Size Exclusion Chromatography for the Quantitative Profiling of the Enzyme-Catalyzed Hydrolysis of Xylo-oligosaccharides

Abstract

High-performance size exclusion chromatography (HPSEC) is a widely used method for the qualitative profiling of oligosaccharide mixtures, including, for example, enzymatic hydrolysates of plant biomass materials. A novel method employing HPSEC for the quantitative analytical profiling of the progress of enzymatic hydrolysis of different xylan substrates was developed. The method relies on dividing the HPSEC elution profiles into fixed time intervals and utilizing the linear refractive index response (area under the curve) of defined standard compounds. To obtain optimal HPSEC profiles, the method was designed using 0.1 M CH(3)COONa both in the mobile phase and as the sample solution matrix, after systematic evaluation of the influence of the mobile phase, including the type, ionic strength, and pH, on the refractive index detector response. A time study of the enzyme-catalyzed hydrolysis of birchwood xylan and wheat bran by a Bacillus subtilis XynA xylanase (GH 11) was used as an example to demonstrate the workability of the HPSEC method for obtaining progress curves describing the evolution in the product profile during enzyme catalysis.

Validated HPAEC-PAD Method for the Determination of Fully Deacetylated Chitooligosaccharides

“…These elution behaviors are similar to those seen with high-performance size exclusion chromatography (HPSEC), which has been widely used for polysaccharide separation. Separation using HPSEC relies on a differentiation in the time of permeation of the polysaccharides into and out of the pores of the column, and this is related to the molecular size [42]. Vårum reported that when separating COS on a size exclusion column, the one with the highest DP eluted first [43].…”

Structural features underlying prebiotic activity of conventional and potential prebiotic oligosaccharides in food and health

“…Poly/oligosaccharides are characterized by a combination of chemical and spectroscopic techniques. High‐performance size‐exclusion chromatography (SEC) is a popular approach for the qualitative profiling of OS mixtures (Rasmussen & Meyer, ). SEC has been demonstrated to be the most effective among a number of fractionation techniques to obtain prebiotic GaOS (Hernández, Ruiz‐Matute, Olano, Moreno, & Sanz, ).…”

Selection of Bacillus species for targeted in situ release of prebiotic galacto-rhamnogalacturonan from potato pulp in piglets

“…100 mM sodium acetate (pH 6) with 0.02 % sodium azide was used as mobile phase and sample solution matrix and samples were separated on a Shodex SB-806 HQ GPC column 300x8mm) with a Shodex SB-Gguard column (50x6 mm) from Showa Denko K.K. (Tokyo, Japan) as described previously (Rasmussen and Meyer 2010). The precipitated material was dissolved to 5 mg/mL by vigorous shaking for 6 h at 60 °C, centrifuged (8000 g, 4 min) and filtered using a 0.22 μm syringe filter.…”