allowing a very fast differentiation between strains belonging to the same species. This method can become a valuable tool not only in the fast analysis of strain isolates but also in the targeted screening for polysaccharides containing special rare sugars as well in the screening of strain libraries from genetic engineering for altered structures.

“…This method would have the potential to expand the portfolioo fs tructurally divers EPS variants. [71] By using the power of next-generation sequencing technologies [72] in combination with highly exact and fast analytical techniques [73,74] as well as synthetic biological methods, the potentialt ot ailor make EPSs might be fully exploited, and new fields of applications could be opened.…”

Section: Wwwchembiochemorgmentioning

confidence: 99%

Enzymatic Transformations Involved in the Biosynthesis of Microbial Exo‐polysaccharides Based on the Assembly of Repeat Units

2015

ChemBioChem

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Microbial exo-polysaccharides can serve as valuable biopolymers in medicine, food and the feed industry as well as in various technical applications as substitutes of petro-based polymers or with unusual performance. Due to their different natural functions, they have vastly diverse structures, which lead to a very different properties. This structural diversity is brought about by complex biosyntheses based on enzymes whose genes are mostly encoded in clusters within the genomes of the different microbial species. The organisation of the genes and the chemical structures of the corresponding polysaccharides are closely related. Here, we will mainly focus on the genetics and biosynthesis of some major bacterial hetero-polysaccharides that are based on repeat unit assembly and will present specific examples of enzymatic transformation steps. Finally, a short outlook will be given on how in vivo modifications based on enzymatic transformations could be used to engineer these polymers.