Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by β-agarase and HCl in liquid chromatography systems

Abstract

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by β-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 4 U/mg β-agarase and for AOS was 45.6% by 0.4 M 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 1H 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.