Characterization of New Oligosaccharides Obtained by An Enzymatic Cleavage of the Exopolysaccharide Produced by the Deep-Sea Bacterium Alteromonas infernus Using its Cell Extract

Abstract

Bacteria from deep-sea hydrothermal vents constitute an attractive source of bioactive molecules. In particular, exopolysaccharides (EPS) produced by these bacteria become a renewable source of both biocompatible and biodegradable molecules. The low molecular weight (LMW) derivatives of the GY785 EPS produced by the deep-sea hydrothermal vent strain Alteromonas infernus have previously displayed some biological properties, similar to those of glycosaminoglycans (GAG), explored in cancer and tissue engineering. These GAG-mimetic derivatives are obtained through a free radical depolymerization process, which could, however, affect their structural integrity. In a previous study, we have shown that A. infernus produces depolymerizing enzymes active on its own EPS. In the present study, an enzymatic reaction was optimized to generate LMW derivatives of the GY785 EPS, which could advantageously replace the present bioactive derivatives obtained by a chemical process. Analysis by mass spectrometry of the oligosaccharide fractions released after enzymatic treatment revealed that mainly a lyase activity was responsible for the polysaccharide depolymerization. The repeating unit of the GY785 EPS produced by enzyme cleavage was then fully characterized.

Keywords: Alteromonas infernus; deep-sea bacterium; enzymatic depolymerization; exopolysaccharides; glycosaminoglycan-mimetic; mass spectrometry; structural analysis; wild-type strain.

Figure 1

Structure of the GY785 exopolysaccharide (EPS) repeating unit [27].

Figure 2

PAGE analysis of the native HMW GY785 EPS, EPS (A), EPS incubated with soluble cell lysate, L + EPS (B), soluble cell lysate, L (C), EPS incubated with insoluble cell debris, D + EPS (D), insoluble cell debris, D (E) in Tris HCl buffer at different incubation times (0, 3, 22, 27, 48, 74 and 142 h).

Figure 3

High pressure size exclusion chromatography (HPSEC) profiles (refractive index (RI) detection) of the native GY785 EPS incubated with lysate, L + EPS (A), lysate, L (B), EPS incubated with insoluble cell debris, D + EPS (C) and cell debris, D (D) in Tris HCl buffer for different incubation times (T) 0, 3, 22 and 74 h.

Figure 4

High pressure size exclusion chromatography with multi angle light scattering (HPSEC-MALS) profiles of the GY785 EPS derivatives (EPS DR) of weight-average molecular weights of 4000, 11,500, 20,000 and 230,000 g/mol.

Figure 5

SEC fractionation of the native GY785 EPS depolymerized with cell debris, D for 63 h; UV and RI detections are indicated in blue and grey, respectively (A). PAGE analysis of the native GY785 EPS, four EPS derivatives with the weight-average molecular weight of 4000, 11,500, 20,000 and 230,000 g/mol, respectively, and five SEC fractions obtained after the GY785 EPS incubated with cell debris (B).

Figure 6

Helium charge transfer dissociation (He-CTD) tandem MS spectrum of the structure A isolated as a [M − 2H]²⁻ species at m/z 748.4 in negative ionization mode. For better readability, the mass range was split into two parts. Red, fully sulfated fragments; Blue, fragments with one sulfate loss; Triangle, water losses.