Size exclusion chromatography for the quantitative profiling of the enzyme-catalyzed hydrolysis of xylo-oligosaccharides

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 CH3COONa 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.