Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-agarase and HCl in liquid chromatography systems
ABSTRACT A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.
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ABSTRACT: Many researchers have utilised undigested sulfated polysaccharides as effective in vitro antiviral agents; however, their in vivo activity remains controversial. Here we report the utility of novel low-degree-polymerisation (low-DP) sulfated saccharides from two algae, Gracilaria sp. and Monostroma nitidum, in the prevention of Japanese encephalitis virus (JEV) infection both in vitro and in vivo. During in vitro studies performed by MTT or plaque assays, low-DP sulfated saccharides have slightly lower antiviral activities than their undigested polysaccharides (PS). However low-DP sulfated saccharides bind to the JEV envelope protein at least as strongly as PS. In addition, the low-DP sulfated saccharides have a distinctly higher positive effect on survivability in JEV-infected C3H/HeN mice in comparison to PS. The in vivo antiviral activity seems to be connected with better absorption of low-DP sulfated saccharides than undigested PS. Our results point out that the low-DP sulfated saccharides are promising candidates for further development as antiviral agents.Food Chemistry 08/2012; DOI:10.1016/j.foodchem.2012.01.106 · 3.26 Impact Factor
Article: Polysaccharides of the red algae.[Show abstract] [Hide abstract]
ABSTRACT: Red algae (Rhodophyta) are known as the source of unique sulfated galactans, such as agar, agarose, and carrageenans. The wide practical uses of these polysaccharides are based on their ability to form strong gels in aqueous solutions. Gelling polysaccharides usually have molecules built up of repeating disaccharide units with a regular distribution of sulfate groups, but most of the red algal species contain more complex galactans devoid of gelling ability because of various deviations from the regular structure. Moreover, several red algae may contain sulfated mannans or neutral xylans instead of sulfated galactans as the main structural polysaccharides. This chapter is devoted to a description of the structural diversity of polysaccharides found in the red algae, with special emphasis on the methods of structural analysis of sulfated galactans. In addition to the structural information, some data on the possible use of red algal polysaccharides as biologically active polymers or as taxonomic markers are briefly discussed.Advances in Carbohydrate Chemistry and Biochemistry 01/2011; 65:115-217. DOI:10.1016/B978-0-12-385520-6.00004-2 · 3.92 Impact Factor
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ABSTRACT: A series of linear glucan saccharides (GS) with defined quantity and degree of polymerization (DP) were synthesized from α-d-glucose 1-phosphate (α-d-Glc 1-P) by phosphorylase-a. The GS product fractions with average DP 11, 22, 38, 52, 60, 70, and 79 were measured by HPSEC-ELSD system. Then the same seven fractions were resolved into individual peaks with DP: 6–14, 10–32, 27–55, 37–67, 44–75, 49–83 and 53–89 by HPAEC-PAD system. Results showed that measurement of α-d-Glc 1-P amount consuming during GS synthesis by both systems enable calculation of reaction yield. The reaction yield for the 24 h biosynthesis of the GS product was 25.3% (measured by HPSEC-ELSD) or 29.1% (measured by HPAEC-PAD). The HPSEC-ELSD and HPAEC-PAD systems were also successfully used for phosphorylase-a activity measurement in order to perform its kinetic characterization. This study established feasible systems for preparation of various sizes of the GS with defined DP and quantity as well as characterization of phosphorylase-a kinetics.Carbohydrate Polymers 06/2014; 106:209–216. DOI:10.1016/j.carbpol.2014.01.101 · 3.92 Impact Factor