Elsevier

Carbohydrate Research

Volume 345, Issue 9, 16 June 2010, Pages 1163-1173
Carbohydrate Research

Structural investigation of an exopolysaccharide substituted with a lactyl ether group produced by Raoultella terrigena Ez-555-6 isolated in the Chernobyl exclusion zone

https://doi.org/10.1016/j.carres.2010.03.015Get rights and content

Abstract

Raoultella terrigena strain Ez-555-6, isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone, produces a non-referenced high-molecular-mass exopolysaccharide (EPS). The structure of this EPS was determined using a combination approach including monosaccharide composition (GLC–FID, HPAEC–PAD), determination of glycosylation sites (GLC–EIMS) and 1D/2D NMR (1H, 13C) and ESIMS (HR, MS/MS) studies of oligosaccharides obtained from mild acid hydrolysis. The EPS was found to be a charged pentasaccharide with a repeating unit composed of d-galactose, d-glucose, d-mannose and d-glucuronic acid (1:2:1:1). Lactic acid and O-acetyl substituents were localized on galactose and glucose residues, respectively, as presented in the following structure:

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Introduction

Raoultella terrigena,1 previously known as Klebsiella terrigena, because of the environmental origin of the isolates (water, soil and plants),2 is a Gram-negative bacteria of the Enterobacteriaceae family. As all Gram-negative bacteria, R. terrigena produce both lipopolysaccharides (LPS) and exopolysaccharides (EPS). The structure of the O-specific polysaccharide from the lipopolysaccharide of the bacteria has been established.3 To our knowledge, no data on the exopolysaccharide production and composition from R. terrigena are available. A strain of R. terrigena, R. terrigena strain Ez-555-6 was recently isolated from a root nodule of Medicago sativa harvested in the Chernobyl exclusion zone.4 This bacterial strain, which is able to fix nitrogen, produces under laboratory conditions a high-molecular-mass exopolysaccharide.

Exopolysaccharides have found a wide range of applications in the food, cosmetic, pharmaceutical and environmental industries.5, 6 It is well known that among different EPS there are a number of variations in their sugar composition and sugar linkages, chain length, presence of repeated side chains and substitutions. All of these factors determine their technological applications and their biological properties. Therefore, the determination of the chemical composition and structure of a novel EPS is relevant for predicting their potential applications. In order to investigate the biological nature of the EPS secreted by R. terrigena strain Ez-555-6 for pharmaceutical or environmental property applications, we present in this manuscript the elucidation of the molecular structure of this EPS.

Investigations into the composition of EPS were carried out with GLC–FID and GLC–MS. The glycosyl residue sequences were determined through a combination of high-resolution NMR techniques (1D experiments: 1H, 13C and 2D experiments: 1H/1H COSY, 1H/1H TOCSY, 1H/13C HSQC, 1H/13C HMBC)7 and by electrospray-ionization mass spectrometry (ESI-QToF-MS) and low-energy collision tandem mass spectrometry (CID–MS/MS) experiments. The main advantages of ESI-QToF-MS and CID–MS/MS are the capability to assign the sequences, the glycosidic linkages, the branching and sensitivity in assigning the substitution pattern information.8, 9

Section snippets

Results

The analytical strategy described in Scheme 1 was used to determine the molecular structure of the EPS under study. Therefore, the purified native EPS was subjected to monosaccharide identification, glycosidic linkage determination and structural characterization of the oligosaccharides obtained from mild acid hydrolysis.

Isolation and purification of exopolysaccharide (EPS) from bacteria

The R. terrigena Ez-555-6 was isolated from the root nodules of the M. sativa plant harvested in the Chernobyl exclusion zone.4 A three-day-old bacteria culture of bacteria grown in mannitol salt medium (GMS) was centrifuged (14,000g, 30 min), and the crude EPS was precipitated from the supernatant by addition of 3 vol of cold 2-PrOH (overnight, 4 °C). The precipitate was dissolved in deionized water (1/10 of initial volume), dialyzed against deionized water and lyophilized for quantification and

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