Improved isolation of glucuronan from algae and the production of glucuronic acid oligosaccharides using a glucuronan lyase Elboutachfaiti Redouan a, * , Delattre Cedric b,c , Petit Emmanuel a , El Gadda Mohamed d , Courtois Bernard a , Michaud Philippe b , El Modafar Cherkaoui d , Courtois Josiane a a LPMV, Université de Picardie Jules Verne, IUT-GB Amiens, Avenue des facultés, Le Bailly, F-80025 Amiens Cedex 1, France b LGCB, Université Blaise Pascal-Polytech Clermont Ferrand, 24 rue des Landais, F-63174 Aubière, France c GREENTECH, Biopôle Clermont Limagne 63360 Saint Beauzire, France d LBVPA, Université Cadi Ayyad, Faculté des Sciences et Techniques Guéliz B.P. 549, Avenue Abdelkarim El Khattabi 40 000 Marrakech, Maroc article info Article history: Received 9 March 2009 Received in revised form 26 April 2009 Accepted 8 May 2009 Available online 7 June 2009 Keywords: Glucuronan Green seaweed Oligosaccharides Polysaccharide lyase Glucuronic acid oligosaccharides Ulva lactuca abstract A new source for the production of bioactive glucuronic acid oligosaccharides (GlcUAOs) from the depo- lymerization of green seaweed Ulva lactuca glucuronan (Algal glucuronan) has been investigated. Algal glucuronan purification was optimized by the acidic precipitation method which allowed us to separate the polysaccharide mixture extracted from the cell wall of Ulva lactuca using hot water containing sodium oxalate. A series of the GlcUAOs were obtained by enzyme degradation of algal glucuronan with a glucuronan lyase (GL) isolated from Trichoderma strain. The putative bioactive GlcUAOs generated were then purified by size-exclusion chromatography in gram quantity and characterized by 1 H/ 13 C NMR spec- troscopy and ESI-Q/TOF-mass spectrometry. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Homopolyuronides are anionic polysaccharides presenting many biological interests and industrial applications. It is described in the literature that apart from polygalacturonate and alginate, the structural variability of this class of acidic polysaccha- rides is poor and low. Consequently, new generation of the homopolyuronides has been developed. Firstly, the emergence of 2,2,6,6-tetramethylpiperidine-1-oxy (TEMPO) chemistry has led to regioselective oxidation of polysaccharides. 1 When applied to cellulose, this specific oxidation of primary alcohol function gener- ated b-(1,4)-D-polyglucuronic acids (synthetic glucuronans). 2 Secondly, the bacterial strain Sinorhizobium meliloti M5N1CS was isolated for its ability to excrete variably acetylated b-(1,4)-D- polyglucuronic acids called glucuronans (bacterial glucuronans). 3,4 The interest of the scientific community for this new family of acidic polysaccharides was mostly motivated by its rheological and biological properties. Effectively, this glucuronan form may be employed as gelifying, thickening, hydrating, stabilizing, chelating, or flocculating agent, 5 and also as a biologically active polysaccharide in therapy and agronomy. 5–9 In the two latter cases, low-molecular weight glucuronan and glucuronic acid oligosac- charides (GlcUAOs) seem to be more active than the native polymer. This phenomenon is often described for oligosaccharides showing biological activities. 10 It is true that bacterial glucuronans appear to have some advan- tages over the synthetic glucuronans, generally because they are non-toxic, less expensive, and freely available. Moreover, appropri- ate strains can be genetically modified to acquire a product with desired properties. 11 In addition, several bacterial oligo- and poly- saccharides have been reported, while only a few of them have been developed on a commercial scale because of the pathogenic nature of certain producer organisms. 12 For this reason, new sources of natural glucuronan have been explored. Previous studies have revealed the presence of b-(1,4)-D-poly glucuronic acids (algal glucuronans) in the cell walls of a number of green seaweeds. 13 It was reported that the algal glucuronan was co-extracted together with ulvans, major water-soluble polysaccharides usually extracted from the cell wall of Ulva sp. using hot water often containing a calcium chelating agent such as sodium oxalate. 14 This algal glucuronan poses several problems for the fine chemical structure analysis of the ulvans. Thus, in previous studies some authors have successfully used the ion-ex- change chromatography to eliminate the algal glucuronan. 14,15 Nevertheless, these chromatographic techniques, which are labor-intensive and time-consuming, pose real obstacles and limit the industrial scale valorization of algal glucuronan. 0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2009.05.031 * Corresponding author. Tel.: +33 3 2253494; fax: +33 3 22956254. E-mail address: redouan_elboutachfaiti@hotmail.com (E. Redouan). Carbohydrate Research 344 (2009) 1670–1675 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres