New Family of Ulvan Lyases Identified in Three Isolates from the Alteromonadales Order

Ulvan is the main polysaccharide component of the Ulvales (green seaweed) cell wall. It is composed of disaccharide building blocks comprising 3-sulfated rhamnose linked to d-glucuronic acid (GlcUA), l-iduronic acid (IdoUA), or d-xylose (Xyl). The degradation of ulvan requires ulvan lyase, which catalyzes the endolytic cleavage of the glycoside bond between 3-sulfated rhamnose and uronic acid according to a β-elimination mechanism. The first characterized ulvan lyase was identified in Nonlabens ulvanivorans, an ulvanolytic bacterial isolate. In the current study, we have identified and biochemically characterized novel ulvan lyases from three Alteromonadales isolated bacteria. Two homologous ulvan lyases (long and short) were found in each of the bacterial genomes. The protein sequences have no homology to the previously reported ulvan lyases and therefore are the first representatives of a new family of polysaccharide lyases. The enzymes were heterologously expressed in Escherichia coli to determine their mode of action. The heterologous expressed enzymes were secreted into the milieu subsequent to their signal sequence cleavage. An endolytic mode of action was observed and studied using gel permeation chromatography and ¹H NMR. In contrast to N. ulvanivorans ulvan lyase, cleavage occurred specifically at the GlcUA residues. In light of the genomic context and modular structure of the ulvan lyase families identified to date, we propose that two ulvan degradation pathways evolved independently.     

Characterization of an Alteromonas long-type ulvan lyase involved in the degradation of ulvan extracted from Ulva ohnoi

Ulvan is a sulfated polysaccharide found in the cell wall of the green algae Ulva. We first isolated several ulvan-utilizing Alteromonas sp. from the feces of small marine animals. The strain with the highest ulvan-degrading activity, KUL17, was analyzed further. We identified a 55-kDa ulvan-degrading protein secreted by this strain and cloned the gene encoding for it. The deduced amino acid sequence indicated that the enzyme belongs to polysaccharide lyase family 24 and thus the protein was named ulvan lyase. The predicted molecular mass of this enzyme is 110 kDa, which is different from that of the identified protein. By deletion analysis, the catalytic domain was proven to be located on the N-terminal half of the protein. KUL17 contains two ulvan lyases, one long and one short, but the secreted and cleaved long ulvan lyase was demonstrated to be the major enzyme for ulvan degradation.     

Antioxidant activity of different molecular weight sulfated polysaccharides from <Emphasis Type="Italic">Ulva pertusa</Emphasis> Kjellm (Chlorophyta)

Polysaccharides extracted from Ulva pertusa Kjellm (Chlorophyta) are a group of sulfated heteropolysaccharides, the ulvans. In this study, different molecular weight ulvans were prepared by H₂O₂ degradation and their antioxidant activities investigated including superoxide and hydroxyl radical scavenging activity, reducing power and metal chelating ability. The molecular weights of natural and degraded ulvans were 151.7, 64.5, 58.0, and 28.2 kDa, respectively, as determined by high performance gel permeation chromatography. Among the four samples, U₃ (the lowest molecular weight sample) showed significant inhibitory effects on superoxide and hydroxyl radicals with IC₅₀ values of 22.1 μ g mL⁻¹ and 2.8 mg mL⁻¹; its reducing power and metal chelating ability were also the strongest among the four samples. All the other samples also demonstrated strong activity against superoxide radicals. The results indicated that molecular weight had a significant effect on the antioxidant activity of ulvan with low molecular weight ulvan having stronger antioxidant activity.     

SEASONAL VARIABILITY OF PHYSICOCHEMICAL AND RHEOLOGICAL PROPERTIES OF ULVAN IN TWO ULVA SPECIES (CHLOROPHYTA) FROM THE BRITTANY COAST1

The seasonal variability in the extraction yield, physicochemical characteristics, and rheological properties of ulvan from two Ulva species contributing to Brittany “green tides” has been studied. These seaweeds were collected in the water column for Ulva armoricana Dion, de Reviers et Coat and on hard substrata for Ulva rotundata Bliding. The maximum ulvan extraction efficiency was not related to the maximum ulvan content in the seaweeds, but with the active growth period of the seaweeds. Ulvan chemical structure, macromolecular characteristics, and rheological properties were affected by both species and seasons. The proportion of high‐molecular‐weight ulvan was the major factor positively correlated with the gelling properties. Characteristics of ulvan from U. rotundata subjected to tides were more affected by seasons than ulvan from U. armoricana living in a more constant environment. These results point to several useful recommendations concerning Ulva sp. biomass collected with regard to ulvan characteristics and uses.     

Chemical characterisation and gelling properties of cell wall polysaccharides from species of Ulva (Ulvales,Chlorophyta)

Two alkali-soluble polysaccharide fractions from the cell wall of Ulva rigida were determined to be ,-1,4-linked glucoxylans and a -1,4-linked glucuronan by chemical and NMR spectroscopic analysis. The ¹³C NMR spectrum of water-soluble xyloglucuronorhamnan sulfate from Ulva rigida referred to as ulvan is reported and the ¹³C and ¹H NMR chemical shifts of its major repeating unit, the aldobiuronic acid -D-GlcA-(1,4)-L-Rha, are given. The composition and gelling properties of ulvan from Ulva species from green tides are also reported. The thermoreversible gel required both calcium and borate ions and the shear storage modulus G was ion concentration dependent. The mechanism of gelation and the associations of the different Ulva cell wall soluble polysaccharides are discussed.     

A new carbohydrate-active oligosaccharide dehydratase is involved in the degradation of ulvan

Marine algae catalyze half of all global photosynthetic production of carbohydrates. Owing to their fast growth rates, Ulva spp. rapidly produce substantial amounts of carbohydrate-rich biomass and represent an emerging renewable energy and carbon resource. Their major cell wall polysaccharide is the anionic carbohydrate ulvan. Here, we describe a new enzymatic degradation pathway of the marine bacterium Formosa agariphila for ulvan oligosaccharides involving unsaturated uronic acid at the nonreducing end linked to rhamnose-3-sulfate and glucuronic or iduronic acid (Δ-Rha3S-GlcA/IdoA-Rha3S). Notably, we discovered a new dehydratase (P29_PDnc) acting on the nonreducing end of ulvan oligosaccharides, i.e., GlcA/IdoA-Rha3S, forming the aforementioned unsaturated uronic acid residue. This residue represents the substrate for GH105 glycoside hydrolases, which complements the enzymatic degradation pathway including one ulvan lyase, one multimodular sulfatase, three glycoside hydrolases, and the dehydratase P29_PDnc, the latter being described for the first time. Our research thus shows that the oligosaccharide dehydratase is involved in the degradation of carboxylated polysaccharides into monosaccharides.     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis and antihyperlipidemic activity of acetylated derivative of ulvan from Ulva pertusa

In this study, acetylated ulvan (AU) was prepared with acetic anhydride in N,N-dimethylacetamide, and the antihyperlipidemic activity of natural ulvan and its acetylated ulvan derivative (AU) in mice was determined. Obvious differences in antihyperlipidemic activity between natural ulvan and its derivative were observed, moreover, AU showed stronger antihyperlipidemic activity on triglyceride (TG) and low density lipoprotein cholesterol (LDL-C).     

Antihyperlipidemic activity of high sulfate content derivative of polysaccharide extracted from Ulva pertusa (Chlorophyta)

In this study, high sulfate content ulvan (HU) was prepared with sulfur trioxide/N,N-dimethylformamide (SO₃-DMF) in formamide, and the antihyperlipidemic activity of ulvan and HU in mice was determined. Obvious differences in antihyperlipidemic activity between natural ulvan and HU were observed, moreover, the antihyperlipidemic activity of HU-fed 250 mg/kg was the strongest, compared to natural ulvan fed group, triglyceride (TG) and low density lipoprotein cholesterol (LDL-C) concentrations were significantly decreased 28.1% (P < 0.05) and 28.4% (P < 0.01), respectively. It was likely that the sulfate content had significant effect on the antihyperlipidemic activity. On the other hand, the results proved that the antihyperlipidemic activity was not concentration dependent for HU-fed mice.