Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by β-agarase and HCl in liquid chromatography systems
Department of Food Science, Biotechnology Division, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung 20224, Taiwan. Carbohydrate Research
(Impact Factor: 1.93).
10/2008; 343(14):2443-50. DOI: 10.1016/j.carres.2008.06.019
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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- "The developed reaction products were visualized by spraying 10% (v/v) sulfuric acid in ethanol and heating at 90 • C. For quantification, the reaction products were separated and purified to a pure form by using a recycling preparative high-performance liquid chromatography (HPLC) (LC-9104, Japan Analytical Industry Co., Ltd, Japan) with JAI GEL 252W and 251W columns (Japan Analytical Industry Co., Ltd, Japan). Each of the purified samples was identified by TLC again and mass spectrometry (MS) (micrOTOF-Q II, Bruker Daltonik, Germany) and HPLC with a Asahipak NH2P-50 4E (250 mm × 4.6 mm, Shodex, Japan) column and ELSD detector (Sedex 75, Sedere, France) under conditions as previously described  "
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ABSTRACT: The gene of agaG1 from Alteromonas sp. GNUM1 encoding a β-agarase (AgaG1) was heterologously expressed in E. coli BL21 (DE3). The recombinant strain was cultured at 37 °C and then AgaG1 was expressed at 25 °C and 0.5 mM IPTG. The optimum conditions for AgaG1 to hydrolyze agarose were pH 7.0 and 40 °C. The main products of agarose hydrolysis by AgaG1 were confirmed to be neoagarobiose and neoagarotetraose. A new agarose hydrolysis process using AgaG1 was developed, in which the reaction temperature was adjusted stepwise to avoid gelation problem with no chemical pretreatment step. The enzyme AgaG1 was found to be very effective and highly selective. When 10.0 g/L agarose was hydrolyzed, 98% of the agarose added was converted to 3.8 and 6.4 g/L of neoagarobiose and neoagarotetraose, respectively.
Available from: Bartosz Kazłowski
- "ESI-MS: samples were analysed by the VG platform singlequadrupole mass spectrometer (Micromass, Altrincham, UK) equipped with a capillary electrophoresis (CE) interface. The interface utilised a triaxial flow arrangement whereby the CE eluent was mixed with a suitable sheath liquid at the probe tip and then nebulised using N 2 gas (Kazłowski et al., 2008). "
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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.
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ABSTRACT: This study was to investigate near-infrared reflectance spectroscopy (NIRS) application for determination of individual soluble carbohydrate content in soybean seeds for development of high-quality new soybean variety and quality control of soy products. NIRS was used as a rapid and simultaneous analysis method to determine individual soluble carbohydrate content of soybean seeds in Korea. A total of 240 samples (about 2 g) were scanned in the reflectance mode and the reference values for the individual soluble carbohydrate contents were measured by high performance liquid chromatography. Calibration equation for individual soluble carbohydrate contents were developed using modified partial least-squares regression with internal cross validation (n=155). The equation for sucrose content had lower standard error of cross-validation, higher R2 (coefficient of determination in calibration), and higher ratio of unexplained variance divided by variance (1-VR) value than that for raffinose, and stachyose. Prediction of an external validation set (n=85) showed significant correlation between reference value and NIRS estimated value based on the standard error of prediction (SEP), r2 (coefficient of determination in prediction), and the ratio of standard deviation (SD) of reference data to SEP. The model for sucrose had higher value of RSP (ratio of standard deviation for prediction, the ratio of SD of reference data to SEP(C) in the external validation set) and r2 (3.50 and 0.921, respectively), thereby that equation has good quantitative accuracy, whereas those of raffinose, and stachyose had lower value (below 0.89 and 0.45, respectively). These results indicated that NIRS could be used to rapidly determine the content of sucrose in soybean seed samples in the breeding programs for development of high-quality new soybean variety.
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